U.S. patent application number 17/154829 was filed with the patent office on 2021-06-24 for methods and materials for detecting viral or microbial infections.
This patent application is currently assigned to Cascade Biosystems, Inc.. The applicant listed for this patent is Cascade Biosystems, Inc.. Invention is credited to Mariya Smit, Kenneth D. Smith, Nina Yazvenko.
Application Number | 20210189465 17/154829 |
Document ID | / |
Family ID | 1000005444458 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210189465 |
Kind Code |
A1 |
Smith; Kenneth D. ; et
al. |
June 24, 2021 |
METHODS AND MATERIALS FOR DETECTING VIRAL OR MICROBIAL
INFECTIONS
Abstract
This document provides methods and materials for detecting
target nucleic acid. For example, methods and materials for
detecting the presence or absence of target nucleic acid, methods
and materials for detecting the amount of target nucleic acid
present within a sample, kits for detecting the presence or absence
of target nucleic acid, kits for detecting the amount of target
nucleic acid present within a sample, and methods for making such
kits are provided.
Inventors: |
Smith; Kenneth D.; (Colfax,
WI) ; Yazvenko; Nina; (Vancouver, WA) ; Smit;
Mariya; (Vancouver, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cascade Biosystems, Inc. |
Colfax |
WI |
US |
|
|
Assignee: |
Cascade Biosystems, Inc.
Colfax
WI
|
Family ID: |
1000005444458 |
Appl. No.: |
17/154829 |
Filed: |
January 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16146419 |
Sep 28, 2018 |
10927420 |
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17154829 |
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15225365 |
Aug 1, 2016 |
10113204 |
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16146419 |
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14841289 |
Aug 31, 2015 |
9428814 |
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15225365 |
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14080388 |
Nov 14, 2013 |
9150935 |
|
|
14841289 |
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13028021 |
Feb 15, 2011 |
8597886 |
|
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14080388 |
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61304784 |
Feb 15, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/705 20130101;
C12Q 1/689 20130101; C12Q 1/683 20130101; C12Q 1/701 20130101 |
International
Class: |
C12Q 1/689 20060101
C12Q001/689; C12Q 1/683 20060101 C12Q001/683; C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A method for assessing a mammal for an infection, said method
comprising: (a) contacting a sample from said mammal with a probe
nucleic acid comprising an amplifying restriction endonuclease and
a nucleotide sequence complementary to a sequence of a target
nucleic acid present within a microorganism or virus under
conditions wherein, if said target nucleic acid is present in said
sample, at least a portion of said target nucleic acid hybridizes
to at least a portion of said probe nucleic acid to form a
double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site, (b) contacting said double-stranded portion
of nucleic acid with a recognition restriction endonuclease having
the ability to cut said double-stranded portion of nucleic acid at
said restriction endonuclease cut site under conditions wherein
said recognition restriction endonuclease cleaves said
double-stranded portion of nucleic acid at said restriction
endonuclease cut site, thereby separating a portion of said probe
nucleic acid comprising said amplifying restriction endonuclease
from at least another portion of said probe nucleic acid, (c)
contacting said portion of said probe nucleic acid comprising said
amplifying restriction endonuclease with a reporter nucleic acid
comprising a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site of said amplifying restriction
endonuclease under conditions wherein said amplifying restriction
endonuclease cleaves said reporter nucleic acid at said restriction
endonuclease cut site of said amplifying restriction endonuclease,
thereby separating a portion of said reporter nucleic acid from at
least another portion of said reporter nucleic acid, and (d)
determining the presence or absence of said portion of said
reporter nucleic acid, wherein the presence of said portion of said
reporter nucleic acid indicates that said sample contains said
target nucleic acid, thereby indicating that said mammal is
infected with said microorganism or virus, and wherein the absence
of said portion of said reporter nucleic acid indicates that said
sample does not contain said target nucleic acid, thereby
indicating that said mammal is not infected with said microorganism
or virus.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said mammal is a farm animal
selected from the group consisting of bovine, porcine, and equine
species.
4. The method of claim 1, wherein said mammal is a dog or cat.
5. The method of claim 1, wherein said infection is a microbial
infection, and wherein said target nucleic acid is present within a
microorganism.
6. The method of claim 1, wherein said infection is a viral
infection, and wherein said target nucleic acid is present within a
virus.
7. The method of claim 1, wherein said sample comprises a nasal or
throat swab sample.
8. The method of claim 1, wherein said sample is selected from the
group consisting of nasal samples, throat samples, sputum samples,
bronchial lavage samples, tissue samples, cellular samples, and
blood samples.
9. The method of claim 1, wherein, prior to step (a), said sample
was cultured to enrich the population of microorganisms or viruses,
if present, within said sample.
10. The method of claim 9, wherein said sample was cultured for at
least 30 minutes in the presence of enrichment medium.
11. The method of claim 1, wherein, prior to step (a), said sample
was processed to remove non-nucleic acid material from said sample,
thereby increasing the concentration of nucleic acid, if present,
within said sample.
12. The method of claim 11, wherein said sample was subjected to a
nucleic acid extraction technique.
13. The method of claim 1, wherein, prior to step (a), said sample
was subjected to a nucleic acid amplification technique to increase
the concentration of microbial or viral nucleic acid, if present,
within said sample.
14. The method of claim 13, wherein said sample was subjected to a
PCR-based technique designed to amplify said target nucleic
acid.
15. The method of claim 1, wherein, prior to step (a), said method
comprises (i) culturing said sample to enrich the population of
microorganisms or viruses, if present, within said sample and
removing non-nucleic acid material from said sample, thereby
increasing the concentration of nucleic acid, if present, within
said sample or (ii) culturing said sample to enrich the population
of microorganisms or viruses, if present, within said sample,
removing non-nucleic acid material from said sample, thereby
increasing the concentration of nucleic acid, if present, within
said sample, and performing a nucleic acid amplification technique
to increase the concentration of microbial or viral nucleic acid,
if present, within said sample.
16. The method of claim 1, wherein said probe nucleic acid is
single-stranded probe nucleic acid.
17. The method of claim 1, wherein said determining step comprises
determining the amount of said target nucleic acid present within
said sample.
18. A method for assessing a mammal for an infection, said method
comprising: (a) contacting a sample from said mammal with a probe
nucleic acid comprising an initial amplifying restriction
endonuclease and a nucleotide sequence complementary to a sequence
of a target nucleic acid present within a microorganism or virus
under conditions wherein, if said target nucleic acid is present in
said sample, at least a portion of said target nucleic acid
hybridizes to at least a portion of said probe nucleic acid to form
a double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site, (b) contacting said double-stranded portion
of nucleic acid with a recognition restriction endonuclease having
the ability to cut said double-stranded portion of nucleic acid at
said restriction endonuclease cut site under conditions wherein
said recognition restriction endonuclease cleaves said
double-stranded portion of nucleic acid at said restriction
endonuclease cut site, thereby separating a portion of said probe
nucleic acid comprising said initial amplifying restriction
endonuclease from at least another portion of said probe nucleic
acid, (c) contacting said portion of said probe nucleic acid
comprising said initial amplifying restriction endonuclease with a
first nucleic acid comprising a secondary amplifying restriction
endonuclease and a double-stranded portion of nucleic acid
comprising a restriction endonuclease cut site of said initial
amplifying restriction endonuclease under conditions wherein said
initial amplifying restriction endonuclease cleaves said first
nucleic acid at said restriction endonuclease cut site of said
initial amplifying restriction endonuclease, thereby separating a
portion of said first nucleic acid comprising said secondary
amplifying restriction endonuclease from at least another portion
of said first nucleic acid, (d) contacting said portion of said
first nucleic acid comprising said secondary amplifying restriction
endonuclease with a second nucleic acid comprising said initial
amplifying restriction endonuclease and a double-stranded portion
of nucleic acid comprising a restriction endonuclease cut site of
said secondary amplifying restriction endonuclease under conditions
wherein said secondary amplifying restriction endonuclease cleaves
said second nucleic acid at said restriction endonuclease cut site
of said secondary amplifying restriction endonuclease, thereby
separating a portion of said second nucleic acid comprising said
initial amplifying restriction endonuclease from at least another
portion of said second nucleic acid, (e) contacting said portion of
said second nucleic acid comprising said initial amplifying
restriction endonuclease with a reporter nucleic acid comprising a
double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site of said initial amplifying restriction
endonuclease under conditions wherein said initial amplifying
restriction endonuclease cleaves said reporter nucleic acid at said
restriction endonuclease cut site of said initial amplifying
restriction endonuclease, thereby separating a portion of said
reporter nucleic acid from at least another portion of said
reporter nucleic acid, and (f) determining the presence or absence
of said portion of said reporter nucleic acid, wherein the presence
of said portion of said reporter nucleic acid indicates that said
sample contains said target nucleic acid, thereby indicating that
said mammal is infected with said microorganism or virus, and
wherein the absence of said portion of said reporter nucleic acid
indicates that said sample does not contain said target nucleic
acid, thereby indicating that said mammal is not infected with said
microorganism or virus.
19. A method for assessing a mammal for an infection, said method
comprising: (a) contacting a sample from said mammal with a probe
nucleic acid comprising an initial amplifying restriction
endonuclease and a nucleotide sequence complementary to a sequence
of a target nucleic acid present within a microorganism or virus
under conditions wherein, if said target nucleic acid is present in
said sample, at least a portion of said target nucleic acid
hybridizes to at least a portion of said probe nucleic acid to form
a double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site, (b) contacting said double-stranded portion
of nucleic acid with a recognition restriction endonuclease having
the ability to cut said double-stranded portion of nucleic acid at
said restriction endonuclease cut site under conditions wherein
said recognition restriction endonuclease cleaves said
double-stranded portion of nucleic acid at said restriction
endonuclease cut site, thereby separating a portion of said probe
nucleic acid comprising said initial amplifying restriction
endonuclease from at least another portion of said probe nucleic
acid, (c) contacting said portion of said probe nucleic acid
comprising said initial amplifying restriction endonuclease with a
first reporter nucleic acid comprising a secondary amplifying
restriction endonuclease and a double-stranded portion of nucleic
acid comprising a restriction endonuclease cut site of said initial
amplifying restriction endonuclease under conditions wherein said
initial amplifying restriction endonuclease cleaves said first
reporter nucleic acid at said restriction endonuclease cut site of
said initial amplifying restriction endonuclease, thereby
separating a portion of said first nucleic acid comprising said
secondary amplifying restriction endonuclease from at least another
portion of said first nucleic acid, (d) contacting said portion of
said first reporter nucleic acid comprising said secondary
amplifying restriction endonuclease with a second reporter nucleic
acid comprising said initial amplifying restriction endonuclease
and a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site of said secondary amplifying
restriction endonuclease under conditions wherein said initial
amplifying restriction endonuclease cleaves said second nucleic
acid at said restriction endonuclease cut site of said secondary
amplifying restriction endonuclease, thereby separating a portion
of said second nucleic acid comprising said initial amplifying
restriction endonuclease from at least another portion of said
second nucleic acid, and (e) determining the presence or absence of
said portion of said first reporter nucleic acid, said second
reporter nucleic acid, or both said first reporter nucleic acid and
said second reporter nucleic acid, wherein said presence indicates
that said sample contains said target nucleic acid, thereby
indicating that said mammal is infected with said microorganism or
virus, and wherein said absence indicates that said sample does not
contain said target nucleic acid, thereby indicating that said
mammal is not infected with said microorganism or virus.
20. A kit for assessing a mammal for an infection, said kit
comprises a probe nucleic acid comprising an amplifying restriction
endonuclease and a nucleotide sequence complementary to a sequence
of a target nucleic acid present in a microorganism or virus,
wherein at least a portion of said target nucleic acid is capable
of hybridizing to at least a portion of said probe nucleic acid to
form a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 16/146,419, filed Sep. 28, 2018, which is a continuation of
U.S. application Ser. No. 15/225,365, filed Aug. 1, 2016 (now U.S.
Pat. No. 10,113,204), which is a continuation of U.S. application
Ser. No. 14/841,289, filed Aug. 31, 2015 (now U.S. Pat. No.
9,428,814), which is a continuation of U.S. application Ser. No.
14/080,388, filed Nov. 14, 2013 (now U.S. Pat. No. 9,150,935),
which is a continuation of U.S. application Ser. No. 13/028,021,
filed Feb. 15, 2011 (now U.S. Pat. No. 8,597,886), which claims the
benefit of priority of U.S. Provisional Application Ser. No.
61/304,784, filed Feb. 15, 2010. The disclosures of the prior
applications are considered part of (and are incorporated by
reference in) the disclosure of this application.
BACKGROUND
1. Technical Field
[0002] This document relates to methods and materials involved in
detecting viral and/or microbial infections. For example, this
document relates to methods and materials involved in using an
enzymatic amplification cascade of restriction endonucleases to
detect nucleic acid of a virus or microbe (e.g., a pathogen) within
a sample (e.g., a biological sample such as a nasal swab sample)
being tested, thereby assessing a mammal for a possible
infection.
2. Background
[0003] Many different viruses and microbes can infect mammals and
cause harmful infections. For example, bacteria such as
Staphylococcus, Streptococcus, and Haemophilus species as well as
viruses such as influenza virus A and B, adenovirus 4, respiratory
syncytial virus (RSV), and parainfluenza types 1, 2, and 3 can
cause upper respiratory infections in humans with varying degrees
of clinical symptoms. In some cases, if left undiagnosed and/or
untreated, such infections may increase is duration and/or
severity.
SUMMARY
[0004] This document provides methods and materials for detecting
viral and/or microbial infections. For example, this document
provides methods and materials related to the use of an enzymatic
amplification cascade of restriction endonucleases to detect
nucleic acid of a virus or microbe (e.g., a pathogen) within a
sample (e.g., a biological sample such as a blood sample, mucus
sample, or saliva sample) being tested, thereby assessing a mammal
for a possible infection. In some cases, this document provides
methods and materials for detecting a target microorganism's or
virus' nucleic acid. For example, this document provides methods
and materials for detecting the presence or absence of target
nucleic acid (e.g., a target pathogen's nucleic acid) within a
sample (e.g., a biological sample), methods and materials for
detecting the amount of target nucleic acid (e.g., a target
pathogen's nucleic acid) present within a sample (e.g., a
biological sample), kits for detecting the presence or absence of
target nucleic acid (e.g., a target pathogen's nucleic acid) within
a sample (e.g., a biological sample), kits for detecting the amount
of target nucleic acid (e.g., a target pathogen's nucleic acid)
present within a sample (e.g., a biological sample), and methods
for making such kits.
[0005] In general, the methods and materials provided herein can
include performing an enzymatic amplification cascade of
restriction endonucleases as described herein to detect a target
microorganism's or virus's nucleic acid (e.g., a target pathogen's
nucleic acid) in a sample (e.g., a biological sample) in a manner
that is rapid, inexpensive, sensitive, and specific. For example, a
biological sample can be obtained from a mammal (e.g., a human)
and/or processed such that target microbial or viral nucleic acid
(e.g., target pathogen nucleic acid), if present within the sample,
is capable of hybridizing to probe nucleic acid of an enzymatic
amplification cascade of restriction endonucleases described
herein. In some cases, such an obtained and/or processed biological
sample can be assessed for the presence, absence, or amount of
target microbial or viral nucleic acid (e.g., target pathogen
nucleic acid) using an enzymatic amplification cascade of
restriction endonucleases described herein without using a nucleic
acid amplification technique (e.g., a PCR-based nucleic acid
technique). Assessing samples (e.g., biological samples) for the
presence, absence, or amount of target nucleic acid using an
enzymatic amplification cascade of restriction endonucleases
described herein without using a nucleic acid amplification
technique can allow patients as well as medical, laboratory, or
veterinarian personnel (e.g., clinicians, physicians, physician's
assistants, laboratory technicians, research scientists, and
veterinarians) to test mammals for possible infections using a
nucleic acid-based assay without the need for potentially expensive
thermal cycling devices and potentially time consuming thermal
cycling techniques. In addition, the methods and materials provided
herein can allow patients as well as medical, laboratory, or
veterinarian personnel to detect an infection by any type of
microbial organism (e.g., a microbial pathogen) or virus (e.g., a
viral pathogen) suspected of infecting a mammal. For example, the
methods and materials provided herein can be used to detect the
presence or absence of a Staphylococcus aureus infection in a
human.
[0006] In general, one aspect of this document features a method
for assessing a mammal for an infection. The method comprises, or
consists essentially of, (a) contacting a sample from the mammal
with a probe nucleic acid comprising an amplifying restriction
endonuclease and a nucleotide sequence complementary to a sequence
of a target nucleic acid present within a microorganism or virus
under conditions wherein, if the target nucleic acid is present in
the sample, at least a portion of the target nucleic acid
hybridizes to at least a portion of the probe nucleic acid to form
a double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site, (b) contacting the double-stranded portion
of nucleic acid with a recognition restriction endonuclease having
the ability to cut the double-stranded portion of nucleic acid at
the restriction endonuclease cut site under conditions wherein the
recognition restriction endonuclease cleaves the double-stranded
portion of nucleic acid at the restriction endonuclease cut site,
thereby separating a portion of the probe nucleic acid comprising
the amplifying restriction endonuclease from at least another
portion of the probe nucleic acid, (c) contacting the portion of
the probe nucleic acid comprising the amplifying restriction
endonuclease with a reporter nucleic acid comprising a
double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site of the amplifying restriction endonuclease
under conditions wherein the amplifying restriction endonuclease
cleaves the reporter nucleic acid at the restriction endonuclease
cut site of the amplifying restriction endonuclease, thereby
separating a portion of the reporter nucleic acid from at least
another portion of the reporter nucleic acid, and (d) determining
the presence or absence of the portion of the reporter nucleic
acid, wherein the presence of the portion of the reporter nucleic
acid indicates that the sample contains the target nucleic acid,
thereby indicating that the mammal is infected with the
microorganism or virus, and wherein the absence of the portion of
the reporter nucleic acid indicates that the sample does not
contain the target nucleic acid, thereby indicating that the mammal
is not infected with the microorganism or virus. The mammal can be
a human. The mammal can be a farm animal selected from the group
consisting of bovine, porcine, and equine species. The mammal can
be a dog or cat. The infection can be a microbial infection, and
wherein the target nucleic acid is present within a microorganism.
The infection can be a viral infection, and the target nucleic acid
can be present within a virus. The sample can comprise a nasal or
throat swab sample. The sample can be selected from the group
consisting of nasal samples, throat samples, sputum samples,
bronchial lavage samples, tissue samples, cellular samples, and
blood samples. Prior to step (a), the sample can be a sample that
was cultured to enrich the population of microorganisms or viruses,
if present, within the sample. The sample can be a sample that was
cultured for at least 30 minutes in the presence of enrichment
medium. Prior to step (a), the sample can be a sample that was
processed to remove non-nucleic acid material from the sample,
thereby increasing the concentration of nucleic acid, if present,
within the sample. The sample can be a sample that was subjected to
a nucleic acid extraction technique. Prior to step (a), the sample
can be a sample that was subjected to a nucleic acid amplification
technique to increase the concentration of microbial or viral
nucleic acid, if present, within the sample. The sample can be a
sample that was subjected to a PCR-based technique designed to
amplify the target nucleic acid. Prior to step (a), the method can
comprise culturing the sample to enrich the population of
microorganisms or viruses, if present, within the sample. The
culturing can comprise culturing the sample for at least 30 minutes
in the presence of enrichment medium. Prior to step (a), the method
can comprise removing non-nucleic acid material from the sample,
thereby increasing the concentration of nucleic acid, if present,
within the sample. The removing can comprise performing a nucleic
acid extraction technique. Prior to step (a), the method can
comprise performing a nucleic acid amplification technique to
increase the concentration of microbial or viral nucleic acid, if
present, within the sample. The nucleic acid amplification
technique can comprise a PCR-based technique designed to amplify
the target nucleic acid. Prior to step (a), the method can comprise
(i) culturing the sample to enrich the population of microorganisms
or viruses, if present, within the sample and removing non-nucleic
acid material from the sample, thereby increasing the concentration
of nucleic acid, if present, within the sample or (ii) culturing
the sample to enrich the population of microorganisms or viruses,
if present, within the sample, removing non-nucleic acid material
from the sample, thereby increasing the concentration of nucleic
acid, if present, within the sample, and performing a nucleic acid
amplification technique to increase the concentration of microbial
or viral nucleic acid, if present, within the sample. The probe
nucleic acid can be single-stranded probe nucleic acid. The probe
nucleic acid can be attached to a solid support. The probe nucleic
acid can be directly attached to a solid support. The portion of
the probe nucleic acid comprising the amplifying restriction
endonuclease can be released from the solid support via the step
(b). Step (a) and step (b) can be performed in the same
compartment, or step (a), step (b), and step (c) can be performed
in the same compartment, or step (a), step (b), step (c), and step
(d) can be performed in the same compartment. Step (a) and step (b)
can be performed in a first compartment, and step (c) can be
performed in a second compartment. Step (a) and step (b) can be
performed by adding the sample to a compartment comprising the
probe nucleic acid and the recognition restriction endonuclease.
The probe nucleic acid can comprise (i) a single-stranded portion
comprising the nucleotide sequence complementary to the sequence of
the target nucleic acid and (ii) a double-stranded portion. The
probe nucleic acid can comprise a first nucleic acid strand
comprising the nucleotide sequence complementary to the sequence of
the target nucleic acid hybridized to a second nucleic acid strand
comprising the amplifying restriction endonuclease. The first
nucleic acid strand can be attached to a solid support. The first
nucleic acid strand can be directly attached to a solid support. A
portion of the second nucleic acid strand can hybridize with the
first nucleic acid strand to form the double-stranded portion. The
portion of the probe nucleic acid comprising the amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can comprise
a portion of the first nucleic acid strand and all of the second
strand. The portion of the probe nucleic acid comprising the
amplifying restriction endonuclease that is separated from the at
least another portion of the probe nucleic acid in step (b) can
comprise at least a portion of the target nucleic acid.
[0007] In some cases, the method can comprise using a plurality of
the probe nucleic acid in the step (a). The method can comprise
using a plurality of the reporter nucleic acid in the step (c). The
reporter nucleic acid in the step (c) can be in molar excess of the
portion of the probe nucleic acid comprising the amplifying
restriction endonuclease from the step (b). The number of molecules
of the portion of the probe nucleic acid comprising the amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can be in an
essentially linear relationship to the number of molecules of the
target nucleic acid present in the sample. The reporter nucleic
acid can be attached to a solid support. The reporter nucleic acid
can be directly attached to a solid support. The reporter nucleic
acid can comprise a single-stranded portion of nucleic acid. The
reporter nucleic acid can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. The portion of the reporter nucleic acid that is separated
from the at least another portion of the reporter nucleic acid can
comprise the label. The reporter nucleic acid can comprise a first
nucleic acid strand comprising the label hybridized to a second
nucleic acid strand. The second nucleic acid strand can be attached
to a solid support. The second nucleic acid strand can be directly
attached to a solid support. A portion of the first nucleic acid
strand can hybridize with the second nucleic acid strand to form
the double-stranded portion of nucleic acid comprising the
restriction endonuclease cut site of the amplifying restriction
endonuclease. The reporter nucleic acid can comprise a third
nucleic acid strand. The third nucleic acid strand can hybridize
with the second nucleic acid strand to form the double-stranded
portion of nucleic acid comprising the restriction endonuclease cut
site of the amplifying restriction endonuclease. The reporter
nucleic acid can be attached to a solid support, and the portion of
the reporter nucleic acid that is separated from the at least
another portion of the reporter nucleic acid and that comprises the
label can be released from the solid support via the step (c). The
determining step (d) can comprise detecting the label. The label
can be a fluorescent label, and the determining step (d) comprises
detecting the fluorescent label. The determining step (d) can
comprise detecting the portion of the reporter nucleic acid
separated from the at least another portion of the reporter nucleic
acid using a capillary electrophoresis technique. Steps (a), (b),
and (c) can be performed without nucleic acid amplification, or
steps (a), (b), (c), and (d) can be performed without nucleic acid
amplification. The determining step can comprise determining the
amount of the target nucleic acid present within the sample.
[0008] In another aspect, this document features a method for
assessing a mammal for an infection. The method comprises, or
consists essentially of, (a) contacting a sample from the mammal
with a probe nucleic acid comprising an initial amplifying
restriction endonuclease and a nucleotide sequence complementary to
a sequence of a target nucleic acid present within a microorganism
or virus under conditions wherein, if the target nucleic acid is
present in the sample, at least a portion of the target nucleic
acid hybridizes to at least a portion of the probe nucleic acid to
form a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site, (b) contacting the
double-stranded portion of nucleic acid with a recognition
restriction endonuclease having the ability to cut the
double-stranded portion of nucleic acid at the restriction
endonuclease cut site under conditions wherein the recognition
restriction endonuclease cleaves the double-stranded portion of
nucleic acid at the restriction endonuclease cut site, thereby
separating a portion of the probe nucleic acid comprising the
initial amplifying restriction endonuclease from at least another
portion of the probe nucleic acid, (c) contacting the portion of
the probe nucleic acid comprising the initial amplifying
restriction endonuclease with a first nucleic acid comprising a
secondary amplifying restriction endonuclease and a double-stranded
portion of nucleic acid comprising a restriction endonuclease cut
site of the initial amplifying restriction endonuclease under
conditions wherein the initial amplifying restriction endonuclease
cleaves the first nucleic acid at the restriction endonuclease cut
site of the initial amplifying restriction endonuclease, thereby
separating a portion of the first nucleic acid comprising the
secondary amplifying restriction endonuclease from at least another
portion of the first nucleic acid, (d) contacting the portion of
the first nucleic acid comprising the secondary amplifying
restriction endonuclease with a second nucleic acid comprising the
initial amplifying restriction endonuclease and a double-stranded
portion of nucleic acid comprising a restriction endonuclease cut
site of the secondary amplifying restriction endonuclease under
conditions wherein the secondary amplifying restriction
endonuclease cleaves the second nucleic acid at the restriction
endonuclease cut site of the secondary amplifying restriction
endonuclease, thereby separating a portion of the second nucleic
acid comprising the initial amplifying restriction endonuclease
from at least another portion of the second nucleic acid, (e)
contacting the portion of the second nucleic acid comprising the
initial amplifying restriction endonuclease with a reporter nucleic
acid comprising a double-stranded portion of nucleic acid
comprising a restriction endonuclease cut site of the initial
amplifying restriction endonuclease under conditions wherein the
initial amplifying restriction endonuclease cleaves the reporter
nucleic acid at the restriction endonuclease cut site of the
initial amplifying restriction endonuclease, thereby separating a
portion of the reporter nucleic acid from at least another portion
of the reporter nucleic acid, and (f) determining the presence or
absence of the portion of the reporter nucleic acid, wherein the
presence of the portion of the reporter nucleic acid indicates that
the sample contains the target nucleic acid, thereby indicating
that the mammal is infected with the microorganism or virus, and
wherein the absence of the portion of the reporter nucleic acid
indicates that the sample does not contain the target nucleic acid,
thereby indicating that the mammal is not infected with the
microorganism or virus. The mammal can be a human. The mammal can
be a farm animal selected from the group consisting of bovine,
porcine, and equine species. The mammal can be a dog or cat. The
infection can be a microbial infection, and the target nucleic acid
can be present within a microorganism. The infection can be a viral
infection, and the target nucleic acid can be present within a
virus. The sample can comprise a nasal or throat swab sample. The
sample can be selected from the group consisting of nasal samples,
throat samples, sputum samples, bronchial lavage samples, tissue
samples, cellular samples, and blood samples. Prior to step (a),
the sample can be a sample that was cultured to enrich the
population of microorganisms or viruses, if present, within the
sample. The sample can be a sample that was cultured for at least
30 minutes in the presence of enrichment medium. Prior to step (a),
the sample can be a sample that was processed to remove non-nucleic
acid material from the sample, thereby increasing the concentration
of nucleic acid, if present, within the sample. The sample can be a
sample that was subjected to a nucleic acid extraction technique.
Prior to step (a), the sample can be a sample that was subjected to
a nucleic acid amplification technique to increase the
concentration of microbial or viral nucleic acid, if present,
within the sample. The sample can be a sample that was subjected to
a PCR-based technique designed to amplify the target nucleic acid.
Prior to step (a), the method can comprise culturing the sample to
enrich the population of microorganisms or viruses, if present,
within the sample. The culturing can comprise culturing the sample
for at least 30 minutes in the presence of enrichment medium. Prior
to step (a), the method can comprise removing non-nucleic acid
material from the sample, thereby increasing the concentration of
nucleic acid, if present, within the sample. The removing can
comprise performing a nucleic acid extraction technique. Prior to
step (a), the method can comprise performing a nucleic acid
amplification technique to increase the concentration of microbial
or viral nucleic acid, if present, within the sample. The nucleic
acid amplification technique can comprise a PCR-based technique
designed to amplify the target nucleic acid. Prior to step (a), the
method can comprise (i) culturing the sample to enrich the
population of microorganisms or viruses, if present, within the
sample and removing non-nucleic acid material from the sample,
thereby increasing the concentration of nucleic acid, if present,
within the sample or (ii) culturing the sample to enrich the
population of microorganisms or viruses, if present, within the
sample, removing non-nucleic acid material from the sample, thereby
increasing the concentration of nucleic acid, if present, within
the sample, and performing a nucleic acid amplification technique
to increase the concentration of microbial or viral nucleic acid,
if present, within the sample. The probe nucleic acid can be
single-stranded probe nucleic acid. The probe nucleic acid can be
attached to a solid support. The probe nucleic acid can be directly
attached to a solid support. The portion of the probe nucleic acid
comprising the initial amplifying restriction endonuclease can be
released from the solid support via the step (b). Step (a) and step
(b) can be performed in the same compartment, step (a), step (b),
and step (c) can be performed in the same compartment, step (a),
step (b), step (c), and step (d) can be performed in the same
compartment, step (a), step (b), step (c), step (d), and step (e)
can be performed in the same compartment, or step (a), step (b),
step (c), step (d), step (e), and step (f) can be performed in the
same compartment. Step (c) and step (d) can be performed in the
same compartment. Step (a) and step (b) can be performed in a first
compartment, and step (c) and step (d) can be performed in a second
compartment. Step (a) and step (b) can be performed by adding the
sample to a compartment comprising the probe nucleic acid and the
recognition restriction endonuclease. Step (c) and step (d) can be
performed by adding the portion of the probe nucleic acid
comprising the initial amplifying restriction endonuclease to a
compartment comprising the first nucleic acid and the second
nucleic acid. The probe nucleic acid can comprise (i) a
single-stranded portion comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid and (ii) a
double-stranded portion. The probe nucleic acid can comprise a
first nucleic acid strand comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid hybridized
to a second nucleic acid strand comprising the initial amplifying
restriction endonuclease. The first nucleic acid strand can be
attached to a solid support. The first nucleic acid strand can be
directly attached to a solid support. A portion of the second
nucleic acid strand can hybridize with the first nucleic acid
strand to form the double-stranded portion. The portion of the
probe nucleic acid comprising the initial amplifying restriction
endonuclease that is separated from the at least another portion of
the probe nucleic acid in step (b) can comprise a portion of the
first nucleic acid strand and all of the second strand. The portion
of the probe nucleic acid comprising the initial amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can comprise
at least a portion of the target nucleic acid.
[0009] In some cases, the method can comprise using a plurality of
the probe nucleic acid in the step (a). The method can comprise
using a plurality of the reporter nucleic acid in the step (e). The
reporter nucleic acid in the step (e) can be in molar excess of the
portion of the probe nucleic acid comprising the initial amplifying
restriction endonuclease from the step (b). The number of molecules
of the portion of the probe nucleic acid comprising the initial
amplifying restriction endonuclease that is separated from the at
least another portion of the probe nucleic acid in step (b) can be
in an essentially linear relationship to the number of molecules of
the target nucleic acid present in the sample. The first nucleic
acid and the second nucleic acid can be attached to a solid
support. The first nucleic acid and the second nucleic acid can be
directly attached to a solid support. The first nucleic acid and
the second nucleic acid can be attached to a solid support in the
same compartment. The portion of the first nucleic acid comprising
the secondary amplifying restriction endonuclease can be released
from the solid support via the step (c). The portion of the second
nucleic acid comprising the initial amplifying restriction
endonuclease can be released from the solid support via the step
(d). The first nucleic acid can comprise a first nucleic acid
strand comprising the secondary amplifying restriction endonuclease
hybridized to a second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the initial amplifying restriction
endonuclease. The first nucleic acid strand can be attached to a
solid support. The first nucleic acid strand can be directly
attached to a solid support. The second nucleic acid strand can be
attached to a solid support. The second nucleic acid strand can be
directly attached to a solid support. The second nucleic acid can
comprise a first nucleic acid strand comprising the initial
amplifying restriction endonuclease hybridized to a second nucleic
acid strand to form the double-stranded portion of nucleic acid
comprising the restriction endonuclease cut site of the secondary
amplifying restriction endonuclease. The first nucleic acid strand
can be attached to a solid support. The first nucleic acid strand
can be directly attached to a solid support. The second nucleic
acid strand can be attached to a solid support. The second nucleic
acid strand can be directly attached to a solid support. The
reporter nucleic acid can be attached to a solid support. The
reporter nucleic acid can be directly attached to a solid support.
The reporter nucleic acid can comprise a single-stranded portion of
nucleic acid. The reporter nucleic acid can comprise a label. The
label can be a fluorescent label, a radioactive label, an enzyme
label, or a redox label. The portion of the reporter nucleic acid
that is separated from the at least another portion of the reporter
nucleic acid can comprise the label. The reporter nucleic acid can
comprise a first nucleic acid strand comprising the label
hybridized to a second nucleic acid strand. The second nucleic acid
strand can be attached to a solid support. The second nucleic acid
strand can be directly attached to a solid support. A portion of
the first nucleic acid strand can hybridize with the second nucleic
acid strand to form the double-stranded portion of nucleic acid
comprising the restriction endonuclease cut site of the initial
amplifying restriction endonuclease. The reporter nucleic acid can
comprise a third nucleic acid strand. The third nucleic acid strand
can hybridize with the second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the initial amplifying restriction
endonuclease.
[0010] The reporter nucleic acid can be attached to a solid
support, and the portion of the reporter nucleic acid that is
separated from the at least another portion of the reporter nucleic
acid and that comprises the label can be released from the solid
support via the step (e). The determining step (f) can comprise
detecting the label. The label can be a fluorescent label, and the
determining step (f) can comprise detecting the fluorescent label.
The determining step (f) can comprise detecting the portion of the
reporter nucleic acid separated from the at least another portion
of the reporter nucleic acid using a capillary electrophoresis
technique. Steps (a), (b), (c), (d), and (e) can be performed
without nucleic acid amplification, or steps (a), (b), (c), (d),
(e), and (f) can be performed without nucleic acid amplification.
The determining step can comprise determining the amount of the
target nucleic acid present within the sample.
[0011] In another aspect, this document features a method for
assessing a mammal for an infection. The method comprises, or
consists essentially of, (a) contacting a sample from the mammal
with a probe nucleic acid comprising an initial amplifying
restriction endonuclease and a nucleotide sequence complementary to
a sequence of a target nucleic acid present within a microorganism
or virus under conditions wherein, if the target nucleic acid is
present in the sample, at least a portion of the target nucleic
acid hybridizes to at least a portion of the probe nucleic acid to
form a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site, (b) contacting the
double-stranded portion of nucleic acid with a recognition
restriction endonuclease having the ability to cut the
double-stranded portion of nucleic acid at the restriction
endonuclease cut site under conditions wherein the recognition
restriction endonuclease cleaves the double-stranded portion of
nucleic acid at the restriction endonuclease cut site, thereby
separating a portion of the probe nucleic acid comprising the
initial amplifying restriction endonuclease from at least another
portion of the probe nucleic acid, (c) contacting the portion of
the probe nucleic acid comprising the initial amplifying
restriction endonuclease with a first reporter nucleic acid
comprising a secondary amplifying restriction endonuclease and a
double-stranded portion of nucleic acid comprising a restriction
endonuclease cut site of the initial amplifying restriction
endonuclease under conditions wherein the initial amplifying
restriction endonuclease cleaves the first reporter nucleic acid at
the restriction endonuclease cut site of the initial amplifying
restriction endonuclease, thereby separating a portion of the first
nucleic acid comprising the secondary amplifying restriction
endonuclease from at least another portion of the first nucleic
acid, (d) contacting the portion of the first reporter nucleic acid
comprising the secondary amplifying restriction endonuclease with a
second reporter nucleic acid comprising the initial amplifying
restriction endonuclease and a double-stranded portion of nucleic
acid comprising a restriction endonuclease cut site of the
secondary amplifying restriction endonuclease under conditions
wherein the initial amplifying restriction endonuclease cleaves the
second nucleic acid at the restriction endonuclease cut site of the
secondary amplifying restriction endonuclease, thereby separating a
portion of the second nucleic acid comprising the initial
amplifying restriction endonuclease from at least another portion
of the second nucleic acid, and (e) determining the presence or
absence of the portion of the first reporter nucleic acid, the
second reporter nucleic acid, or both the first reporter nucleic
acid and the second reporter nucleic acid, wherein the presence
indicates that the sample contains the target nucleic acid, thereby
indicating that the mammal is infected with the microorganism or
virus, and wherein the absence indicates that the sample does not
contain the target nucleic acid, thereby indicating that the mammal
is not infected with the microorganism or virus. The mammal can be
a human. The mammal can be a farm animal selected from the group
consisting of bovine, porcine, and equine species. The mammal can
be a dog or cat. The infection can be a microbial infection, and
the target nucleic acid can be present within a microorganism. The
infection can be a viral infection, and the target nucleic acid can
be present within a virus. The sample can comprise a nasal or
throat swab sample. The sample can be selected from the group
consisting of nasal samples, throat samples, sputum samples,
bronchial lavage samples, tissue samples, cellular samples, and
blood samples. Prior to step (a), the sample can be a sample that
was cultured to enrich the population of microorganisms or viruses,
if present, within the sample. The sample can be a sample that was
cultured for at least 30 minutes in the presence of enrichment
medium. Prior to step (a), the sample can be a sample that was
processed to remove non-nucleic acid material from the sample,
thereby increasing the concentration of nucleic acid, if present,
within the sample. The sample can be a sample that was subjected to
a nucleic acid extraction technique. Prior to step (a), the sample
can be a sample that was subjected to a nucleic acid amplification
technique to increase the concentration of microbial or viral
nucleic acid, if present, within the sample. The sample can be a
sample that was subjected to a PCR-based technique designed to
amplify the target nucleic acid. Prior to step (a), the method can
comprise culturing the sample to enrich the population of
microorganisms or viruses, if present, within the sample. The
culturing can comprise culturing the sample for at least 30 minutes
in the presence of enrichment medium. Prior to step (a), the method
can comprise removing non-nucleic acid material from the sample,
thereby increasing the concentration of nucleic acid, if present,
within the sample. The removing can comprise performing a nucleic
acid extraction technique. Prior to step (a), the method can
comprise performing a nucleic acid amplification technique to
increase the concentration of microbial or viral nucleic acid, if
present, within the sample. The nucleic acid amplification
technique can comprise a PCR-based technique designed to amplify
the target nucleic acid. Prior to step (a), the method can comprise
(i) culturing the sample to enrich the population of microorganisms
or viruses, if present, within the sample and removing non-nucleic
acid material from the sample, thereby increasing the concentration
of nucleic acid, if present, within the sample or (ii) culturing
the sample to enrich the population of microorganisms or viruses,
if present, within the sample, removing non-nucleic acid material
from the sample, thereby increasing the concentration of nucleic
acid, if present, within the sample, and performing a nucleic acid
amplification technique to increase the concentration of microbial
or viral nucleic acid, if present, within the sample. The probe
nucleic acid can be single-stranded probe nucleic acid. The probe
nucleic acid can be attached to a solid support. The probe nucleic
acid can be directly attached to a solid support. The portion of
the probe nucleic acid comprising the initial amplifying
restriction endonuclease can be released from the solid support via
the step (b). Step (a) and step (b) can be performed in the same
compartment, step (a), step (b), and step (c) can be performed in
the same compartment, step (a), step (b), step (c), and step (d)
can be performed in the same compartment, or step (a), step (b),
step (c), step (d), and step (e) can be performed in the same
compartment. Step (c) and step (d) can be performed in the same
compartment. Step (a) and step (b) can be performed in a first
compartment, and step (c) and step (d) can be performed in a second
compartment. Step (a) and step (b) can be performed by adding the
sample to a compartment comprising the probe nucleic acid and the
recognition restriction endonuclease. Step (c) and step (d) can be
performed by adding the portion of the probe nucleic acid
comprising the initial amplifying restriction endonuclease to a
compartment comprising the first reporter nucleic acid and the
second reporter nucleic acid. The probe nucleic acid can comprise
(i) a single-stranded portion comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid and (ii) a
double-stranded portion. The probe nucleic acid can comprise a
first nucleic acid strand comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid hybridized
to a second nucleic acid strand comprising the initial amplifying
restriction endonuclease. The first nucleic acid strand can be
attached to a solid support. The first nucleic acid strand can be
directly attached to a solid support. A portion of the second
nucleic acid strand can hybridize with the first nucleic acid
strand to form the double-stranded portion. The portion of the
probe nucleic acid comprising the initial amplifying restriction
endonuclease that is separated from the at least another portion of
the probe nucleic acid in step (b) can comprise a portion of the
first nucleic acid strand and all of the second strand. The portion
of the probe nucleic acid comprising the initial amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can comprise
at least a portion of the target nucleic acid.
[0012] In some cases, the method can comprise using a plurality of
the probe nucleic acid in the step (a). The method can comprise
using a plurality of the first reporter nucleic acid in the step
(c). The first reporter nucleic acid in the step (c) can be in
molar excess of the portion of the probe nucleic acid comprising
the initial amplifying restriction endonuclease from the step (b).
The method can comprise using a plurality of the second reporter
nucleic acid in the step (d). The second reporter nucleic acid in
the step (d) can be in molar excess of the portion of the probe
nucleic acid comprising the initial amplifying restriction
endonuclease from the step (b). The number of molecules of the
portion of the probe nucleic acid comprising the initial amplifying
restriction endonuclease that is separated from the at least
another portion of the probe nucleic acid in step (b) can be in an
essentially linear relationship to the number of molecules of the
target nucleic acid present in the sample. The first reporter
nucleic acid and the second reporter nucleic acid can be attached
to a solid support. The first reporter nucleic acid and the second
reporter nucleic acid can be directly attached to a solid support.
The first reporter nucleic acid and the second reporter nucleic
acid can be attached to a solid support in the same compartment.
The portion of the first reporter nucleic acid comprising the
secondary amplifying restriction endonuclease can be released from
the solid support via the step (c). The portion of the second
reporter nucleic acid comprising the initial amplifying restriction
endonuclease can be released from the solid support via the step
(d). The first reporter nucleic acid can comprise a label. The
label can be a fluorescent label, a radioactive label, an enzyme
label, or a redox label. The second reporter nucleic acid can
comprise a label. The label can be a fluorescent label, a
radioactive label, an enzyme label, or a redox label. The first
reporter nucleic acid and the second reporter nucleic acid can
comprise a label. The first reporter nucleic acid and the second
reporter nucleic acid can comprise the same label. The label can be
a fluorescent label, a radioactive label, an enzyme label, or a
redox label. The first reporter nucleic acid can be attached to a
solid support, the portion of the first reporter nucleic acid that
is separated from the at least another portion of the first
reporter nucleic acid can comprise a label, and the portion of the
first reporter nucleic acid that is separated from the at least
another portion of the first reporter nucleic acid and that
comprises the label can be released from the solid support via the
step (c). The first reporter nucleic acid can comprise a first
nucleic acid strand comprising the secondary amplifying restriction
endonuclease hybridized to a second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the initial amplifying restriction
endonuclease. The first nucleic acid strand can be attached to a
solid support. The first nucleic acid strand can be directly
attached to a solid support. The second nucleic acid strand can be
attached to a solid support. The second nucleic acid strand can be
directly attached to a solid support. The first nucleic acid strand
can comprise a label. The label can be a fluorescent label, a
radioactive label, an enzyme label, or a redox label. The second
nucleic acid strand can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. The second reporter nucleic acid can be attached to a solid
support, the portion of the second reporter nucleic acid that is
separated from the at least another portion of the second reporter
nucleic acid can comprise a label, and the portion of the second
reporter nucleic acid that is separated from the at least another
portion of the second reporter nucleic acid and that comprises the
label can be released from the solid support via the step (d). The
second reporter nucleic acid can comprise a first nucleic acid
strand comprising the initial amplifying restriction endonuclease
hybridized to a second nucleic acid strand to form the
double-stranded portion of nucleic acid comprising the restriction
endonuclease cut site of the secondary amplifying restriction
endonuclease. The first nucleic acid strand can be attached to a
solid support. The first nucleic acid strand can be directly
attached to a solid support. The second nucleic acid strand can be
attached to a solid support. The second nucleic acid strand can be
directly attached to a solid support. The first nucleic acid strand
can comprise a label. The label can be a fluorescent label, a
radioactive label, an enzyme label, or a redox label. The second
nucleic acid strand can comprise a label. The label can be a
fluorescent label, a radioactive label, an enzyme label, or a redox
label. The portion of the first reporter nucleic acid separated
from the at least another portion of the first reporter nucleic
acid can comprise a fluorescent label, the portion of the second
reporter nucleic acid separated from the at least another portion
of the second reporter nucleic acid can comprise a fluorescent
label, and the determining step (e) can comprise detecting the
fluorescent label. The determining step (e) can comprise detecting
the portion of the first reporter nucleic acid separated from the
at least another portion of the first reporter nucleic acid using a
capillary electrophoresis technique. The determining step (e) can
comprise detecting the portion of the second reporter nucleic acid
separated from the at least another portion of the second reporter
nucleic acid using a capillary electrophoresis technique. Steps
(a), (b), (c), and (d) can be performed without nucleic acid
amplification, or steps (a), (b), (c), (d), and (e) can be
performed without nucleic acid amplification. The determining step
can comprise determining the amount of the target nucleic acid
present within the sample.
[0013] In another aspect, this document features a kit for
assessing a mammal for an infection. The kit comprises, or consists
essentially of, a probe nucleic acid comprising an amplifying
restriction endonuclease and a nucleotide sequence complementary to
a sequence of a target nucleic acid present in a microorganism or
virus, wherein at least a portion of the target nucleic acid is
capable of hybridizing to at least a portion of the probe nucleic
acid to form a double-stranded portion of nucleic acid comprising a
restriction endonuclease cut site. The probe nucleic acid can be
single-stranded probe nucleic acid. The kit can comprise a solid
support, and wherein the probe nucleic acid can be attached to the
solid support. A portion of the probe nucleic acid comprising the
amplifying restriction endonuclease can be releasable from the
solid support via cleavage with a recognition restriction
endonuclease having the ability to cleave at the restriction
endonuclease cut site. The kit can further comprise the recognition
restriction endonuclease. The probe nucleic acid can comprise (i) a
single-stranded portion comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid and (ii) a
double-stranded portion. The probe nucleic acid can comprise a
first nucleic acid strand comprising the nucleotide sequence
complementary to the sequence of the target nucleic acid hybridized
to a second nucleic acid strand comprising the amplifying
restriction endonuclease. The kit can further comprise a reporter
nucleic acid comprising a double-stranded portion of nucleic acid
comprising a restriction endonuclease cut site of the amplifying
restriction endonuclease. The kit can comprise a solid support, and
the reporter nucleic acid can be attached to the solid support. The
reporter nucleic acid can be directly attached to the solid
support. The reporter nucleic acid can comprise a single-stranded
portion of nucleic acid. The reporter nucleic acid can comprise a
label. The label can be a fluorescent label, a radioactive label,
an enzyme label, or a redox label. A portion of the reporter
nucleic acid comprising the label can be capable of being separated
from at least another portion of the reporter nucleic acid via
cleavage by the amplifying restriction endonuclease. The reporter
nucleic acid can comprise a first nucleic acid strand comprising
the label hybridized to a second nucleic acid strand. The kit can
further comprise: (a) a first signal expansion nucleic acid
comprising a secondary amplifying restriction endonuclease and a
double-stranded section having a restriction endonuclease cut site
for the amplifying restriction endonuclease, and (b) a second
signal expansion nucleic acid comprising the amplifying restriction
endonuclease and a double-stranded section having a restriction
endonuclease cut site for the secondary amplifying restriction
endonuclease. The probe nucleic acid can be lyophilized. All the
ingredients of the kit can be lyophilized or dry.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references (e.g., GenBank.RTM.
records) mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0015] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic depicting an exemplary method for
detecting target nucleic acid using probe nucleic acid, a
recognition restriction endonuclease, and reporter nucleic
acid.
[0017] FIG. 2 is a schematic of an exemplary configuration of probe
nucleic acid that can be used with the methods and materials
provided herein for detecting target nucleic acid.
[0018] FIG. 3 is a schematic depicting an exemplary method for
detecting target nucleic acid using probe nucleic acid, a
recognition restriction endonuclease, first signal expansion
nucleic acid, second signal expansion nucleic acid, and reporter
nucleic acid.
[0019] FIG. 4 is a schematic of an exemplary configuration of first
signal expansion nucleic acid and second signal expansion nucleic
acid that can be used with the methods and materials provided
herein for detecting target nucleic acid. Such first signal
expansion nucleic acid and second signal expansion nucleic acid can
be used with or without reporter nucleic acid. When used without a
separate reporter nucleic acid step, such signal expansion nucleic
acid can be referred to as reporter nucleic acid.
[0020] FIG. 5 is a schematic of an exemplary configuration of first
signal expansion nucleic acid and second signal expansion nucleic
acid that can be used with the methods and materials provided
herein for detecting target nucleic acid. Such first signal
expansion nucleic acid and second signal expansion nucleic acid can
be used with or without reporter nucleic acid. When used without a
separate reporter nucleic acid step, such signal expansion nucleic
acid can be referred to as reporter nucleic acid.
[0021] FIG. 6 contains line graphs demonstrating the effect of
target oligonucleotide concentration (A) and recognition
restriction endonuclease concentration (B) on the cleavage of
HRP-labeled nucleic acid as detected by the formation of colored
reaction product.
[0022] FIG. 7 is a schematic of an exemplary configuration for a
single-use, pen-style point of care device.
DETAILED DESCRIPTION
[0023] This document provides methods and materials for detecting
viral and/or microbial infections. For example, this document
provides methods and materials related to the use of an enzymatic
amplification cascade of restriction endonucleases to detect
nucleic acid of a virus or microbe (e.g., a pathogen) within a
sample (e.g., a biological sample such as a blood sample, mucus
sample, or saliva sample) being tested, thereby assessing a mammal
for a possible infection. In some cases, this document provides
methods and materials for detecting a target microorganism's or
virus's nucleic acid (e.g., a target pathogen's nucleic acid). For
example, this document provides methods and materials for detecting
the presence or absence of target nucleic acid (e.g., a target
microorganism's or virus's nucleic acid) within a sample (e.g., a
biological sample), methods and materials for detecting the amount
of target nucleic acid (e.g., a target microorganism's or virus's
nucleic acid) present within a sample (e.g., a biological sample),
kits for detecting the presence or absence of target nucleic acid
(e.g., a target microorganism's or virus's nucleic acid) within a
sample (e.g., a biological sample), kits for detecting the amount
of target nucleic acid (e.g., a target microorganism's or virus's
nucleic acid) present within a sample (e.g., a biological sample),
and methods for making such kits.
[0024] Any type of mammal can be assessed using the methods and
materials provided herein to determine whether or not the mammal
has a viral and/or microbial infection. For example, humans, dogs,
cats, cows, horses, pigs, sheep, goats, monkeys, buffalo, bears,
whales, and dolphins can be assessed for a viral and/or microbial
infection as described herein. Any type of biological sample can be
used with the methods and materials provided herein to assess a
mammal for a viral and/or microbial infection. For example, nasal
samples (e.g., nasal swab samples), throat samples (e.g., throat
swab samples), sputum samples, bronchial lavage samples, tissue
samples (e.g., tissue biopsy samples), cellular samples, and blood
samples can be collected from a mammal and assessed to determine
whether or not the mammal has a viral or microbial infection as
described herein.
[0025] The methods and materials provided herein can be used to
assess a mammal for any type of viral and/or microbial infection.
Examples of potentially infecting viruses include, without
limitation, influenza virus A and B, adenovirus 4, RSV,
parainfluenza types 1, 2, and 3, human coronaviruses OC43, 229E and
HK, human metapneumovirus, rhinoviruses, enteroviruses, Hepatitis
A, B, C and E viruses, rotavirus, human papillomavirus, measles
viruses, caliciviruses, astrovirus, West Nile virus, Ebola virus,
Dengue fever virus, African swine fever, and human immunodeficiency
virus (HIV1 and 2). Examples of potentially infecting
microorganisms include, without limitation, bacterial
microorganisms such as Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Mycoplasma pneumoniae,
Haemophilus influenzae, Chlamydia pneumoniae, Bordetella pertussis,
Mycobacterium tuberculosis, E. coli (e.g., enterohaemorrhagic E.
coli such as O157:H7 E. coli or enteropathogenic E. coli),
Salmonella species (e.g., Salmonella enterica), Listeria
monocytogenes, Acinetobacter baumanni, Klebsiella oxytoca, Giardia
intestinalis, Sarcoptes scabiei, and Treponema pallidum, fungal
microorganisms such as Aspergillus species (e.g., A. flavus, A.
fumigatus, and A. niger), yeast (e.g., Candida norvegensis and C.
albicans), Penicillium species, Rhizopus species, and Alternaria
species, and protozoan microorganisms such as Cryptosporidium
parvum, Giardia lamblia, and Toxoplasma gondii. In some cases, a
mammal can be assessed for one or more of the viruses or
microorganisms listed in Table 1 using the methods and materials
provided herein. When designing a method for detecting a virus or
microorganism listed in Table 1, a probe nucleic acid can be
designed that is complementary to a portion of any of the indicated
sequences from Table 1. For example, when designing a method for
detecting influenza virus A, a probe nucleic acid can be designed
that is complementary to a portion of the influenza A sequence set
forth in GenBank.RTM. GI number 8486122.
TABLE-US-00001 TABLE 1 Types of infections that can be detected.
Genomic sequence Mammal Infection (GenBank.sup..RTM. gi number)
Sample Human Upper respiratory viral infection: Refseq: NC_002023,
GenBank.sup..RTM.: Nasal swab influenza virus A V00603 or mucus
influenza virus B Refseq: NC_002209, GenBank.sup..RTM.: J02095
adenovirus 4 Refseq: NC_003266; GenBank.sup..RTM.: 51527264 RSV
NC_001781, GenBank.sup..RTM.: AF013254 parainfluenza type 1 Refseq:
NC_003461, GenBank.sup..RTM.: AF457102 parainfluenza type 2 Refseq:
NC_003443, GenBank.sup..RTM.: X57559 parainfluenza type 3 Refseq:
NC_001796, GenBank.sup..RTM.: AB012132 human coronavirus OC43
Refseq: NC_012920, GenBank.sup..RTM.: J01415 human coronavirus 229E
Refseq: NC_002645, GenBank.sup..RTM.: AF304460 human coronavirus HK
Refseq: NC_012951, GenBank.sup..RTM.: FJ938052 human
metapneumovirus Refseq: NC_004148, GenBank.sup..RTM.: AY297749
rhinoviruses Refseq: NC_001490, GenBank.sup..RTM.: K02121
enteroviruses Refseq: NC_013115, GenBank.sup..RTM.: AB426609 Upper
respiratory microbial infection: Refseq: NZ_ACOT00000000,
Staphylococcus aureus GenBank.sup..RTM.: ACOT00000000 Streptococcus
pyogenes Refseq: NZ_AAFV00000000, GenBank.sup..RTM.: AAFV00000000
Streptococcus pneumoniae Refseq: NZ_ACJP00000000,
GenBank.sup..RTM.: ACJP00000000 Mycoplasma pneumoniae Refseq:
NC_000912, GenBank.sup..RTM.: U00089 Haemophilus influenzae Refseq:
NZ_ABWV00000000, GenBank.sup..RTM.: ABWV00000000 Chlamydi
Chlamydophila Refseq: NC_005043, pneumoniae TW-183
GenBank.sup..RTM.: AE009440 Bordetella pertussis Refseq: NC_008459,
GenBank.sup..RTM.: AB237782 Mycobacterium tuberculosis T46 Refseq:
NZ_ACHO00000000, GenBank.sup..RTM.: ACHO00000000 Human Human
immunodeficiency Refseq: NC_001722, Blood virus (HIV)
GenBank.sup..RTM.: M30502 sample Human Rabies Refseq: NC_001542,
GenBank.sup..RTM.: M13215 Human Lymes disease Bat Rabies Refseq:
NC_009528, GenBank: EF157977 Dog Lymes disease Cat Rabies Refseq:
NC_001542, GenBank.sup..RTM.: M13215 Cat Leptospira Refseq:
NCO10846, GenBank.sup..RTM.: CP000779 Cat Leukemia Virus (FELV)
Bovine Bovine herpesvirus 1 Refseq: NC_001847, GenBank.sup..RTM.:
AJ004801 Bovine Foot-and-Mouth Disease Refseq: NC_011452,
GenBank.sup..RTM.: AY593850 Horse Equine Encephalomyelitis Refseq:
NC_003908, (sleeping sickness) GenBank.sup..RTM.: AF214040 Horse
Strangles (shipping fever) Refseq: NC_012471, GenBank.sup..RTM.:
FM204883
[0026] In some cases, nucleic acid sequences of viruses and
microorganisms known to infect the upper respiratory tract of
mammals (e.g., humans) can be used to design probe nucleic acids
for detecting upper respiratory tract infections. For example,
probe nucleic acids having the sequences set forth in Table 2 can
be used with the indicated recognition restriction endonuclease to
detect the indicated target nucleic acids of the indicated
pathogens. In some cases, a single kit can be designed as described
herein to detect one or more of the indicated pathogens of Table
2.
TABLE-US-00002 TABLE 2 Target nucleic acids, recognition
restriction endonucleases, and probe nucleic acids for detecting
the indicated pathogens. Recongition Target Nucleic Restriction
Sequence for Probe Pathogen Acid Endonuclease Nucleic Acid
Staphylococcus gyrB (DNA EcoRV (gatatc) TGATCTAGCGAAAGCAAGATA
aureus gyrase subunit B) TCACAAAATCGTCATTATG (SEQ ID NO: 1)
methicillin- mecA (penicillin PstI (ctgcag) ATTGGCAAATCCGGTACTGCA
resistant binding protein 2) GAACTCAAAATGAAACAAG (MRSA) (SEQ ID NO:
2) Streptococcus ply PstI (ctgcag) AACAGAGAGGAATTTCTGCAG pneumoniae
(pneumolysin) AGCGTCCTTTGGTCTATAT (SEQ ID NO: 3) Streptococcus speA
(exotoxin BstEII ATATTTTCTTTATGAGGGTGA pyogenes type A precursor)
(ggtgacc) CCCTGTTACTCACGAGAAT (SEQ ID NO: 4) Mycobacterium rpoB
(RNA HincII (gttgac) AACAACCCGCTGTCGGGGTTG tuberculosis polymerase
ACCCACAAGCGCCGACTGT subunit beta) (SEQ ID NO: 5) Influenza A M1
(matrix PstI (ctgcag) ACCGTGCCCAGTGAGCGAGGA virus protein)
CTGCAGCGTAGACGCTTTG (SEQ ID NO: 6) Influenza B M1 (matrix HindIII
(aagctt) AATGAGAAGATGTGTAAGCTT virus protein) TCATGAAGCATTTGAAATA
(SEQ ID NO: 7) Adenovirus 4 gp 12 BglII (agatct)
CCAACTCGCCGGATCGGGAAG (E) (glycoprotein 12) ATCTTCCTTCACGCCTCGT
(SEQ ID NO: 8) Respiratory M2 (matrix EcoRV (gatatc)
CCATAAAAACCACATTGGATA syncytial virus protein) TCCACAAGAGCATAACCAT
(SEQ ID NO: 9)
[0027] In some cases, an enzymatic amplification cascade can be
used to assess the presence or absence of microorganisms and
viruses associated with sexually transmitted infections (STIs).
Millions of STIs occur every year in the United States, and if
untreated or allowed to proceed to advanced stages, they have
severe consequences (e.g., infertility, blindness, or brain
damage). Common STIs include, without limitation, gonorrhea,
Chlamydia, syphilis, and genital herpes. Gonorrhea, Chlamydia, and
syphilis are bacterial, while genital herpes is viral. An enzymatic
amplification cascade can be used to detect, for example, N.
gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, or a herpes
simplex virus such as HSV-2. Probe nucleic acids can be designed to
contain nucleic acid sequences from the microorganism or virus of
interest, as described herein. The type of sample used for the
reaction can vary depending on the target of interest. For example,
urine samples or urethral or endocervical swab samples can be
tested for the presence or absence of gonorrhea and/or Chlamydia.
Blood, plasma, or lesion swab samples can be tested for the
presence or absence of syphilis, and genital sore swabs can be
tested for the presence or absence of HSV2.
[0028] In one embodiment, a method for assessing a mammal for a
viral and/or microbial infection can include detecting a target
virus's and/or microorganism's nucleic acid (e.g., a target nucleic
acid) within a biological sample obtained from the mammal. For
example, a biological sample (e.g., a blood sample to be tested)
can be placed in contact with probe nucleic acid. The probe nucleic
acid can be designed to have a single-stranded portion with a
nucleotide sequence that is complementary to at least a portion of
the target nucleic acid to be detected. In this case, target
nucleic acid present within the sample can hybridize with the
complementary sequence of this single-stranded portion of the probe
nucleic acid to form a double-stranded section with one strand
being target nucleic acid and the other strand being probe nucleic
acid. In addition, the single-stranded portion of the probe nucleic
acid having the nucleotide sequence that is complementary to at
least a portion of the target nucleic acid to be detected can be
designed such that hybridization with the target nucleic acid
creates a restriction endonuclease cut site. Thus, target nucleic
acid present within the sample can hybridize with the complementary
sequence of the single-stranded portion of the probe nucleic acid
to form a double-stranded section that creates a cut site for a
restriction endonuclease. This cut site created by the
hybridization of target nucleic acid to probe nucleic acid can be
referred to as a recognition restriction endonuclease cut site. In
addition, a restriction endonuclease that cleaves nucleic acid at
such a recognition restriction endonuclease cut site can be
referred to as a recognition restriction endonuclease.
[0029] The probe nucleic acid also can be designed to contain a
restriction endonuclease. This restriction endonuclease, which can
be a component of the probe nucleic acid, can be referred to as an
amplifying restriction endonuclease. An amplifying restriction
endonuclease is typically a different restriction endonuclease than
the restriction endonuclease that is used as a recognition
restriction endonuclease. For example, when an EcoRI restriction
endonuclease is used as a recognition restriction endonuclease, a
restriction endonuclease other than an EcoRI restriction
endonuclease (e.g., a Hind III restriction endonuclease) is used as
an amplifying restriction endonuclease. Thus, in general, probe
nucleic acid is designed to contain an amplifying restriction
endonuclease and to have a nucleotide sequence such that the target
nucleic acid can hybridize to the probe nucleic acid and create a
recognition restriction endonuclease cut site for a recognition
restriction endonuclease. In some cases, the probe nucleic acid can
be attached to a solid support (e.g., a well of a microtiter
plate). For example, the probe nucleic acid can be attached to a
solid support such that cleavage at the recognition restriction
endonuclease cut site via the recognition restriction endonuclease
releases a portion of the probe nucleic acid that contains the
amplifying restriction endonuclease.
[0030] After contacting the sample (e.g., a biological sample) that
may or may not contain target nucleic acid with the probe nucleic
acid that is attached to a solid support, the target nucleic acid,
if present in the sample, can hybridize to the probe nucleic acid
and create the recognition restriction endonuclease cut site. At
this point, the recognition restriction endonuclease, whether added
to the reaction or already present in the reaction, can cleave the
probe nucleic acid at the recognition restriction endonuclease cut
sites that are formed by the hybridization of target nucleic acid
to the probe nucleic acid, thereby releasing the portion of the
probe nucleic acid that contains the amplifying restriction
endonuclease from the solid support. The number of amplifying
restriction endonuclease-containing portions of the probe nucleic
acid that are released from the solid support can be in an
essentially linear relationship (e.g., essentially a one-for-one
relationship) with the number of target nucleic acid molecules that
hybridize with the probe nucleic acid to form the recognition
restriction endonuclease cut site.
[0031] The portions of the probe nucleic acid containing the
amplifying restriction endonuclease that were released from the
solid support can be collected and placed in contact with reporter
nucleic acid. For example, the released portions of the probe
nucleic acid, if present, can be transferred from one well of a
microtiter plate (e.g., a 96-well plate) that contained the probe
nucleic acid to another well of a microtiter plate that contains
the reporter nucleic acid. The reporter nucleic acid can be
designed to have a double-stranded portion with a restriction
endonuclease cut site for the amplifying restriction endonuclease
of the probe nucleic acid. This restriction endonuclease cut site
for the amplifying restriction endonuclease can be referred to as
an amplifying restriction endonuclease cut site. If portions of the
probe nucleic acid containing the amplifying restriction
endonuclease are present and placed in contact with the reporter
nucleic acid, then the reporter nucleic acid can be cleaved at the
amplifying restriction endonuclease cut site by the amplifying
restriction endonuclease. Since the amplifying restriction
endonucleases of the released portions of the probe nucleic acid
are free to carry out repeated cleavage events, the number of
reporter nucleic acid molecules that are cleaved can greatly exceed
the number of amplifying restriction endonucleases present in the
reaction. For example, the number of cleaved reporter nucleic acid
molecules can greatly exceed (e.g., exponentially exceed) the
number of amplifying restriction endonucleases present in the
reaction and therefore can greatly exceed (e.g., exponentially
exceed) the number of target nucleic acid molecules that were
present in the sample contacted with the probe nucleic acid. Such a
greatly expanded relationship (e.g., an exponential relationship)
can allow very small amounts of target nucleic acid present in the
sample to be readily detected.
[0032] After the released portions of the probe nucleic acid, if
present, are contacted with the reporter nucleic acid, the presence
or absence of cleaved reporter nucleic acid can be determined. The
presence of cleaved reporter nucleic acid can indicate that the
sample contained the target nucleic acid, thereby indicating that
the sample contained the target virus or microorganism for which
the sample is being tested, while the absence of cleaved reporter
nucleic acid can indicate that the sample lacked the target nucleic
acid, thereby indicating that the sample lacked the target virus or
microorganism for which the sample is being tested. In some cases,
the amount of cleaved reporter nucleic acid can be determined. In
such cases, the amount of cleaved reporter nucleic acid can
indicate the amount of target nucleic acid present in the sample,
which can indicated the degree or level of infection by the target
virus or microorganism for which the sample is being tested. A
standard curve using known amounts of target nucleic acid or known
amounts target viruses or microorganisms can be used to aid in the
determination of the amount of target nucleic acid or target
viruses or microorganisms present within a sample.
[0033] In some cases, the reporter nucleic acid can contain a label
to aid in the detection of cleaved reporter nucleic acid. For
example, reporter nucleic acid can contain a fluorescent label and
a quencher such that cleaved reporter nucleic acid provides a
fluorescent signal and uncleaved reporter nucleic acid does not
provide a fluorescent signal. In some cases, the reporter nucleic
acid can contain a label (e.g., a colorimetric label, a fluorescent
label or an enzyme (e.g., a redox enzyme) such as horse radish
peroxidase) and can be attached to a solid support (e.g., a well of
a microtiter plate). For example, the reporter nucleic acid can be
attached to a solid support such that cleavage at the amplifying
restriction endonuclease cut site by the amplifying restriction
endonuclease releases a portion of the reporter nucleic acid that
contains the label. The resulting reaction mixture can be collected
and assessed for the presence, absence, or amount of released
portions of the reporter nucleic acid using the label. For example,
the released portions of the reporter nucleic acid, if present, can
be transferred from one well of a microtiter plate (e.g., a 96-well
plate) that contained the reporter nucleic acid to another well of
a microtiter plate, where the transferred material can be assessed
for a signal from the label.
[0034] One example of a method of detecting target nucleic acid
that includes using probe nucleic acid and reporter nucleic acid is
set forth in FIG. 1. With reference to FIG. 1, first reaction
chamber 100 (e.g., a microtiter plate well) can contain probe
nucleic acid 101. Probe nucleic acid 101 can be attached (e.g.,
immobilized) to solid support 102 and can include amplifying
restriction endonuclease 103 (Ra). Probe nucleic acid 101 can be
attached to solid support 102 such that amplifying restriction
endonuclease 103 is released from solid support 102 upon cleavage
of a nucleic acid component of probe nucleic acid 101. Probe
nucleic acid 101 can have a single-stranded section having a
nucleotide sequence that is complementary to at least a portion of
target nucleic acid 104. Probe nucleic acid 101 can be contacted
with a sample that may or may not contain target nucleic acid 104.
If target nucleic acid 104 is present, at least a portion of target
nucleic acid 104 and probe nucleic acid 101 can hybridize to form a
double-stranded section of nucleic acid. Such a double-stranded
section can contain at least one recognition restriction
endonuclease cut site 105. Addition of recognition restriction
endonuclease 106 (Rr) to first reaction chamber 100 can result in
the cleave of probe nucleic acid 101 at recognition restriction
endonuclease cut site 105 formed by one strand of probe nucleic
acid and one strand of target nucleic acid, thereby releasing
portion 107 of probe nucleic acid 101 from solid support 102.
Portion 107 can include amplifying restriction endonuclease
103.
[0035] The reaction product from first reaction chamber 100
containing released portion 107, if target nucleic acid 104 was
present, can be transferred (e.g., manually or automatically) to
second reaction chamber 120. Second reaction chamber 120 can
contain reporter nucleic acid 121. Reporter nucleic acid 121 can be
attached (e.g., immobilized) to solid support 122 and can include
marker (e.g., a label) 123 (M). Reporter nucleic acid 121 can be
attached to solid support 122 such that marker 123 is released from
solid support 122 upon cleavage of a nucleic acid component of
reporter nucleic acid 121. Reporter nucleic acid 121 can have at
least one double-stranded portion that contains at least one
amplifying restriction endonuclease cut site 124. Addition of the
reaction product from first reaction chamber 100 to second reaction
chamber 120 can result in the cleavage of reporter nucleic acid 121
at amplifying restriction endonuclease cut site 124 if the reaction
product contains portion 107. Such cleavage of reporter nucleic
acid 121 can result in the release of portion 127 from solid
support 122. Portion 127 can include marker 123.
[0036] The reaction product from second reaction chamber 120 can be
assessed to determine the presence, absence, or amount of portion
127. The presence of portion 127 can indicate that the sample
contained target nucleic acid 104, while the absence of portion 127
can indicate that the sample lacked target nucleic acid 104. In
some cases, the amount of portion 127 can be determined. In such
cases, the amount of portion 127 can indicate the amount of target
nucleic acid 104 present in the sample. The presence, absence, or
amount of portion 127 can be determined using marker 123, and
portion 127 having marker 123 can be distinguished from uncleaved
reporter nucleic acid 121 having marker 123 since, in this example,
portion 127 is released from solid support 122, while uncleaved
reporter nucleic acid 121 remains attached to solid support 122.
For example, in some cases, the reaction product from second
reaction chamber 120 can be transferred to third reaction chamber
where the presence or absence of portion 127 via marker 123 is
assessed. If portion 127 is present, the amount of portion 127
present can be quantified.
[0037] Probe nucleic acid 101 and reporter nucleic acid 121 can
have various configurations. For example, with reference to FIG. 1,
probe nucleic acid 101 can be designed to have a single nucleic
acid strand such that the entire nucleic acid component of probe
nucleic acid 101 is single-stranded prior to contact with target
nucleic acid 104. In another example, with reference to FIG. 2,
probe nucleic acid 101 can be designed to have first strand 128 and
second strand 108. First strand 128 can be attached to solid
support 102 and can be designed to have a single-stranded section
having a nucleotide sequence that is complementary to at least a
portion of target nucleic acid 104. Second strand 108 can include
amplifying restriction endonuclease 103 and can have a
single-stranded section having a nucleotide sequence that can
hybridize to first strand 128. In some cases, first strand 128 and
second strand 108 can be synthesized or obtained separately and
then mixed together to form probe nucleic acid 101. For example,
first strand 128 can be synthesized, biotinylated, and attached to
a streptavidin-coated solid support. After synthesizing the nucleic
acid component of second strand 108 and attaching amplifying
restriction endonuclease 103 to the synthesized nucleic acid
component, second strand 108 can be incubated with first strand 128
to form nucleic acid probe 101. In some cases, probe nucleic acid
101 can contain more than two strands. For example, probe nucleic
acid can include first strand 150, second strand 152, and third
strand 154. In this case, first strand 150 can be attached to solid
support 102, second strand 152 can be hybridized to first strand
150 and can include a single-stranded section having a nucleotide
sequence that is complementary to at least a portion of target
nucleic acid 104, and third strand 154 can be hybridized to second
strand 152 and can be attached to amplifying restriction
endonuclease 103. Similar one, two, three, or more strand
configurations can be used to make reporter nucleic acid.
[0038] In another embodiment, a method for detecting target nucleic
acid can include contacting a sample (e.g., a biological sample to
be tested) with probe nucleic acid. The probe nucleic acid can be
designed to have a single-stranded portion with a nucleotide
sequence that is complementary to at least a portion of the target
nucleic acid to be detected. In this case, target nucleic acid
present within the sample can hybridize with the complementary
sequence of this single-stranded portion of the probe nucleic acid
to form a double-stranded section with one strand being target
nucleic acid and the other strand being probe nucleic acid. In
addition, the single-stranded portion of the probe nucleic acid
having the nucleotide sequence that is complementary to at least a
portion of the target nucleic acid to be detected can be designed
such that hybridization with the target nucleic acid creates a
recognition restriction endonuclease cut site. Thus, target nucleic
acid present within the sample can hybridize with the complementary
sequence of the single-stranded portion of the probe nucleic acid
to form a double-stranded section that creates a recognition
restriction endonuclease cut site for a recognition restriction
endonuclease. The probe nucleic acid also can be designed to
contain an amplifying restriction endonuclease. Since this method
includes the use of two or more different amplifying restriction
endonucleases, the amplifying restriction endonuclease that is a
component of the probe nucleic acid can be referred to as a first
or an initial amplifying restriction endonuclease, with additional
amplifying restriction endonucleases being referred to as second,
third, and so on or secondary, tertiary, and so on amplifying
restriction endonucleases. This initial amplifying restriction
endonuclease is typically a different restriction endonuclease than
the restriction endonuclease that is used as a recognition
restriction endonuclease. For example, when an EcoRI restriction
endonuclease is used as a recognition restriction endonuclease, a
restriction endonuclease other than an EcoRI restriction
endonuclease (e.g., a Hind III restriction endonuclease) is used as
an initial amplifying restriction endonuclease. Thus, in general,
probe nucleic acid is designed to contain an initial amplifying
restriction endonuclease and to have a nucleotide sequence such
that the target nucleic acid can hybridize to the probe nucleic
acid and create a recognition restriction endonuclease cut site for
a recognition restriction endonuclease. In some cases, the probe
nucleic acid can be attached to a solid support (e.g., a well of a
microtiter plate). For example, the probe nucleic acid can be
attached to a solid support such that cleavage at the recognition
restriction endonuclease cut site via the recognition restriction
endonuclease releases a portion of the probe nucleic acid that
contains the initial amplifying restriction endonuclease.
[0039] After contacting the sample that may or may not contain
target nucleic acid with the probe nucleic acid that is attached to
a solid support, the target nucleic acid, if present in the sample,
can hybridize to the probe nucleic acid and create the recognition
restriction endonuclease cut site. At this point, the recognition
restriction endonuclease, whether added to the reaction or already
present in the reaction, can cleave the probe nucleic acid at the
recognition restriction endonuclease cut sites that are formed by
the hybridization of target nucleic acid to the probe nucleic acid,
thereby releasing the portion of the probe nucleic acid that
contains the initial amplifying restriction endonuclease from the
solid support. The number of initial amplifying restriction
endonuclease-containing portions of the probe nucleic acid that are
released from the solid support can be in an essentially linear
relationship (e.g., essentially a one-for-one relationship) with
the number of target nucleic acid molecules that hybridize with the
probe nucleic acid to form the recognition restriction endonuclease
cut site.
[0040] The portions of the probe nucleic acid containing the
initial amplifying restriction endonuclease that were released from
the solid support can be collected and placed in contact with first
signal expansion nucleic acid and second signal expansion nucleic
acid. The first signal expansion nucleic acid can be designed to
have a double-stranded portion with a restriction endonuclease cut
site for the initial amplifying restriction endonuclease of the
probe nucleic acid. This restriction endonuclease cut site for the
initial amplifying restriction endonuclease can be referred to as
an initial amplifying restriction endonuclease cut site. The first
signal expansion nucleic acid also can be designed to contain a
secondary amplifying restriction endonuclease. The second signal
expansion nucleic acid can be designed to have a double-stranded
portion with a restriction endonuclease cut site for the secondary
amplifying restriction endonuclease of the first signal expansion
nucleic acid. This restriction endonuclease cut site for the
secondary amplifying restriction endonuclease can be referred to as
a secondary amplifying restriction endonuclease cut site. The
second signal expansion nucleic acid also can be designed to
contain an initial amplifying restriction endonuclease. For
example, when an EcoRI restriction endonuclease is used as a
recognition restriction endonuclease and a HindIII restriction
endonuclease is used as an initial amplifying restriction
endonuclease of the probe nucleic acid, a SmaI restriction
endonuclease can be used as a secondary amplifying restriction
endonuclease of the first signal expansion nucleic acid and a
HindIII restriction endonuclease can be used as the initial
amplifying restriction endonuclease of the second signal expansion
nucleic acid.
[0041] In some cases, the first signal expansion nucleic acid and
second signal expansion nucleic acid can be attached to a solid
support (e.g., a well of a microtiter plate). For example, the
first signal expansion nucleic acid can be attached to a solid
support such that cleavage at the initial amplifying restriction
endonuclease cut site via the initial amplifying restriction
endonuclease releases a portion of the first signal expansion
nucleic acid that contains the secondary amplifying restriction
endonuclease, and the second signal expansion nucleic acid can be
attached to a solid support such that cleavage at the secondary
amplifying restriction endonuclease cut site via the secondary
amplifying restriction endonuclease releases a portion of the
second signal expansion nucleic acid that contains the initial
amplifying restriction endonuclease. The first signal expansion
nucleic acid can be attached to the same solid support (e.g., two
different sub-compartments of a larger compartment) that contains
the second signal expansion nucleic acid provided that the
secondary amplifying restriction endonuclease of uncleaved first
signal expansion nucleic acid is unable to cleave the second signal
expansion nucleic acid and provided that the initial amplifying
restriction endonuclease of uncleaved second signal expansion
nucleic acid is unable to cleave the first signal expansion nucleic
acid. In some cases, the first signal expansion nucleic acid can be
attached to the same solid support within a joint compartment such
that the first signal expansion nucleic acid is within a first
compartment of the joint compartment and the second signal
expansion nucleic acid is within a second compartment of the joint
compartment. In such cases, the secondary amplifying restriction
endonuclease of uncleaved first signal expansion nucleic acid in
the first compartment is unable to cleave the second signal
expansion nucleic acid located in the second compartment, while the
secondary amplifying restriction endonuclease of cleaved first
signal expansion nucleic acid is capable of moving (e.g.,
diffusing) from the first compartment to the second compartment to
cleave the second signal expansion nucleic acid located in the
second compartment. In addition, the initial amplifying restriction
endonuclease of uncleaved second signal expansion nucleic acid in
the second compartment is unable to cleave the first signal
expansion nucleic acid located in the first compartment, while the
initial amplifying restriction endonuclease of cleaved second
signal expansion nucleic acid is capable of moving (e.g.,
diffusing) from the second compartment to the first compartment to
cleave the first signal expansion nucleic acid located in the first
compartment.
[0042] If portions of the probe nucleic acid containing the initial
amplifying restriction endonuclease are present and placed in
contact with the first signal expansion nucleic acid, then the
first signal expansion nucleic acid can be cleaved at the initial
amplifying restriction endonuclease cut site by the initial
amplifying restriction endonuclease, thereby releasing a portion of
the first signal expansion nucleic acid that contains the secondary
amplifying restriction endonuclease from the solid support. The
released portions of the first signal expansion nucleic acid
containing the secondary amplifying restriction endonuclease can be
free to cleave the second signal expansion nucleic acid at the
secondary amplifying restriction endonuclease cut site, thereby
releasing a portion of the second signal expansion nucleic acid
that contains the initial amplifying restriction endonuclease from
the solid support. Since the initial amplifying restriction
endonucleases of the released portions of the probe nucleic acid,
the initial amplifying restriction endonucleases of the released
portions of the second signal expansion nucleic acid, and the
secondary amplifying restriction endonucleases of the released
portions of the first signal expansion nucleic acid are free to
carry out repeated cleavage events, the number of released portions
containing the initial amplifying restriction endonucleases is
greatly increased from the number that were released by the
recognition restriction endonuclease. For example, the number of
cleaved first signal expansion nucleic acid molecules can greatly
exceed (e.g., exponentially exceed) the number of released portions
of the probe nucleic acid, and the number of cleaved second signal
expansion nucleic acid molecules can greatly exceed (e.g.,
exponentially exceed) the number of released portions of the probe
nucleic acid. Such a greatly expanded relationship (e.g., an
exponential relationship) can allow very small amounts of target
nucleic acid present in the sample to be readily detected.
[0043] In some cases, this method can be performed with the first
signal expansion nucleic acid being attached to a solid support
that is different from the solid support that contains the second
signal expansion nucleic acid. For example, the first signal
expansion nucleic acid can be attached to one well of a microtiter
plate, while the second signal expansion nucleic acid can be
attached to a different well of a microtiter plate. In this case,
the resulting reaction material from the well with the first signal
expansion nucleic acid can be collected and transferred to the well
containing the second signal expansion nucleic acid.
[0044] The portions of the second signal expansion nucleic acid
containing the initial amplifying restriction endonuclease that
were released from the solid support containing the second signal
expansion nucleic acid along with any other released portions in
this reaction (e.g., the released portions of the probe nucleic
acid containing the initial amplifying restriction endonuclease and
the released portions of the first signal expansion nucleic acid
containing the secondary amplifying restriction endonuclease) can
be collected and placed in contact with reporter nucleic acid. For
example, the released portions, if present, can be transferred from
one well of a microtiter plate (e.g., a 96-well plate) that
contained the second signal expansion nucleic acid to another well
of a microtiter plate that contains the reporter nucleic acid. The
reporter nucleic acid can be designed to have a double-stranded
portion with a restriction endonuclease cut site for the initial
amplifying restriction endonuclease. If released portions
containing the initial amplifying restriction endonuclease are
present and placed in contact with the reporter nucleic acid, then
the reporter nucleic acid can be cleaved at the initial amplifying
restriction endonuclease cut site by the initial amplifying
restriction endonuclease. Since the initial amplifying restriction
endonucleases of the released portions are free to carry out
repeated cleavage events, the number of reporter nucleic acid
molecules that are cleaved can greatly exceed the number of initial
amplifying restriction endonucleases present in the reaction. For
example, the number of cleaved reporter nucleic acid molecules can
greatly exceed (e.g., exponentially exceed) the number of initial
amplifying restriction endonucleases present in the reaction and
therefore can greatly exceed (e.g., exponentially exceed) the
number of target nucleic acid molecules that were present in the
sample contacted with the probe nucleic acid. Such a greatly
expanded relationship (e.g., an exponential relationship) can allow
very small amounts of target nucleic acid present in the sample to
be readily detected.
[0045] After the released portions containing the initial
amplifying restriction endonuclease, if present, are contacted with
the reporter nucleic acid, the presence or absence of cleaved
reporter nucleic acid can be determined. The presence of cleaved
reporter nucleic acid can indicate that the sample contained the
target nucleic acid, thereby indicating that the sample contained
the target virus or microorganism for which the sample is being
tested, while the absence of cleaved reporter nucleic acid can
indicate that the sample lacked the target nucleic acid, thereby
indicating that the sample lacked the target virus or microorganism
for which the sample is being tested.
[0046] In some cases, the amount of cleaved reporter nucleic acid
can be determined. In such cases, the amount of cleaved reporter
nucleic acid can indicate the amount of target nucleic acid present
in the sample, which can indicated the degree or level of infection
by the target virus or microorganism for which the sample is being
tested. A standard curve using known amounts of target nucleic acid
or known amounts target viruses or microorganisms can be used to
aid in the determination of the amount of target nucleic acid or
target viruses or microorganisms present within a sample.
[0047] In some cases, the reporter nucleic acid can contain a label
to aid in the detection of cleaved reporter nucleic acid. For
example, reporter nucleic acid can contain a fluorescent label and
a quencher such that cleaved reporter nucleic acid provides a
fluorescent signal and uncleaved reporter nucleic acid does not
provide a fluorescent signal. In some cases, the reporter nucleic
acid can contain a label (e.g., a colorimetric label, fluorescent
label or an enzyme such as horse radish peroxidase) and can be
attached to a solid support (e.g., a well of a microtiter plate).
For example, the reporter nucleic acid can be attached to a solid
support such that cleavage at the initial amplifying restriction
endonuclease cut site by the initial amplifying restriction
endonuclease releases a portion of the reporter nucleic acid that
contains the label. The resulting reaction mixture can be collected
and assessed for the presence, absence, or amount of released
portions of the reporter nucleic acid using the label. For example,
the released portions of the reporter nucleic acid, if present, can
be transferred from one well of a microtiter plate (e.g., a 96-well
plate) that contained the reporter nucleic acid to another well of
a microtiter plate, where the transferred material can be assessed
for a signal from the label.
[0048] In some cases, the presence or absence of cleaved first
signal expansion nucleic acid, cleaved second signal expansion
nucleic acid, or both can be determined. The presence of such
cleaved nucleic acid can indicate that the sample contained the
target nucleic acid, thereby indicating that the sample contained
the target virus or microorganism for which the sample is being
tested, while the absence of such cleaved nucleic acid can indicate
that the sample lacked the target nucleic acid, thereby indicating
that the sample lacked the target virus or microorganism for which
the sample is being tested. In some cases, the amount of cleaved
first signal expansion nucleic acid, cleaved second signal
expansion nucleic acid, or both can be determined. In such cases,
the amount of cleaved nucleic acid can indicate the amount of
target nucleic acid present in the sample, which can indicate the
degree or level of infection by the target virus or microorganism
for which the sample is being tested. In these cases, the use of
cleaved first signal expansion nucleic acid, cleaved second signal
expansion nucleic acid, or both to assess the sample for target
nucleic acid can be in addition to the use of a separate reporter
nucleic acid step or can replace the use of a separate reporter
nucleic acid step. In some cases, the first signal expansion
nucleic acid, the second signal expansion nucleic acid, or both can
be labeled in a manner similar to that described herein for the
reporter nucleic acid to aid in detection. When the presence,
absence, or amount of cleaved first signal expansion nucleic acid,
cleaved second signal expansion nucleic acid, or both are
determined to assess the sample for target nucleic acid, the first
signal expansion nucleic acid can be referred to as a first
reporter nucleic acid and the second signal expansion nucleic acid
can be referred to as a second reporter nucleic acid even though
they include amplifying restriction endonucleases. A standard curve
using known amounts of target nucleic acid or known amounts of
target viruses or microorganisms can be used to aid in the
determination of the amount of target nucleic acid or target
viruses or microorganisms present within a sample.
[0049] Examples of a method of detecting target nucleic acid that
includes using probe nucleic acid, first signal expansion nucleic
acid, second signal expansion nucleic acid, and reporter nucleic
acid are set forth in FIGS. 3-5. With reference to FIG. 3, first
reaction chamber 200 (e.g., a microtiter plate well) can contain
probe nucleic acid 201. Probe nucleic acid 201 can be attached
(e.g., immobilized) to solid support 202 and can include initial
amplifying restriction endonuclease 203 (Ra). Probe nucleic acid
201 can be attached to solid support 202 such that initial
amplifying restriction endonuclease 203 is released from solid
support 202 upon cleavage of a nucleic acid component of probe
nucleic acid 201. Probe nucleic acid 201 can have a single-stranded
section having a nucleotide sequence that is complementary to at
least a portion of target nucleic acid 204. Probe nucleic acid 201
can be contacted with a sample that may or may not contain target
nucleic acid 204. If target nucleic acid 204 is present, at least a
portion of target nucleic acid 204 and probe nucleic acid 201 can
hybridize to form a double-stranded section of nucleic acid. Such a
double-stranded section can contain at least one recognition
restriction endonuclease cut site 205. Addition of recognition
restriction endonuclease 206 (Rr) to first reaction chamber 200 can
result in the cleavage of probe nucleic acid 201 at recognition
restriction endonuclease cut site 205 formed by one strand of probe
nucleic acid and one strand of target nucleic acid, thereby
releasing portion 207 of probe nucleic acid 201 from solid support
202. Portion 207 can include initial amplifying restriction
endonuclease 203.
[0050] The reaction product from first reaction chamber 200
containing released portion 207, if target nucleic acid 204 was
present, can be transferred (e.g., manually or automatically) to
second reaction chamber 220. Second reaction chamber 220 can
contain first signal expansion nucleic acid 226 and second signal
expansion nucleic acid 225. First signal expansion nucleic acid 226
can have at least one double-stranded portion that contains at
least one initial amplifying restriction endonuclease cut site 230.
First signal expansion nucleic acid 226 can be attached (e.g.,
immobilized) to solid support 222 and can include secondary
amplifying restriction endonuclease 223 (Rb). First signal
expansion nucleic acid 226 can be attached to solid support 222
such that portion 234 containing secondary amplifying restriction
endonuclease 223 is released from solid support 222 upon cleavage
of first signal expansion nucleic acid 226 at initial amplifying
restriction endonuclease cut site 230. For clarity, frame E of FIG.
3 omits depicting one strand from the cleaved versions of first
signal expansion nucleic acid 226 and second signal expansion
nucleic acid 225.
[0051] Second signal expansion nucleic acid 225 can have at least
one double-stranded portion that contains at least one secondary
amplifying restriction endonuclease cut site 232. Second signal
expansion nucleic acid 225 can be attached (e.g., immobilized) to
solid support 222 and can include initial amplifying restriction
endonuclease 224. Second signal expansion nucleic acid 225 can be
attached to solid support 222 such that portion 236 containing
initial amplifying restriction endonuclease 224 is released from
solid support 222 upon cleavage of second signal expansion nucleic
acid 225 at secondary amplifying restriction endonuclease cut site
232. Initial amplifying restriction endonuclease 203 of probe
nucleic acid 201 and initial amplifying restriction endonuclease
224 of second signal expansion nucleic acid 225 can be the same
restriction endonuclease. For example, both can be an EcoRI
restriction endonuclease.
[0052] Addition of the reaction product from first reaction chamber
200 to second reaction chamber 220 can result in the cleavage of
first signal expansion nucleic acid 226 at initial amplifying
restriction endonuclease cut site 230 if the reaction product
contains portion 207. Such cleavage of first signal expansion
nucleic acid 226 can result in the release of portion 234 from
solid support 222. Portion 234, which can include secondary
amplifying restriction endonuclease 223, can result in the cleavage
of second signal expansion nucleic acid 225 at secondary amplifying
restriction endonuclease cut site 232. Such cleavage of second
signal expansion nucleic acid 225 can result in the release of
portion 236 from solid support 222. Thus, this reaction can result
in the accumulation of released portions 234 and 236.
[0053] The reaction product from second reaction chamber 220
containing released portion 207, released portion 234, and released
portion 236, if target nucleic acid 204 was present, can be
transferred (e.g., manually or automatically) to third reaction
chamber 240. Third reaction chamber 240 can contain reporter
nucleic acid 241. Reporter nucleic acid 241 can be attached (e.g.,
immobilized) to solid support 242 and can include marker (e.g., a
label) 243 (M). Reporter nucleic acid 241 can be attached to solid
support 242 such that marker 243 is released from solid support 242
upon cleavage of a nucleic acid component of reporter nucleic acid
241. Reporter nucleic acid 241 can have at least one
double-stranded portion that contains at least one initial
amplifying restriction endonuclease cut site 246. Addition of the
reaction product from second reaction chamber 220 to third reaction
chamber 240 can result in the cleavage of reporter nucleic acid 241
at initial amplifying restriction endonuclease cut site 246 if the
reaction product contains portion 207 and portion 236. In some
cases, reporter nucleic acid 241 can include at least one
double-stranded portion that contains at least one cut site for
secondary amplifying restriction endonuclease 223. In such cases,
addition of the reaction product from second reaction chamber 220
to third reaction chamber 240 can result in the cleavage of
reporter nucleic acid 241 at the cut site for secondary amplifying
restriction endonuclease 223 if the reaction product contains
portion 234. Cleavage of reporter nucleic acid 241 can result in
the release of portion 247 from solid support 242. Portion 247 can
include marker 243.
[0054] The reaction product from third reaction chamber 240 can be
assessed to determine the presence, absence, or amount of portion
247. The presence of portion 247 can indicate that the sample
contained target nucleic acid 204, while the absence of portion 247
can indicate that the sample lacked target nucleic acid 204. In
some cases, the amount of portion 247 can be determined. In such
cases, the amount of portion 247 can indicate the amount of target
nucleic acid 204 present in the sample. The presence, absence, or
amount of portion 247 can be determined using marker 243, and
portion 247 having marker 243 can be distinguished from uncleaved
reporter nucleic acid 241 having marker 243 since, in this example,
portion 247 is released from solid support 242, while uncleaved
reporter nucleic acid 241 remains attached to solid support 242.
For example, in some cases, the reaction product from third
reaction chamber 24 can be transferred to fourth reaction chamber
where the presence or absence of portion 247 via marker 243 is
assessed. If portion 347 is present, the amount of portion 247
present can be quantified.
[0055] In some cases and with reference to FIGS. 4 and 5, first
signal expansion nucleic acid 226 can include marker (e.g., a
label) 243 (M) and second signal expansion nucleic acid 225 can
include marker (e.g., a label) 243 (M). In such cases, cleavage of
first signal expansion nucleic acid 226 and cleavage of second
signal expansion nucleic acid 225 can be assessed using marker 243
to determine the presence, absence, or amount of target nucleic
acid within a sample. For example, detector 250 can be used to
detect marker 243 released from solid support 222.
[0056] Probe nucleic acid 201, first signal expansion nucleic acid
226, second signal expansion nucleic acid 225, and reporter nucleic
acid 241 can have various configurations. For example, with
reference to FIG. 3, probe nucleic acid 201 can be designed to have
a single nucleic acid strand such that the entire nucleic acid
component of probe nucleic acid 201 is single-stranded prior to
contact with target nucleic acid 204. In another example, probe
nucleic acid 201 can be designed in a manner like probe nucleic
acid 101 to have two or more strands. See, e.g., FIG. 2. For
example, probe nucleic acid 201 can have a first strand and a
second strand. The first strand can be attached to a solid support
and can be designed to have a single-stranded section having a
nucleotide sequence that is complementary to at least a portion of
target nucleic acid. The second strand can include an initial
amplifying restriction endonuclease and can have a single-stranded
section having a nucleotide sequence that can hybridize to the
first strand. In some cases, the first strand and second strand can
be synthesized or obtained separately and then mixed together to
form probe nucleic acid 201. For example, the first strand can be
synthesized, biotinylated, and attached to a streptavidin-coated
solid support. After synthesizing the nucleic acid component of the
second strand and attaching an initial amplifying restriction
endonuclease to the synthesized nucleic acid component, the second
strand can be incubated with the first strand to form nucleic acid
probe 201. In some cases, probe nucleic acid 201 can contain more
than two strands. For example, probe nucleic acid can include a
first strand, a second strand, and a third strand. In this case,
the first strand can be attached to a solid support, the second
strand can be hybridized to the first strand and can include a
single-stranded section having a nucleotide sequence that is
complementary to at least a portion of target nucleic acid, and the
third strand can be hybridized to the second strand and can be
attached to an initial amplifying restriction endonuclease. Similar
one, two, three, or more strand configurations can be used to make
first signal expansion nucleic acid, second signal expansion
nucleic acid, or reporter nucleic acid. For example, first signal
expansion nucleic acid and second signal expansion nucleic acid can
be designed to have a configuration as shown in FIG. 4 or 5.
[0057] Probe nucleic acid described herein typically includes at
least one single-stranded DNA section that is designed to hybridize
with a desired target nucleic acid and thereby create a recognition
restriction endonuclease cut site. The other portions of the probe
nucleic acid can include DNA, RNA, or other molecules. For example,
probe nucleic acid can include biotin such that the probe nucleic
acid can be attached to a streptavidin-coated solid support. In
some cases, the single-stranded section of the probe nucleic acid
that is designed to hybridize with a desired target nucleic acid
and create a recognition restriction endonuclease cut site can be
RNA or a nucleic acid analog (e.g., a peptide nucleic acid (PNA))
provided that such a single-stranded section can (i) hybridize with
the desired target nucleic acid and (ii) create a recognition
restriction endonuclease cut site with the complementary target
nucleic acid sequence that is capable of being cleaved by the
recognition restriction endonuclease. Examples of restriction
endonucleases that can be used as recognition restriction
endonucleases to cleave a recognition restriction endonuclease cut
site that is created between an RNA section of the probe nucleic
acid and a DNA section of the target nucleic acid include, without
limitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI
restriction endonucleases.
[0058] Probe nucleic acid described herein can be any length
provided that the single-stranded section of the probe nucleic acid
that is designed to hybridize with a desired target nucleic acid is
capable of hybridizing to the target nucleic acid and provided that
the amplifying restriction endonuclease of the probe nucleic acid
is capable of cleaving its amplifying restriction endonuclease cut
site after the probe nucleic acid is cleaved by a recognition
restriction endonuclease. In general, the single-stranded section
of the probe nucleic acid that is designed to hybridize with a
desired target nucleic acid can be between about 10 and about 500
or more nucleotides (e.g., between about 10 and about 400
nucleotides, between about 10 and about 300 nucleotides, between
about 10 and about 200 nucleotides, between about 10 and about 100
nucleotides, between about 10 and about 50 nucleotides, between
about 10 and about 25 nucleotides, between about 20 and about 500
nucleotides, between about 30 and about 500 nucleotides, between
about 40 and about 500 nucleotides, between about 50 and about 500
nucleotides, between about 15 and about 50 nucleotides, between
about 15 and about 25 nucleotides, between about 20 and about 50
nucleotides, between about 18 and about 25 nucleotides, between
about 20 and about 60 nucleotides, between about 25 and about 55
nucleotides, between about 30 and about 50 nucleotides, between
about 35 and about 45 nucleotides, or between about 38 and about 42
nucleotides) in length. The recognition restriction endonuclease
cut site that will be created by the hybridization of target
nucleic acid to this single-stranded section of the probe nucleic
acid can be located at any position alone the single-stranded
section. For example, the recognition restriction endonuclease cut
site to be created can be towards the 5' end, towards the '3 end,
or near the center of the single-stranded section of the probe
nucleic acid. In general, the overall length of the probe nucleic
acid described herein can be between about 10 and about 2500 or
more nucleotides (e.g., between about 10 and about 2000
nucleotides, between about 10 and about 1000 nucleotides, between
about 10 and about 500 nucleotides, between about 10 and about 400
nucleotides, between about 10 and about 300 nucleotides, between
about 10 and about 200 nucleotides, between about 10 and about 100
nucleotides, between about 10 and about 50 nucleotides, between
about 10 and about 25 nucleotides, between about 20 and about 500
nucleotides, between about 30 and about 500 nucleotides, between
about 40 and about 500 nucleotides, between about 50 and about 500
nucleotides, between about 75 and about 500 nucleotides, between
about 100 and about 500 nucleotides, between about 150 and about
500 nucleotides, between about 15 and about 50 nucleotides, between
about 15 and about 25 nucleotides, between about 20 and about 50
nucleotides, between about 18 and about 25 nucleotides, between
about 20 and about 60 nucleotides, between about 25 and about 55
nucleotides, between about 30 and about 50 nucleotides, between
about 35 and about 45 nucleotides, or between about 38 and about 42
nucleotides) in length.
[0059] The recognition restriction endonuclease cut site to be
created by hybridization of target nucleic acid to the probe
nucleic acid can be a cut site of any type of restriction
endonuclease. In addition, any type of restriction endonuclease can
be used as a recognition restriction endonuclease to cleave probe
nucleic acid upon target nucleic acid hybridization. Examples of
restriction endonucleases that can be used as recognition
restriction endonucleases include, without limitation, EcoRI,
EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI,
HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI,
SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II,
NlaIV, RsaI, TaiI, AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I,
I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI,
Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,
AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI,
AccI, BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauI
restriction endonucleases. In some cases, nucleic acid encoding a
naturally-occurring restriction endonuclease can be genetically
engineered to create a modified restriction endonuclease that has
the ability to recognize a particular cut site. Common computer
algorithms can be used to locate restriction endonuclease cut sites
along the nucleotide sequence of any desired target nucleic acid.
Once located, the sequence of the restriction endonuclease cut site
along with additional flanking sequence (e.g., 5' flanking
sequence, 3' flanking sequence, or both 5' and 3' flanking
sequence) can be used to design the complementary sequence of the
probe nucleic acid that is used to hybridize to the target nucleic
acid and create the recognition restriction endonuclease cut site
upon target nucleic acid hybridization. In some cases, a probe
nucleic acid can be designed to have the restriction endonuclease
cut site located in the middle or near the middle such that the
restriction endonuclease cut site has both 5' and 3' flanking
sequences that are complementary to the target nucleic acid.
[0060] In general, probe nucleic acid can be designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form a single recognition restriction
endonuclease cut site upon target nucleic acid hybridization. In
some cases, probe nucleic acid can be designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form more than one (e.g., two, three,
four, five, six, seven, eight, nine, ten, or more) recognition
restriction endonuclease cut site upon target nucleic acid
hybridization. When more than one recognition restriction
endonuclease cut site is used, the multiple recognition restriction
endonuclease cut sites can be cut sites for the same restriction
endonuclease or cut sites for different restriction endonucleases.
For example, probe nucleic acid can be designed to have a
single-stranded section that is designed to hybridize with desired
target nucleic acid and to form one recognition restriction
endonuclease cut site for an EcoRI recognition restriction
endonuclease and one recognition restriction endonuclease cut site
for an XbaI recognition restriction endonuclease upon target
nucleic acid hybridization. In such cases, each recognition
restriction endonuclease can be used individually or in combination
(e.g., as a mixture) to cleave probe nucleic acid that hybridized
to target nucleic acid and formed the corresponding recognition
restriction endonuclease cut site via such hybridization.
[0061] Probe nucleic acid can be designed such that any target
nucleic acid of a target virus or microorganism suspected of
infecting a mammal (e.g., a human) can be detected. Examples of
target nucleic acid that can be detected using the methods and
materials provided herein include, without limitation, viral DNA or
RNA, microbial DNA or RNA (e.g., bacterial, fungal, or protozoan
DNA or RNA), and methylated microbial DNA. In some cases such as
those involving assessing a biological sample for an RNA virus, the
target nucleic acid can be an RNA or a cDNA generated from an RNA.
When detecting an RNA target nucleic acid, restriction
endonucleases having the ability to cleave a recognition
restriction endonuclease cut site that is created between a DNA
section of the probe nucleic acid and the RNA target nucleic acid
can be used as recognition restriction endonucleases. Examples of
such restriction endonucleases include, without limitation, HhaI,
AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI restriction
endonucleases. When detecting methylated target nucleic acid (e.g.,
a methylated target nucleic acid of a bacterial organism),
restriction endonucleases having the ability to cleave a
recognition restriction endonuclease cut site that includes a
methylated nucleotide to be assessed can be used as recognition
restriction endonucleases. Examples of restriction endonucleases
having the ability to recognize methylated nucleotides include,
without limitation, DpnI, GlaI, HpaII, MspI, AciI, HhaI, and SssI
restriction endonucleases. In such cases, a control can include
detecting the same target nucleic acid without the methylated
nucleotide. In some cases, a combination of methylation insensitive
and methylation sensitive restriction endonucleases can be used to
assess a sample for methylated target nucleic acid. For example,
similar generation of cleavage products using both methylation
insensitive and methylation sensitive restriction endonucleases
designed for the same site can indicate that the target nucleic
acid lacks methylation at that site, while an increased level of
cleavage products using a methylation insensitive restriction
endonuclease as compared to the level generated using a methylation
sensitive restriction endonuclease designed for the same site can
indicate that the target nucleic acid is methylated at that
site.
[0062] The nucleotide sequence of target nucleic acid to be
detected can be obtained from, for example, common nucleic acid
sequence databases such as GenBank.RTM.. A portion of target
nucleic acid sequence can be selected using a computer-based
program. For example, a computer-based program can be used to
detect restriction endonuclease cut sites within a portion of
target nucleic acid. Such information can be used to design probe
nucleic acid such that the single-stranded section creates at least
one recognition restriction endonuclease cut site upon
hybridization of the target nucleic acid. In some cases,
bioinformatics computer-based programs and tools can be used to
assist in the design of probe nucleic acid. For example, computer
programs (e.g., BLAST.RTM. and alignment programs) and computer
databases (e.g., GenBank.RTM.) can be used to indentify nucleic
acid sequences from particular viruses or microorganisms and can be
used to identify regions of high sequence similarity among various
strains or variants of particular viruses or microorganisms. In
addition, computer programs such as CLC Workbench or Vector NTI
(Invitrogen) can be used to identify the location of restriction
endonuclease cut sites within a particular nucleic acid sequence.
In some cases, sequence analysis computer programs can be used to
identify sequences with limited or an absence of repeats, a
presence of high sequence complexity of a potential recognition
restriction endonuclease cut site, and/or limited or an absence of
hairpin structures. Identification of such sequences can help
reduce the risk of probe self-hybridization and potentially
unintended cutting by a recognition endonuclease.
[0063] Any appropriate method can be used to obtain the nucleic
acid component of the probe nucleic acid. For example, common
molecular cloning and chemical nucleic acid synthesis techniques
can be used to obtain the nucleic acid component of the probe
nucleic acid. In some cases, the nucleic acid component of the
probe nucleic acid can be synthesized using commercially available
automated oligonucleotide synthesizers such as those available from
Applied Biosystems (Foster City, Calif.). In some cases, probe
nucleic acids can be synthesized de novo using any of a number of
procedures widely available in the art. Examples of such methods of
synthesis include, without limitation, the P-cyanoethyl
phosphoramidite method (Beaucage et al., Tet. Let., 22:1859-1862
(1981)) and the nucleoside H-phosphonate method (Garegg et al.,
Tet. Let., 27:4051-4054 (1986); Froehler et al., Nucl. Acid Res.,
14:5399-5407 (1986); Garegg et al., Tet. Let., 27:4055-4058 (1986);
and Gaffney et al., Tet. Let., 29:2619-2622 (1988)). These methods
can be performed by a variety of commercially-available automated
oligonucleotide synthesizers. In some cases, recombinant nucleic
acid techniques such as PCR and those that include using
restriction enzyme digestion and ligation of existing nucleic acid
sequences (e.g., genomic DNA or cDNA) can be used to obtain the
nucleic acid component of the probe nucleic acid.
[0064] Probe nucleic acid described herein can be attached to a
solid support. Examples of solid supports include, without
limitation, a well of a microtiter plate (e.g., a 96-well
microtiter plate or ELISA plate), beads (e.g., magnetic, glass,
plastic, or gold-coated beads), slides (e.g., glass or gold-coated
slides), micro- or nano-particles (e.g., carbon nanotubes),
platinum solid supports, palladium solid supports, and a surface of
a chamber or channel within a microfluidic device. In some cases, a
solid support can be a silicon oxide-based solid support, a plastic
polymer-based solid support (e.g., a nylon, nitrocellulose, or
polyvinylidene fluoride-based solid support), or a biopolymer-based
(e.g., a cross-linked dextran or cellulose-based solid support)
solid support. Probe nucleic acid can be directly or indirectly
attached to a solid support. For example, biotin can be a component
of the probe nucleic acid, and the probe nucleic acid containing
biotin can be indirectly attached to a solid support that is coated
with streptavidin via a biotin-streptavidin interaction. In some
cases, probe nucleic acid can be attached to a solid support via a
covalent or non-covalent interaction. For example, probe nucleic
acid can be covalently attached to magnetic beads as described
elsewhere (Albretsen et al., Anal. Biochem., 189(1):40-50
(1990)).
[0065] Probe nucleic acid can be designed to contain any type of
restriction endonuclease as an amplifying restriction endonuclease.
In general, an amplifying restriction endonuclease of the probe
nucleic acid is typically a different restriction endonuclease than
the restriction endonuclease that is used as a recognition
restriction endonuclease. For example, when an EcoRI restriction
endonuclease is used as a recognition restriction endonuclease, a
restriction endonuclease other than an EcoRI restriction
endonuclease (e.g., a HindIII restriction endonuclease) is used as
an amplifying restriction endonuclease. Examples of restriction
endonucleases that can be used as amplifying restriction
endonucleases include, without limitation, EcoRI, EcoRII, BamHI,
HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI,
EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI, XbaI,
AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI,
AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,
TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,
BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,
BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI,
BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauI restriction
endonucleases. Any number of molecules of the same amplifying
restriction endonuclease can be attached to one probe nucleic acid
molecule. For example, a single probe nucleic acid molecule can
contain one, two, three, four, five, or more EcoRI amplifying
restriction endonuclease molecules. In some cases, a single probe
nucleic acid molecule can contain two or more (e.g., two, three,
four, five, or more) different types of amplifying restriction
endonucleases. For example, a single probe nucleic acid molecule
can contain three EcoRI amplifying restriction endonuclease
molecules and two BanII amplifying restriction endonuclease
molecules.
[0066] Any appropriate method can be used to attach an amplifying
restriction endonuclease to a nucleic acid component of the probe
nucleic acid. In some cases, an amplifying restriction endonuclease
can be attached by an ionic or covalent attachment. For example,
covalent bonds such as amide bonds, disulfide bonds, and thioether
bonds, or bonds formed by crosslinking agents can be used. In some
cases, a non-covalent linkage can be used. The attachment can be a
direct attachment or an indirect attachment. For example, a linker
can be used to attach an amplifying restriction endonuclease to a
nucleic acid component of the probe nucleic acid. In some cases,
nucleic acid can include a thiol modification, and a restriction
endonuclease can be conjugated to the thiol-containing nucleic acid
based on succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) using
techniques similar to those described elsewhere (Dill et al.,
Biosensors and Bioelectronics, 20:736-742 (2004)). In some cases, a
biotinylated nucleic acid and a streptavidin-containing restriction
endonuclease can be attached to one another via a
biotin-streptavidin interaction. A restriction endonuclease can be
conjugated with streptavidin using, for example, sulfosuccinimidyl
6-(3'-[2-pyridyldithio]-propionamido)hexanoate. An amplifying
restriction endonuclease can be attached at any location of a
nucleic acid component of the probe nucleic acid. For example, an
amplifying restriction endonuclease can be attached at an end
(e.g., a 5' end or 3' end) of a nucleic acid component, in the
middle of a nucleic acid component, or at any position along the
length of a nucleic acid component.
[0067] Signal expansion nucleic acid (e.g., first signal expansion
nucleic acid and second signal expansion nucleic acid) and reporter
nucleic acid described herein typically include at least one
double-stranded DNA section that includes an amplifying restriction
endonuclease cut site (e.g., an initial amplifying restriction
endonuclease cut site, a secondary amplifying restriction
endonuclease cut site, or a tertiary amplifying restriction
endonuclease cut site). The other portions of the signal expansion
nucleic acid or reporter nucleic acid can include DNA, RNA, or
other molecules. For example, reporter nucleic acid can include
biotin such that the reporter nucleic acid can be attached to a
streptavidin-coated solid support. In some cases, one or both
strands of the double-stranded section of the signal expansion
nucleic acid or the reporter nucleic acid that contains an
amplifying restriction endonuclease cut site can be RNA or a
nucleic acid analog (e.g., a peptide nucleic acid (PNA)) provided
that such a double-stranded section is capable of being cleaved by
the amplifying restriction endonuclease. Examples of restriction
endonucleases that can be used as amplifying restriction
endonucleases to cleave a DNA:RNA hybrid section of signal
expansion nucleic acid or reporter nucleic acid include, without
limitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindII, SalI, and MspI
restriction endonucleases.
[0068] Signal expansion nucleic acid or reporter nucleic acid
described herein can be any length provided that the
double-stranded section that contains the amplifying restriction
endonuclease cut site is capable of being cleaved by the amplifying
restriction endonuclease. In general, the double-stranded section
of signal expansion nucleic acid or reporter nucleic acid can be
between about 10 and about 500 or more nucleotides (e.g., between
about 10 and about 400 nucleotides, between about 10 and about 300
nucleotides, between about 10 and about 200 nucleotides, between
about 10 and about 100 nucleotides, between about 10 and about 50
nucleotides, between about 10 and about 25 nucleotides, between
about 20 and about 500 nucleotides, between about 30 and about 500
nucleotides, between about 40 and about 500 nucleotides, between
about 50 and about 500 nucleotides, between about 15 and about 50
nucleotides, between about 15 and about 25 nucleotides, between
about 20 and about 50 nucleotides, or between about 18 and about 25
nucleotides, between about 20 and about 60 nucleotides, between
about 25 and about 55 nucleotides, between about 30 and about 50
nucleotides, between about 35 and about 45 nucleotides, or between
about 38 and about 42 nucleotides) in length. In some cases, the
double-stranded section of signal expansion nucleic acid or
reporter nucleic acid can be between 5 and 50 nucleotides in
length. The amplifying restriction endonuclease cut site of the
signal expansion nucleic acid or the reporter nucleic acid can be
located at any position alone the double-stranded section. For
example, the amplifying restriction endonuclease cut site can be
towards the 5' end, towards the '3 end, or near the center of the
double-stranded section of the signal expansion nucleic acid or the
reporter nucleic acid. In general, the overall length of signal
expansion nucleic acid or reporter nucleic acid described herein
can be between about 10 and about 2500 or more nucleotides (e.g.,
between about 10 and about 2000 nucleotides, between about 10 and
about 1000 nucleotides, between about 10 and about 500 nucleotides,
between about 10 and about 400 nucleotides, between about 10 and
about 300 nucleotides, between about 10 and about 200 nucleotides,
between about 10 and about 100 nucleotides, between about 10 and
about 50 nucleotides, between about 10 and about 25 nucleotides,
between about 20 and about 500 nucleotides, between about 30 and
about 500 nucleotides, between about 40 and about 500 nucleotides,
between about 50 and about 500 nucleotides, between about 75 and
about 500 nucleotides, between about 100 and about 500 nucleotides,
between about 150 and about 500 nucleotides, between about 15 and
about 50 nucleotides, between about 15 and about 25 nucleotides,
between about 20 and about 50 nucleotides, between about 18 and
about 25 nucleotides, between about 20 and about 60 nucleotides,
between about 25 and about 55 nucleotides, between about 30 and
about 50 nucleotides, between about 35 and about 45 nucleotides, or
between about 38 and about 42 nucleotides) in length.
[0069] The amplifying restriction endonuclease cut site of signal
expansion nucleic acid or reporter nucleic acid described herein
can be a cut site of any type of restriction endonuclease. In
addition, any type of restriction endonuclease can be used as an
amplifying restriction endonuclease to cleave signal expansion
nucleic acid or reporter nucleic acid. Examples of restriction
endonucleases that can be used as amplifying restriction
endonucleases include, without limitation, EcoRI, EcoRII, BamHI,
HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI,
EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI, XbaI,
AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI,
AasI, BbsI, BseJI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,
TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,
BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,
BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI,
BsrGI, CspI, Esp3I, KspAI, Nsil, SapI, and TauI restriction
endonucleases.
[0070] In general, signal expansion nucleic acid or reporter
nucleic acid can be designed to have a double-stranded section that
contains a single amplifying restriction endonuclease cut site. In
some cases, signal expansion nucleic acid or reporter nucleic acid
provided herein can be designed to have a double-stranded section
that contains more than one (e.g., two, three, four, five, six,
seven, eight, nine, ten, or more) amplifying restriction
endonuclease cut site. When more than one amplifying restriction
endonuclease cut site is used, the multiple amplifying restriction
endonuclease cut sites can be cut sites for the same restriction
endonuclease or cut sites for different restriction endonucleases.
For example, reporter nucleic acid can be designed to have a
double-stranded section that contains one initial amplifying
restriction endonuclease cut site for an EcoRI initial amplifying
restriction endonuclease and one secondary amplifying restriction
endonuclease cut site for an Xbal secondary amplifying restriction
endonuclease.
[0071] Any appropriate method can be used to obtain the nucleic
acid component of signal expansion nucleic acid or reporter nucleic
acid. For example, common molecular cloning and chemical nucleic
acid synthesis techniques can be used to obtain the nucleic acid
component of signal expansion nucleic acid or reporter nucleic
acid. In some cases, the nucleic acid component of signal expansion
nucleic acid or reporter nucleic acid can be synthesized using
commercially available automated oligonucleotide synthesizers such
as those available from Applied Biosystems (Foster City, Calif.).
In some cases, signal expansion nucleic acid or reporter nucleic
acid can be synthesized de novo using any of a number of procedures
widely available in the art. Examples of such methods of synthesis
include, without limitation, the .beta.-cyanoethyl phosphoramidite
method (Beaucage et al., Tet. Let., 22:1859-1862 (1981)) and the
nucleoside H-phosphonate method (Garegg et al., Tet. Let.,
27:4051-4054 (1986); Froehler et al., Nucl. Acid Res., 14:5399-5407
(1986); Garegg et al., Tet. Let., 27:4055-4058 (1986); and Gaffney
et al., Tet. Let., 29:2619-2622 (1988)). These methods can be
performed by a variety of commercially-available automated
oligonucleotide synthesizers. In some cases, recombinant nucleic
acid techniques such as PCR and those that include using
restriction enzyme digestion and ligation of existing nucleic acid
sequences (e.g., genomic DNA or cDNA) can be used to obtain the
nucleic acid component of signal expansion nucleic acid or reporter
nucleic acid.
[0072] Signal expansion nucleic acid or reporter nucleic acid
described herein can be attached to a solid support. Examples of
solid supports include, without limitation, a well of a microtiter
plate (e.g., a 96-well microtiter plate or ELISA plate), beads
(e.g., magnetic, glass, plastic, or gold-coated beads), slides
(e.g., glass or gold-coated slides), micro- or nano-particles
(e.g., carbon nanotubes), platinum solid supports, palladium solid
supports, and a surface of a chamber or channel within a
microfluidic device. In some cases, a solid support can be a
silicon oxide-based solid support, a plastic polymer-based solid
support (e.g., a nylon, nitrocellulose, or polyvinylidene
fluoride-based solid support) or a biopolymer-based (e.g., a
cross-linked dextran or cellulose-based solid support) solid
support.
[0073] Signal expansion nucleic acid or reporter nucleic acid can
be directly or indirectly attached to a solid support. For example,
biotin can be a component of signal expansion nucleic acid or
reporter nucleic acid, and the signal expansion nucleic acid or the
reporter nucleic acid containing biotin can be indirectly attached
to a solid support that is coated with streptavidin via a
biotin-streptavidin interaction. In some cases, signal expansion
nucleic acid or reporter nucleic acid can be attached to a solid
support via a covalent or non-covalent interaction. For example,
signal expansion nucleic acid or reporter nucleic acid can be
covalently attached to magnetic beads as described elsewhere
(Albretsen et al., Anal. Biochem., 189(1):40-50 (1990)).
[0074] Signal expansion nucleic acid can be designed to contain any
type of restriction endonuclease as an amplifying restriction
endonuclease (e.g., an initial amplifying restriction endonuclease,
a secondary amplifying restriction endonuclease, or a tertiary
amplifying restriction endonuclease). In general, an amplifying
restriction endonuclease of signal expansion nucleic acid is
typically a different restriction endonuclease than the restriction
endonuclease that is used as a recognition restriction
endonuclease. For example, when an EcoRI restriction endonuclease
is used as a recognition restriction endonuclease, a restriction
endonuclease other than an EcoRI restriction endonuclease (e.g., a
HeaIII restriction endonuclease) is used as an amplifying
restriction endonuclease. Examples of restriction endonucleases
that can be used as amplifying restriction endonucleases include,
without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,
HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I,
KpnI, PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI,
BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseJI,
BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI,
BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I,
Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI,
KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI, CspI, Esp3I,
KspAI, NsiI, SapI, and TauI restriction endonucleases. Any number
of molecules of the same amplifying restriction endonuclease can be
attached to one signal expansion nucleic acid molecule. For
example, a single signal expansion nucleic acid molecule can
contain one, two, three, four, five, or more EcoRI amplifying
restriction endonuclease molecules. In some cases, a single signal
expansion nucleic acid molecule can contain two or more (e.g., two,
three, four, five, or more) different types of amplifying
restriction endonucleases. For example, a single signal expansion
nucleic acid molecule can contain three BanII amplifying
restriction endonuclease molecules and two SacII amplifying
restriction endonuclease molecules.
[0075] Reporter nucleic acid can be designed to contain a label to
aid in the detection of cleaved reporter nucleic acid. In some
cases, signal expansion nucleic acid can be designed to contain a
label. In such cases, signal expansion nucleic acid containing a
label can be used in addition to reporter nucleic acid or in place
of reporter nucleic acid to detect target nucleic acid. Examples of
labels that can be a component of reporter nucleic acid or signal
expansion nucleic acid include, without limitation, fluorescent
labels (with or without the use of quenchers), dyes, antibodies,
radioactive material, enzymes (e.g., horse radish peroxidase,
alkaline phosphatase, laccase, galactosidase, or luciferase), redox
labels (e.g., ferrocene redox labels), metallic particles (e.g.,
gold nanoparticles), and green fluorescent protein-based labels. In
some cases, for a redox label, such as ferrocene, the detector can
be an electrode for amperometric assay of redox molecules. For
example, if the redox label is present in a reduced form of
ferrocene, then the electrode at high electrode potential can
provide an oxidation of the reduced form of ferrocene, thereby
converting it to an oxidized form of ferrocene. The generated
current can be proportional to the concentration of ferrocene label
in the solution.
[0076] In one embodiment, reporter nucleic acid or signal expansion
nucleic acid can contain a fluorescent label and a quencher such
that cleaved reporter nucleic acid provides a fluorescent signal
and uncleaved reporter nucleic acid does not provide a fluorescent
signal. In some cases, the reporter nucleic acid or signal
expansion nucleic acid can contain a label (e.g., a fluorescent
label or an enzyme such as horse radish peroxidase) and can be
attached to a solid support (e.g., a well of a microtiter plate).
For example, the reporter nucleic acid or signal expansion nucleic
acid can be attached to a solid support such that cleavage at the
amplifying restriction endonuclease cut site by the amplifying
restriction endonuclease releases a portion of the reporter nucleic
acid or the signal expansion nucleic acid that contains the label.
The resulting reaction mixture can be collected and assessed for
the presence, absence, or amount of released portions of the
reporter nucleic acid or signal expansion nucleic acid using the
label. For example, the released portions of the reporter nucleic
acid or the signal expansion nucleic acid, if present, can be
transferred from one well of a microtiter plate (e.g., a 96-well
plate) that contained the reporter nucleic acid or the signal
expansion nucleic acid to another well of a microtiter plate, where
the transferred material can be assessed for a signal from the
label. Any number of molecules of a label can be attached to one
reporter nucleic acid molecule or one signal expansion nucleic acid
molecule. For example, a reporter nucleic acid molecule or a single
signal expansion nucleic acid molecule can contain one, two, three,
four, five, or more fluorescent molecules.
[0077] Any appropriate method can be used to attach a label to a
nucleic acid component of reporter nucleic acid or signal expansion
nucleic acid. In some cases, a label can be attached by an ionic or
covalent attachment. For example, covalent bonds such as amide
bonds, disulfide bonds, and thioether bonds, or bonds formed by
crosslinking agents can be used. In some cases, a non-covalent
linkage can be used. The attachment can be a direct attachment or
an indirect attachment. For example, a linker can be used to attach
a label to a nucleic acid component of reporter nucleic acid or
signal expansion nucleic acid. In some cases, nucleic acid can
include a thiol modification, and a label can be conjugated to the
thiol-containing nucleic acid based on succinimidyl
4-[N-maleimidomethyl]cyclo-hexane-1-carboxylate (SMCC) using
techniques similar to those described elsewhere (Dill et al.,
Biosensors and Bioelectronics, 20:736-742 (2004)). In some cases, a
biotinylated nucleic acid and a streptavidin-containing label can
be attached to one another via a biotin-streptavidin interaction. A
label can be conjugated with streptavidin using, for example,
sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate. A
label can be attached at any location of a nucleic acid component
of reporter nucleic acid or signal expansion nucleic acid. For
example, a label can be attached at an end (e.g., a 5' end or 3'
end) of a nucleic acid component, in the middle of a nucleic acid
component, or at any position along the length of a nucleic acid
component of reporter nucleic acid or signal expansion nucleic
acid.
[0078] As described herein, the methods and materials provided
herein can be used to detect target nucleic acid of a target virus
or microorganism in any type of sample (e.g., a biological sample).
For example, a nasal or mucus sample can be collected from a mammal
and assessed for target nucleic acid to determined if the mammal
has an infection. Once obtained, a sample to be assessed can be
processed to obtain nucleic acid. For example, a nucleic acid
extraction can be performed on a blood sample to obtain a sample
that is enriched for nucleic acid. In some cases, a sample can be
heated or treated with a cell lysis agent to release nucleic acid
from cells present in the sample.
[0079] As described herein, a sample (e.g., a biological sample)
can be assessed for the presence, absence, or amount of target
viral and/or microbial nucleic acid (e.g., target pathogen nucleic
acid) using an enzymatic amplification cascade of restriction
endonucleases described herein without using a nucleic acid
amplification technique (e.g., a PCR-based nucleic acid technique).
Assessing samples (e.g., biological samples) for the presence,
absence, or amount of target nucleic acid using an enzymatic
amplification cascade of restriction endonucleases described herein
without using a nucleic acid amplification technique can allow
patients as well as medical, laboratory, or veterinarian personnel
(e.g., clinicians, physicians, physician's assistants, laboratory
technicians, research scientists, and veterinarians) to test for an
infection without the need for potentially expensive thermal
cycling devices and potentially time consuming thermal cycling
techniques. In some cases, the methods and materials provided
herein can be used in combination with a PCR-based nucleic acid
technique. For example, a PCR-based nucleic acid technique can be
performed to amplify nucleic acid (e.g., a target pathogen's
nucleic acid) present within a biological sample, and the resulting
amplification material can be assessed using an enzymatic
amplification cascade of restriction endonucleases described herein
to detect the presence, absence, or amount of a particular nucleic
acid (e.g., a target pathogen's nucleic acid). In some cases, a
limited PCR-based nucleic acid technique can be performed to
amplify a target nucleic acid to a point where the amount of
amplified target nucleic acid is increased only slightly over the
amount of target nucleic acid originally present within the
biological sample. For example, a two to twelve cycle PCR technique
(e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycle PCR technique)
can be performed to slightly increase the amount of amplified
target nucleic acid as compared to the amount of unamplified target
nucleic acid originally present within the biological sample. Such
limited PCR-based nucleic acid techniques, when used in combination
with an enzymatic amplification cascade of restriction
endonucleases described herein, can allow medical, laboratory, or
veterinarian personnel to test mammals with a potentially increased
level of sensitivity and/or specificity without the potentially
lengthy time involved in thermal cycling techniques that include a
greater number of cycles. This increased level of sensitivity
and/or specificity can be over the high level of sensitivity and
specificity of a comparable testing procedure that includes an
enzymatic amplification cascade of restriction endonucleases
described herein without the limited PCR-based nucleic acid
technique. In some cases, the PCR-based nucleic acid technique can
be performed to amplify a target nucleic acid to a point where the
amount of amplified target nucleic acid is easily detectable (e.g.,
visually detectable using gel electrophoresis and ethidium bromide
staining). For example, a 15 or more cycle PCR technique (e.g., a
20 cycle PCR technique) can be performed to produce at least ng
amounts (e.g., greater than 1 ng, 10 ng, 100 ng, 1 .mu.g, 10 .mu.g,
or more) of amplified nucleic acid. Such PCR-based nucleic acid
techniques, when used in combination with an enzymatic
amplification cascade of restriction endonucleases described
herein, can allow medical, laboratory, or veterinarian personnel to
test mammals with a potentially increased level of sensitivity
and/or specificity. This increased level of sensitivity and/or
specificity can be over the high level of sensitivity and
specificity of a comparable testing procedure that includes an
enzymatic amplification cascade of restriction endonucleases
described herein without the PCR-based nucleic acid technique.
[0080] In some cases, a sample (e.g. a biological sample) can be
obtained and subjected to a culturing technique. For example, a
mucus sample can be collected and cultured with medium (e.g.,
enrichment medium or broth with or without the ability to promote
selective growth) to enrich the sample such that the number of
viruses or microorganisms present in the sample can increase.
Examples of enrichment media include, without limitation, blood
agar, selenite-cystine broth, and tetrathionate. In some cases, the
culture medium can contain a nutrient, ingredient, or drug that
prevents certain microbial species or strains from replicating
while allowing other microbial species or strains to replicate. In
some cases, the culturing technique can include incubating a sample
at an appropriate temperature (e.g. between 15.degree. C. and
45.degree. C., between 20.degree. C. and 45.degree. C., between
25.degree. C. and 45.degree. C., between 30.degree. C. and
45.degree. C., between 30.degree. C. and 40.degree. C., between
35.degree. C. and 45.degree. C., or between 35.degree. C. and
40.degree. C.) for an appropriate period of time (e.g., between
about 0.5 hours and 48 hours, between about 0.5 hours and 36 hours,
between about 0.5 hours and 24 hours, between about 0.5 hours and
12 hours, between about 0.5 hours and 8 hours, between about 0.5
hours and 6 hours, between about 0.5 hours and 5 hours, between
about 0.5 hours and 4 hours, between about 0.5 hours and 3 hours,
between about 0.5 hours and 2 hours, between about 1 hour and 4
hours, or between about 2 hours and 4 hours). For example, a sample
can be obtained and cultured in enrichment medium at 37.degree. C.
for 2 to 6 hours. Examples of culture techniques that can be used
as described herein include, without limitation, those described
elsewhere (Schrank et al., Vet. Micro., 82:45-53 (2001); and Black,
J. G. Microbiology. Principles and Applications, Third Edition,
144-148 (1996)).
[0081] In some cases, a sample, obtained and subjected to a
culturing technique or not, can be processed, for example, to
remove non-nucleic acid material, to disrupt cell membranes to
release nucleic acid, and/or to collect or extract nucleic acid,
such that nucleic acid of the sample, if present within the sample,
is available for hybridization to probe nucleic acid. For example,
a blood or nasal swab sample can be treated with a lysis buffer and
subjected to nucleic acid extraction such that a major component of
the sample is nucleic acid. In some cases, a sample can be
homogenized and treated to disrupt cells including microbial cells
that are present in the sample. For example, a mucus sample can be
subjected to high speed mechanical homogenization with
glass/silica/zirconium/stainless steel beads, can be subjected to
high temperature (e.g., boiling or autoclaving), can be subjected
to chemical lysis with detergents and/or surfactants (e.g., sodium
dodecyl sulfate, cetyltrimethylammonium bromide, or sodium lauroyl
sarcosin), can be subjected to one or more freeze-thaw cycles
using, e.g., liquid nitrogen or dry ice, can be subjected to
sonication, or can be subjected to combinations thereof. The
resulting sample can be subjected to a standard nucleic acid
extraction technique such as those described elsewhere (e.g.,
Sambrook and Russell, (2001) Molecular Cloning: A Laboratory
Manual, Third Edition, Cold Spring Harbor Press) or a nucleic acid
extraction technique that includes the use of magnetic beads or
selective DNA-binding membranes (see, e.g., QIAGEN DNeasy.RTM.
Blood & Tissue Kit, or Mo Bio PowerFood.TM. Microbial DNA
Isolation Kit). For example, the blood sample can be contacted with
magnetic beads that bind nucleic acid, the beads can be removed,
and bound nucleic acid can be eluted into an appropriate buffer to
form a processed sample for further analysis using the methods and
materials provide herein. Such a process can be carried out using a
variety of kits including, without limitation, Qiagen BioSprint 96
One-For-All Vet Kit (a rapid and economical automated purification
of viral nucleic acid and/or bacterial nucleic acid from samples
based on magnetic beads) and Chemicell geneMAG-PCR cleanup kit.
[0082] In some cases, a sample can be processed in a manner
designed to fragment any nucleic acid present within the sample.
For example, genomic or large pieces of nucleic acid present within
a sample can be subjected to a sonication technique and/or
restriction digestion with a restriction endonuclease such as DpnIl
or CviJI to generate nucleic acid fragments. Such fragmentation can
be performed using restriction endonucleases that are different
from those used as recognition or amplifying restriction
endonucleases to assess the sample as described herein.
[0083] In some cases, the sample can be treated such that any
double-stranded nucleic acid present within the sample is
separated. For example, a biological sample can be heated and then
snap-cooled or can be subjected to chemical (e.g., sodium
hydroxide) denaturation. In some cases, when the sample is
subjected to a PCR-based technique, certain primer or reaction
modifications can be used to generate preferentially
single-stranded product. For example, unidirectional DNA polymerase
reaction can be performed with a single specific primer. In some
cases, the strands of nucleic acid can be separated, and the strand
of interest can be enrichment using specific biotinylated primers
and streptavidin-conjugated magnetic beads. In some cases,
selective digestion of one of the strands can be accomplished using
lambda exonucleases.
[0084] As described herein, a sample (e.g., a biological sample)
can be subjected to a nucleic acid amplification technique. For
example, a tissue sample containing extracted nucleic acid can be
subjected to a quick PCR-based amplification of one or more
specific targets (e.g., 1 hour, end-point PCR) or to a whole genome
amplification technique (e.g., Qiagen REPLI-g Screening Kit for
high-throughput manual or automated whole genome
amplification).
[0085] Once obtained, a sample to be assessed, whether subjected to
a PCR-based nucleic acid technique or not, can be contacted with a
probe nucleic acid as described herein. This contacting step can be
carried out for any period of time and at any temperature that
allows target nucleic acid to hybridize with probe nucleic acid.
For example, this step can be performed between 10 seconds and 24
hours (e.g., between 30 seconds and 12 hours, between 30 seconds
and 8 hours, between 30 seconds and 4 hours, between 30 seconds and
2 hours, between 30 seconds and 1 hour, between 1 minute and 24
hours, between 1 minute and 12 hours, between 1 minute and 8 hours,
between 1 minute and 4 hours, between 1 minute and 2 hours, between
1 minute and 1 hour, between 5 minutes and 1 hour, between 10
minutes and 1 hour, between 15 minutes and 1 hour, or between 30
minutes and 1 hour). The initial temperature can be between
15.degree. C. and 100.degree. C. (e.g., between 23.degree. C. and
98.degree. C., between 23.degree. C. and 90.degree. C., between
23.degree. C. and 85.degree. C., between 23.degree. C. and
75.degree. C., between 23.degree. C. and 65.degree. C., between
23.degree. C. and 55.degree. C., between 23.degree. C. and
45.degree. C., between 23.degree. C. and 35.degree. C., between
30.degree. C. and 95.degree. C., between 30.degree. C. and
85.degree. C., between 30.degree. C. and 75.degree. C., between
30.degree. C. and 65.degree. C., between 30.degree. C. and
55.degree. C., between 30.degree. C. and 45.degree. C., between
20.degree. C. and 40.degree. C., between 20.degree. C. and
30.degree. C., and between 25.degree. C. and 35.degree. C.). The
temperature during this contacting step can remain constant or can
be increased or decreased. For example, the initial temperature can
be between about 40.degree. C. and about 85.degree. C., and then
the temperature can be allowed to decrease to room temperature over
a period of about 30 seconds to about 30 minutes (e.g., between
about 30 seconds and about 15 minutes, between about 30 seconds and
about 10 minutes, between about 1 minute and about 30 minutes,
between about 1 minute and about 15 minutes, or between about 1
minute and about 5 minutes).
[0086] Contact of the sample (e.g., a biological sample to be
tested) with probe nucleic acid can occur in the presence of the
recognition restriction endonucleases, or a separate step of adding
the recognition restriction endonucleases to the reaction can be
performed. The recognition restriction endonuclease step can be
carried out for any period of time and at any temperature that
allows the recognition restriction endonuclease to cleave
recognition restriction endonuclease cut sites formed by the
hybridization of target nucleic acid to the probe nucleic acid. For
example, this step can be performed between one second and 24 hours
(e.g., between one second and 30 minutes, between one second and
one hour, between five seconds and one hour, between 30 seconds and
24 hours, between 30 seconds and 12 hours, between 30 seconds and 8
hours, between 30 seconds and 4 hours, between 30 seconds and 2
hours, between 30 seconds and 1 hour, between 1 minute and 24
hours, between 1 minute and 12 hours, between 1 minute and 8 hours,
between 1 minute and 4 hours, between 1 minute and 2 hours, between
1 minute and 1 hour, between 5 minutes and 1 hour, between 10
minutes and 1 hour, between 15 minutes and 1 hour, or between 30
minutes and 1 hour). The temperature can be between 15.degree. C.
and 75.degree. C. (e.g., between 15.degree. C. and 75.degree. C.,
between 15.degree. C. and 65.degree. C., between 15.degree. C. and
55.degree. C., between 15.degree. C. and 45.degree. C., between
15.degree. C. and 35.degree. C., between 15.degree. C. and
30.degree. C., between 23.degree. C. and 55.degree. C., between
23.degree. C. and 45.degree. C., between 30.degree. C. and
65.degree. C., between 30.degree. C. and 55.degree. C., between
30.degree. C. and 45.degree. C., between 30.degree. C. and
40.degree. C., between 35.degree. C. and 40.degree. C., and between
36.degree. C. and 38.degree. C.). Any appropriate concentration of
recognition restriction endonuclease can be used. For example,
between about 0.001 units and 1000 units (e.g., between about 0.001
units and 750 units, between about 0.001 units and 500 units,
between about 0.001 units and 250 units, between about 0.001 units
and 200 units, between about 0.001 units and 150 units, between
about 0.001 units and 100 units, between about 0.001 units and 50
units, between about 0.001 units and 25 units, between about 0.001
units and 10 units, between about 0.001 units and 1 unit, between
about 0.001 units and 0.1 units, between about 0.01 units and 1000
units, between about 0.1 units and 1000 units, between about 1 unit
and 1000 units, between about 10 units and 1000 units, between
about 50 units and 1000 units, between about 0.5 units and 100
units, or between about 1 unit and 100 units) of restriction
endonuclease can be used. Other restriction endonuclease reaction
conditions such as salt conditions can be used according to
manufacture's instructions.
[0087] When one step of a method provided herein is completed, the
resulting reaction product containing cleaved nucleic acid can be
used in the next step. For example, cleaved nucleic acid of a
reaction product can be removed from uncleaved nucleic acid and
used in the next step of the method. For example, when probe
nucleic acid is attached to a solid support, the released portions
of probe nucleic acid that contain an amplifying restriction
endonuclease can be collected and placed in contact with reporter
nucleic acid or signal expansion nucleic acid as described herein.
The resulting reaction products of a particular step can be
manually or automatically (e.g., robotically) transferred to a
location containing nucleic acid for the next step (e.g., reporter
nucleic acid or signal expansion nucleic acid), which nucleic acid
can be attached or not attached to a solid support. In some cases,
one reaction of a method described herein can be carried out at one
location (e.g., a chamber) of a microfluidic device or blister
package device, and the reaction products that are generated can be
moved to another location (e.g., another chamber) that contains
nucleic acid for the next step (e.g., reporter nucleic acid or
signal expansion nucleic acid) via a channel. In some cases,
cleaved nucleic acid of a reaction product can be used in the next
step of the method by removing the uncleaved nucleic acid from the
reaction product. For example, when magnetic beads are used as a
solid support, a magnetic force can be used to remove the magnetic
beads and any attached uncleaved nucleic acid from the reaction
product. In some cases, two or more reactions of a method provided
herein can be carried out at one location (e.g., a single well of a
microtiter plate or a single chamber of a microfluidic device). For
example, a single compartment can have one region that contains
immobilized probe nucleic acid and another region that contains
immobilized reporter nucleic acid provided that the amplifying
restriction endonuclease of the immobilized probe nucleic acid is
not capable of cleaving the amplifying restriction endonuclease cut
site of the reporter nucleic acid unless target nucleic acid
hybridizes to the probe nucleic acid and the recognition
restriction endonuclease cleaves the probe nucleic acid, thereby
releasing a portion of the probe nucleic acid that contains the
amplifying restriction endonuclease so that it is capable of
cleaving the reporter nucleic acid. In another example, a single
compartment can have one region that contains immobilized probe
nucleic acid, other regions that contain immobilized signal
expansion nucleic acid (e.g., one region that contains a first
signal expansion nucleic acid and another region that contains a
second signal expansion nucleic acid), and another region that
contains immobilized reporter nucleic acid provided that the
amplifying restriction endonucleases of immobilized probe nucleic
acid and signal expansion nucleic acid are not capable of cleaving
their intended amplifying restriction endonuclease cut sites until
they are released as described herein. Such single compartments can
be made using partitions or sub-compartments within the single
compartment. For example, a sample to be tested can be placed into
a single well of a microtiter plate that contains probe nucleic
acid, recognition restriction endonucleases, first and second
signal expansion nucleic acid, and reporter nucleic acid such that
cleaved reporter nucleic acid and/or signal expansion nucleic acid
is produced as described herein when target nucleic acid is present
in the sample being tested and little or no cleaved reporter
nucleic acid and/or signal expansion nucleic acid is produced when
target nucleic acid is not present in the sample being tested.
[0088] Any appropriate method can be used to detect cleaved
reporter nucleic acid and/or signal expansion nucleic acid to
determine the presence, absence, or amount of target nucleic acid
in a sample, which can indicate the presence, absence, or amount of
a target virus or microorganism. For example, size separation
techniques can be used to assess reaction products for cleaved
reporter nucleic acid and/or signal expansion nucleic acid.
Examples of such size separation techniques include, without
limitation, gel electrophoresis and capillary electrophoresis
techniques. In some cases, a melt curve analysis can be performed
to assess reaction products for cleaved reporter nucleic acid
and/or signal expansion nucleic acid. As described herein, a label
can be used to aid in the detection of cleaved nucleic acid (e.g.,
reporter nucleic acid and/or signal expansion nucleic acid).
Examples of labels that can be used include, without limitation,
fluorescent labels (with or without the use of quenchers), dyes,
antibodies, radioactive material, enzymes (e.g., horse radish
peroxidase, alkaline phosphatase, laccase, galactosidase, or
luciferase), redox labels (e.g., ferrocene redox labels), metallic
particles (e.g., gold nanoparticles), and green fluorescent protein
based labels. For example, the release of fluorescently labeled
portions of reporter nucleic acid and/or signal expansion nucleic
acid from a solid support can be assessed using common fluorescent
label detectors. In some cases, cleaved reporter nucleic acid
and/or signal expansion nucleic acid can be detected
electrochemically. For electrochemical detection, the reporter
nucleic acid and/or signal expansion nucleic acid can include a
ferrocene redox label. Reporter nucleic acid and/or signal
expansion nucleic acid containing ferrocene can be obtained by
coupling ferrocene carboxylic acid with an amino-modified
oligonucleotide using the carbodiimide reaction in the presence of
an excess of ferrocene carboxylic acid. In one embodiment, for a
redox label, such as ferrocene, the detector can be an electrode
for amperometric assay of redox molecules. For example, if the
redox label is present in a reduced form of ferrocene, then the
electrode at high electrode potential can provide an oxidation of
the reduced form of ferrocene, thereby converting it to an oxidized
form of ferrocene. The generated current can be proportional to the
concentration of ferrocene label in the solution.
[0089] The methods and materials provided herein can be used to
assess one or more samples for target nucleic acid in real-time.
For example, a fluorescent label/quencher system or an
electrochemical redox label system can be used to detect cleavage
of reporter nucleic acid and/or signal expansion nucleic acid in
real time.
[0090] The methods and materials provided herein can be used to
assess one or more samples (e.g., two, three, four, five, six,
seven, eight, nine, ten, 20, 50, 100, 500, 1000, or more) for a
single type of target nucleic acid. For example, 100s of tissue
samples (e.g., tissue biopsy samples) can be assessed for a target
nucleic acid present in particular virus or microbe. In some case,
the methods and materials provided herein can be used in a
multiplex manner to assess one or more samples for more than one
(e.g., two, three, four, five, six, seven, eight, nine, ten, 20,
50, 100, 500, 1000, or more) type of target nucleic acid. For
example, target nucleic acid for ten different sequences (e.g., ten
different sequences from a single bacterial species or strain, or a
different sequence from ten different bacterial species or strains)
can be used to design ten different probe nucleic acid molecules.
In such cases, a different label can be used to correspond to each
probe nucleic acid such that the detected signals can indicate
which of the ten target nucleic acids are being detected. In some
cases, the methods and materials provided herein can be used in a
multiplex manner to assess samples for an infection by a particular
bacterial species that can exist in nature as a heterogeneous
species. For example, the methods and materials provided herein can
be used in a multiplex manner to assess blood samples for infection
by any one of a group of different E. coli strains that exist in
nature. In such cases, many different target nucleic acids can be
designed and included in a particular testing protocol or device
such that the presence of any one of the group of different E. coli
strains are detected.
[0091] This document also provides kits for performing the methods
described herein. For example, a kit provided herein can include
probe nucleic acid with or without being attached to a solid
support and/or reporter nucleic acid with or without being attached
to a solid support. In some cases, such a kit can include a
recognition restriction endonuclease, first signal expansion
nucleic acid, second signal expansion nucleic acid, or a
combination thereof. In some cases, a kit can be configured into a
microfluidic device that allows for the movement of probe nucleic
acid, first signal expansion nucleic acid, second signal expansion
nucleic acid, reporter nucleic acid, or recognition restriction
endonucleases (or any combination thereof) as well as a cleaved
portion of any such nucleic acid in a manner that allows a
detection method provided herein to be carried out with or without
the nucleic acid being attached to a solid support. For example, a
kit provided herein can be a microfluidic device capable of
receiving a sample and contacting that sample with probe nucleic
acid. The probe nucleic acid can be designed to include a length of
nucleotides followed by the sequence complementary to the target
nucleic acid, which can create a recognition restriction
endonuclease cut site, followed by an amplifying restriction
endonuclease. The distance from the recognition restriction
endonuclease cut site to the amplifying restriction endonuclease
can be relatively short (e.g., 100, 50, 25, 10, or less
nucleotides), while the distance from the recognition restriction
endonuclease cut site to the beginning of the length of nucleotides
can be relatively long (e.g., 50, 100, 150, 200, 500, 1000, 2000,
or more). In such cases, cleavage of the probe nucleic acid at the
recognition restriction endonuclease cut site can result in a
relatively small portion that contains the amplifying restriction
endonuclease and is capable of travelling faster than the larger
uncleaved probe nucleic acid. This difference can allow the cleaved
portion containing the amplifying restriction endonuclease to reach
an area of the microfluidic device containing signal expansion
nucleic acid or reporter nucleic acid so that the next reaction can
be carried out without the presence of uncleaved probe nucleic
acid. In some cases, after the smaller portion containing the
amplifying restriction endonuclease enters the area containing
signal expansion nucleic acid or reporter nucleic acid, a valve can
be used to prevent the larger uncleaved probe nucleic acid from
entering. In some cases, a filter can be used to limit the ability
of larger uncleaved probe nucleic acid from proceeding to the next
reaction location. Similar approaches can be used during other
steps of a method provided herein to separate cleaved nucleic acid
from uncleaved nucleic acid.
[0092] In some cases, a kit provided herein can be a portable or
self-contained device, packet, vessel, or container that can be
used, for example, in point of care applications (e.g., in a
clinic, hospital, or other health care facility). For example, such
a kit can be configured to allow a patient or physician's assistant
to insert a sample for analysis. In some cases, a kit can be
designed for use in a home setting or any other setting. Once
inserted, the sample can be heated (e.g., heated to about 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, or more
.degree. C.) and/or cooled by a heating or cooling mechanism
located within the kit. For example, an exothermic or endothermic
chemical reaction can be initiated within the kit to increase,
decrease, or maintain the temperature. Such exothermic or
endothermic chemical reactions can be carried out within the kit
without being in fluid communication with the reactions of the
target nucleic acid detection method. An iron oxidation reaction is
an example of an exothermic chemical reaction that can be used to
heat a kit provided herein. An endothermic chemical reaction that
can be used to cool a kit provided herein can be a reaction that
includes the use of ammonium chloride and water, potassium chloride
and water, or sodium carbonate and ethanoic acid. In general, when
detecting DNA target nucleic acid, the kit can be designed to
generate, if needed, enough heat to denature double stranded DNA
present within the sample. The kit also can be designed to generate
appropriate heating and cooling temperatures to carry out each step
of a detection method provided herein. In some cases, a kit
provided herein can include a temperature indicator (e.g., color
indicator or thermometer) to allows a user to assess
temperature.
[0093] In some cases, a kit can be designed to provide a user with
a "yes" or "no" indication about the presence of target nucleic
acid within a tested sample. For example, a label having the
ability to generate a change in pH can be used, and a visual
indicator (e.g., a pH-based color indicator) can be used to inform
the user of the presence of target nucleic acid based on a change
in pH.
[0094] In some cases, a point of care or home use device can be
designed to carry out the reactions described herein. For example,
point of care or home use device can be designed to include a
series of adjacent chambers. In a relatively simple configuration,
for example, a first "sample" chamber can be configured for sample
insertion, and can contain reagents (e.g., in dry or liquid form)
to effect generation of single stranded nucleic acid fragments. A
second "recognition" chamber can be configured to receive single
stranded nucleic acid fragments from the first chamber, and can
contain probe nucleic acid and recognition restriction endonuclease
(e.g., in dry or liquid form). A third "amplification" chamber can
be configured to receive cleaved portions of probe nucleic acid
from the second chamber, and can contain reporter nucleic acid
(e.g., in dry or liquid form). A fourth "detection" chamber can be
configured to receive cleaved portions of marker nucleic acid from
the third chamber, and can contain a reagent (e.g., in dry or
liquid form) that serves as an indicator of whether or not target
nucleic acid was present in the sample. It is noted that one or
more additional "signal expansion" chambers can be present between
the "recognition" chamber and the "amplification" chamber.
[0095] In some cases, a point of care or home use device can be
configured such the chambers are separated from each other by
membranes that can provide controlled passage of reaction
materials. For example, chambers can be separated by membranes that
are subject to degradation by particular reagents or solutions. In
such cases, a reaction can be confined to a particular chamber
until the membrane separating it from the adjacent chamber
degrades, permitting passage of reaction components there
between.
[0096] In some cases, a point of care or home use device can be
adapted for automatic transfer of the reaction mixture between
chambers. For example, insertion of a sample into the first chamber
can trigger a reaction or provide a reagent that gradually degrades
the membrane separating the first chamber from the second chamber.
Movement of all or a portion of the reaction mixture into the
second chamber can in turn provide a reagent or trigger a reaction
that gradually degrades the membrane separating the second chamber
from the third chamber. For example, if the sample reaction mixture
in the first chamber is an aqueous solution, the reagents in the
second chamber are dry, and the membrane in the second chamber is
degraded by water, movement of the aqueous reaction mixture into
the second chamber can trigger degradation of the membrane
therein.
[0097] In some cases, a point of care or home use device can be
adapted for automatic controlled flow transfer of reaction mixture
between chambers. For example, insertion of a sample into the first
chamber can trigger a reaction or provide a reagent that allows
controlled flow movement of the sample through absorption media.
Movement of all or a portion of the reaction mixture into the
second chamber can in turn provide a reagent or trigger a reaction
that allows controlled flow movement of the sample through
absorption media to a third chamber. In such cases, a reaction can
be confined to a particular chamber until the media separating it
from the adjacent chamber absorbs and permits passage of reaction
components there between.
[0098] In some cases, a point of care or home use device can be
adapted for automatic controlled flow transfer of reaction mixture
between chambers. For example, insertion of a sample into the first
chamber can trigger a reaction or provide a reagent that allows
controlled capillary flow movement of the sample through
micro-fluidic channels. Movement of all or a portion of the
reaction mixture into the second chamber can in turn provide a
reagent or trigger a reaction that allows controlled flow movement
of the sample through micro-fluidic channels to a third chamber. In
such cases, a reaction can be confined to a particular chamber
until the microfluidic channel permits passage of reaction
components there between.
[0099] In some cases, a point of care or home use device can be
adapted for automatic controlled flow transfer of reaction mixture
without chambers. For example, insertion of a sample into the
device can trigger a reaction or provide a reagent that allows
controlled capillary flow movement of the sample through
microfluidic channels. Movement of all or a portion of the reaction
mixture in the microfluidic channel can trigger a reaction that
allows reagents to enter the reaction mixture in a continuous
flow-through manner with no specific chamber for a reaction. In
such cases, a reaction does not need to be confined to a particular
section of the microfluidic channel.
[0100] In some cases, transfer of a reaction mixture from one
chamber to the next can be controlled by a user. An exemplary
user-controlled, pen-style point of care or home use device is
depicted in FIG. 7. Device 300 can include sample collector 310 and
reaction unit 320. Sample collector 310 can have cap 312 with screw
threads 314, shaft 316, and swabber 318. Swabber 318 can be smooth
or rough, and in some cases can have bristles (e.g., smooth or
rough bristles) or a matted texture to facilitate sample collection
from, for example, the inside cheek, throat, or skin of an
individual to be tested.
[0101] Reaction unit 320 can include tube 322, open end 324
reversibly closed by safety cap 326, and closed end 328. Open end
324 can have internal screw threads, and cap 326 can have external
screw threads 329. Screw threads 329 of safety cap 326, as well as
screw threads 314 of sample collector cap 312, can be adapted to
mate with the internal screw threads at open end 324, such that
either safety cap 326 or sample collector 310 can be screwed into
open end 324.
[0102] Tube 322 can contain several chambers, such as lysing and
isolation chamber 330, recognition and amplification chamber 360,
and detection chamber 390. As described herein, the chambers can be
separated from one another to prevent premature mixing of reaction
components. Tube 322 and the chambers contained therein can be made
from, for example, clear plastic (e.g., polycarbonate, acrylic,
nylon, or PVC). Tube 322 also can contain first and second safety
bands 340 and 370, and first and second spring returns 350 and
380.
[0103] Lysing and isolation chamber 330 can be positioned proximal
to open end 324. Lysing and isolation chamber 330 can have proximal
end 332, distal end 334, proximal membrane 336, distal membrane
337, and reaction completion indicator 338. Proximal membrane 336
can be located adjacent to proximal end 332, and distal membrane
337 can be located adjacent to distal end 334. Membranes 336 and
337 can be made from, for example, synthetic rubber, natural latex
rubber, or silicone. Chamber 330 can contain reagents for lysing
cells as well as reagents for cleaving and denaturing cellular
nucleic acids. Reaction completion indicator 338 can be, for
example, a built in timer or stop watch, a built in pH indicator, a
built in color change reagent, or a conductivity probe, and can
indicate when cell lysis and nucleic acid sample generation are
sufficient to proceed to the next step.
[0104] First safety band 340 can be positioned distal to lysing and
isolation chamber 330 within tube 322, and first spring return 350
can be positioned distal to first safety band 340. First safety
band 340 can be, for example, connected to a tab or strap, and can
be moved or removed from reaction unit 320 by pulling on the tab or
strap. First spring return 350 can be made from a shape memory
material that can be compressed and then automatically return to or
toward its original configuration.
[0105] The safety band 340 can be attached to the tube as a secured
ring that can be, for example, over molded as a soft rubber
component or inserted as a spring like split ring component. The
safety band 340 can lock the position of the lysing and isolation
tube chamber 340, preventing linear sliding of the lysing and
isolation chamber 330 to that of the recognition and amplification
chamber 360. Upon removal of safety band 340, the user can actuate
linear movement of the entire device 300 by holding the proximal
end firm and pressing the distal closed end 328 such that both
distal chambers recognition and amplification 360 and detection
chamber 390 are moved toward the lysing and isolation chamber 330.
The needle and sample collector 362 can pierce membrane 337 and
enter the lysing and isolation chamber 330. The user can release a
firm hold on the assembly and spring return 350 can draw the sample
into recognition and amplification chamber 360. After completion of
the reaction, the user can remove safety band 370, and the user can
actuate linear movement of the assembly by holding the recognition
and amplification chamber 360 firm and pressing the distal closed
end 328 such that the detection chamber 390 moves toward the
recognition and amplification chamber 360. The needle and sample
collector 392 can pierce membrane 366. The user can release the
firm hold on the assembly, and spring return 380 can draw the
sample into detection chamber 390.
[0106] Recognition and amplification chamber 360 can be positioned
distal to first spring return 350. Chamber 360 can have proximal
end 361, which in turn can have piercing needle and sample
collector 362, distal end 364, membrane 366, and reaction
completion indicator 368. Recognition and amplification chamber 360
can contain, for example, probe nucleic acid and reporter nucleic
acid and restriction endonucleases for use in enzymatic
amplification cascades as described herein. Piercing needle and
sample collector 362 can have a pointed, beveled, or barbed tip. In
addition, the interior of piercing needle and sample collector 362
can be in fluid communication with the interior of recognition and
amplification chamber 360, such that a nucleic acid test sample can
be collected from lysing and isolation chamber 330 and transferred
to recognition and amplification chamber 360 via collector 362.
Membrane 364 can be located adjacent to distal end 364, and can be
made from, for example, synthetic rubber, natural latex rubber, or
silicone. Reaction completion indicator 368 can be, for example, a
built in timer or stop watch, a built in pH indicator, a built in
color change reagent, or a conductivity probe, and can indicate
when cell lysis and nucleic acid sample generation are sufficient
to proceed to the next step.
[0107] Second safety band 370 can be positioned distal to
recognition and amplification chamber 360 within tube 322, and
second spring return 380 can be positioned distal to second safety
band 370. Second safety band 370 can be, for example, connected to
a tab or strap, and can be moved or removed from reaction unit 320
by pulling on the tab or strap. Second spring return 380 can be
made from a shape memory material (e.g., spring steel, plastic, or
rubber) that can be compressed and then automatically return to or
toward its original configuration.
[0108] Detection chamber 390 can be positioned distal to second
spring return 380, adjacent to closed end 328 of tube 322.
Detection chamber 390 can have proximal end 391, which in turn can
have piercing needle and sample collector 392, and distal end 394.
Piercing needle and sample collector 392 can have a pointed,
beveled, or barbed tip. In addition, the interior of piercing
needle and sample collector 392 can be in fluid communication with
the interior of detection chamber 390, such that a reaction sample
can be collected from recognition and amplification chamber 360 and
transferred to detection chamber 390 via collector 392. Detection
chamber 390 can contain a substrate for an enzyme marker such as a
substrate for horseradish peroxidase (HRP) or alkaline phosphate
(AP).
[0109] Sample collector 310 and reaction unit 320 can be packaged
together and sold as a kit. In use, the sample collector 310 can be
removed from the package, and a swab can be obtained from, for
example, a subject's body. Cap 326 can be removed from open end 324
of tube 322, and sample collector 310 can be screwed into open end
324 such that all or a portion of swabber 318 extends through
proximal membrane 336 and into the interior of lysing and isolation
chamber 330. The sample can be mixed (e.g., by shaking), and the
lysing and nucleic acid preparation can proceed for a particular
length of time, or until reaction completion indicator 338
indicates that the user can proceed to the next reaction step.
[0110] When the nucleic acid sample is ready, the user can remove
first safety band 350 from reaction unit 320, and can actuate
reaction unit 320 such that piercing needle and sample collector
362 moves proximally to penetrate distal membrane 337 of lysing and
isolation chamber 330, collects a sample from chamber 330, and, by
virtue of first spring return 350, moves distally to its original
position. The sample can again be mixed, and the recognition and
amplification steps can proceed for a particular length of time, or
until reaction completion indicator 368 indicates that the user can
proceed to the next reaction step.
[0111] When the reaction sample is ready, the user can remove
second safety band 380 from reaction unit 320, and can actuate
reaction unit 320 such that piercing needle and sample collector
392 moves proximally to penetrate membrane 366 of recognition and
amplification chamber 360, collects a sample from chamber 360, and,
by virtue of second spring return 380, moves distally to its
original position. The sample can again be mixed, and marker
released during the amplification step can be detected (e.g.,
colorimetrically or fluorescently). In some cases, the outer
surface of tube 322 can have a color code printed thereon, so a
user can compare the color of detection chamber 390 with the color
code to determine whether or not the tested sample contains target
nucleic acid.
[0112] Device 300 can have any suitable dimensions. For example,
the size of device 300 can approximate that of a pen or a marker,
which can make it particularly convenient to transport. In some
cases, device 300 can have a diameter at its widest point of about
0.25 to about 2 cm (e.g., 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 0.8, 0.9,
1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 cm), and a
length of about 5 cm to about 200 cm (e.g., 5, 10, 15, 20, 25, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, or 200 cm).
[0113] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Formation and Cleavage of Target-Probe Hybrids
[0114] An oligonucleotide probe (5'-thiol-GGT AGT GCG AAA TGC CAT
TGC TAG TTG TTT-biotin-3'; SEQ ID NO:10) that was modified with a
thiol group at the 5' end and a biotin molecule at the 3' end was
conjugated to horseradish peroxidase (HRP). Conjugation was
performed using the SMCC reagent according to a technique modified
from Dill et al. (Biosensors and Bioelectronics, 20:736-742
(2004)). The HRP conjugate solution was incubated with a
streptavidin-coated ELISA plate to immobilize the
HRP-oligonucleotide probe to the surface via a biotin-streptavidin
interaction. The ELISA plate was then incubated with different
concentrations of a target oligonucleotide (5'-AAA CAA CTA GCA ATG
GCA TTT-3'; SEQ ID NO:11). The target oligonucleotide sequence was
reverse-complementary to the probe sequence to form a
double-stranded hybrid molecule. After washing, the plate was
incubated in a solution containing the restriction endonuclease
BfaI. BfaI specifically recognizes the sequence 5'-CTAG-3' and
cleaves the double-stranded, target-probe hybrids to release the
HRP-oligonucleotide into the reaction solution. After a two-hour
incubation at 37.degree. C., the reaction solution was transferred
to a new ELISA plate. The cleaved HRP-oligonucleotide was contacted
to 3,3',5,5'-tetramethyl benzidine (TMB) to form a colored reaction
product.
[0115] When the restriction endonuclease BfaI was added in excess
to the reaction mixture, a clear direct dependence between the
amount of released HRP-probe and the concentration of
oligonucleotide target was observed (FIG. 6A). The detectable
target concentration was approximately 1 nM. This detection limit
was obtained by direct measurement without any secondary signal
amplification. The addition of a restriction endonuclease signal
amplification cascade as described herein can further improve the
detection limit by several orders of magnitude.
[0116] When the HRP-oligonucleotide probes were pre-incubated with
an excess of target oligonucleotide (500 nM), the amount of cleaved
HRP-oligonucleotide probe was limited by the amount of recognition
restriction endonuclease BfaI (FIG. 6B). Taken together, these data
demonstrate that recognition restriction endonucleases can be used
to initiate the restriction endonuclease cascades described
herein.
Example 2
Detecting Target Nucleic Acid Using Probe Nucleic Acid and Reporter
Nucleic Acid
[0117] A target nucleic acid is selected. Once selected, target
nucleic acid is analyzed using a common genetic database such as
GenBank.RTM. and/or a computer-based sequence analysis program to
identify a portion of the target nucleic acid that contains a cut
site for a restriction endonuclease. Probe nucleic acid is designed
to be complementary to at least a portion of target nucleic acid
that contains a cut site. Once designed and obtained by standard
oligonucleotide synthesis methods, probe nucleic acid is conjugated
to an amplifying restriction endonuclease and immobilized to the
surface of a first well of a microtiter plate. A sample to be
tested is incubated in the first well. If target nucleic acid is
present in the sample, at least a portion of the target nucleic
acid hybridizes to the probe nucleic acid, and thereby forms a
recognition restriction endonuclease cut site. The recognition
restriction endonuclease is added to the first well having the
sample and probe nucleic acid. The microtiter plate is incubated at
37.degree. C. for an appropriate length of time for the cleavage
reaction to proceed.
[0118] Upon cleavage of probe nucleic acid by the recognition
restriction endonuclease, the reaction solution containing the
released portion of the probe nucleic acid is transferred into a
second well. The second well contains reporter nucleic acid that is
immobilized to the surface and contains at least one
double-stranded portion having an amplifying restriction
endonuclease cut site. Reporter nucleic acid also has a fluorescent
label. Upon transfer to the second chamber, the amplifying
restriction endonuclease bound to the released portion of the probe
nucleic acid contacts the reporter nucleic acid. The amplifying
restriction endonuclease cleaves reporter nucleic acid at the
double-stranded amplifying restriction endonuclease cut site to
form at least two portions. The liberated portion of the reporter
nucleic acid having the fluorescent label is moved to a third
microtiter plate well, and a standard fluorescent reader is used to
measure any fluorescent signal.
[0119] A standard curve of known amounts of target nucleic acid is
used to quantify the amount of target nucleic acid in the tested
sample.
Example 3
Detecting Target Nucleic Acid Using Probe Nucleic Acid, First
Signal Expansion Nucleic Acid, Second Signal Expansion Nucleic
Acid, and Reporter Nucleic Acid
[0120] Once selected, target nucleic acid is analyzed using a
common genetic database such as GenBank.RTM. and/or a
computer-based sequence analysis program to identify a portion of
target nucleic acid that contains a cut site for a restriction
endonuclease. Probe nucleic acid is designed based on the desired
target nucleic acid as described herein. Standard oligonucleotide
synthesis methods are used to make the probe nucleic acid, which is
then conjugated to an initial amplifying restriction endonuclease
and immobilized to the surface of a first well of a microtiter
plate. A sample to be tested for the target nucleic acid is
incubated in the first well. If target nucleic acid is present in
the sample, at least a portion of target nucleic acid hybridizes to
probe nucleic acid and thereby forms a recognition restriction
endonuclease cut site. Recognition restriction endonuclease is
added to the first well having the sample and probe nucleic acid.
The microtiter plate is incubated at 37.degree. C. for an
appropriate length of time for the cleavage reaction to
proceed.
[0121] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by the recognition restriction endonuclease, the reaction
solution containing the free portion of probe nucleic acid is
transferred to another well that includes first signal expansion
nucleic acid and second signal expansion nucleic acid. The first
signal expansion nucleic acid and second signal expansion nucleic
acid creates a positive feedback loop that causes an exponential
acceleration of release of initial amplifying restriction enzymes.
The reaction product from this well is transferred to another well
containing reporter nucleic acid, and cleavage of the reporter
nucleic acid is used to determine the presence, absence, or amount
of target nucleic acid in the sample. A standard curve of known
amounts of target nucleic acid is used to quantify the amount of
target nucleic acid in the tested sample.
Example 4
Detecting the Presence or Absence of Bacteria
[0122] The presence or absence of methicillin-resistant
Staphylococcus aureus (MRSA) in a sample is detected using an
enzymatic amplification cascade. A MRSA-specific target nucleic
acid is analyzed using a common genetic database such as GenBank
and/or a computer-based sequence analysis program to identify a
portion of target nucleic acid that contains a cut site for a
restriction endonuclease. Probe nucleic acid is designed to be
complementary to at least a portion of the selected target nucleic
acid. Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A biological sample (e.g.,
tissue sample or nasal swab) that is suspected of having MRSA is
obtained, and the nucleic acid from that sample is incubated in the
first well. If MRSA is present in the sample, at least a portion of
the MRSA-specific nucleic acid hybridizes to the probe nucleic acid
and thereby forms a recognition restriction endonuclease cut site.
Recognition restriction endonuclease is added to the first well
having the sample and probe nucleic acid. The microtiter plate is
incubated at 37.degree. C. for an appropriate length of time for
the cleavage reaction to proceed.
[0123] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by the recognition restriction endonuclease, the reaction
solution in the first well is transferred to a second well
containing reporter nucleic acid that is immobilized to the surface
of the second well and that has at least one double-stranded
portion having an amplifying restriction endonuclease cut site. The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the report nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0124] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time for the
cleavage reaction to proceed. The amplifying restriction
endonucleases cleave reporter nucleic acid at the double-stranded
amplifying restriction endonuclease cut site to form at least two
portions. The reaction solution of the second well is transferred
to a third well for fluorescence detection using a fluorescent
microtiter plate reader. Fluorescence in the third well is
indicative of MRSA-specific nucleic acid present in the sample. If
the sample was obtained from a patient, such a result is indicative
of a MRSA infection in that patient. If no fluorescence is detected
in the third well, such a result is indicative of the absence of
MRSA in the sample.
Example 5
Detecting the Presence or Absence of Staphylococcus Aureus in a
Human Nasal Swab Sample
[0125] The presence or absence of Staphylococcus aureus in a human
nasal swab sample is detected using an enzymatic amplification
cascade. A Staphylococcus aureus nucleic acid (GenBank.RTM.
Accession No. NC_013450; GenBank.RTM. GI No. 269201690) was
analyzed using the GenBank.RTM. genetic database and CLC DNA
Workbench software to identify a portion of target Staphylococcus
aureus nucleic acid that contains a DNA gyrase subunit B gene
(GenBank.RTM. GI No. 269201690:5034-6968) with a cut site for the
EcoRV restriction endonuclease, which cleaves at the 6 bp
nucleotide sequence 5'-GATATC-3'. A 40 nt probe nucleic acid
(5'-TGATCTAGCGAAAGCAAGATATCACAAAATCGTCATTATG-3'; SEQ ID NO:12) was
designed to be complementary to nucleotides 5340 to 5379 of the
selected target nucleic acid.
[0126] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A human nasal swab sample to
be tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If Staphylococcus aureus is present in
the sample, at least a portion of the Staphylococcus aureus nucleic
acid hybridizes to the probe nucleic acid and thereby forms an
EcoRV cut site. EcoRV recognition restriction endonuclease, which
is present within the first well or which is added to the first
well, is allowed to cleave any formed recognition restriction
endonuclease cut sites by incubating the microtiter plate at
37.degree. C. for an appropriate length of time (e.g., 1 minute to
2 hours) for the cleavage reaction to proceed.
[0127] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by EcoRV, the reaction solution in the first well is
transferred to a second well containing reporter nucleic acid that
is immobilized to the surface of the second well and that has at
least one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0128] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of Staphylococcus aureus nucleic acid
present in the sample.
Example 6
Detecting the Presence or Absence of MRSA in a Human Skin Swab
Sample
[0129] The presence or absence of MRSA in a human skin swab sample
is detected using an enzymatic amplification cascade. A MRSA target
nucleic acid (GenBank.RTM. Accession No. NC_002952; GenBank.RTM. GI
No. 49482253) was analyzed using the GenBank.RTM. genetic database
and CLC DNA Workbench software to identify a portion of target MRSA
nucleic acid that contains a penicillin-binding protein 2 (mecA)
gene (GenBank.RTM. GI No. 49482253:c46925-44919) with a cut site
for the PstI restriction endonuclease, which cleaves at the 6 bp
nucleotide sequence 5'-CTGCAG-3'. A 40 nt probe nucleic acid
(5'-ATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACAAG-3'; SEQ ID NO:15) was
designed to be complementary to nucleotides 46702 to 46741 of the
selected target nucleic acid.
[0130] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A human skin swab sample to
be tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If MRSA is present in the sample, at
least a portion of the MRSA nucleic acid hybridizes to the probe
nucleic acid and thereby forms a PstI site. PstI recognition
restriction endonuclease, which is present within the first well or
which is added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0131] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by PstI, the reaction solution in the first well is
transferred to a second well containing reporter nucleic acid that
is immobilized to the surface of the second well and that has at
least one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0132] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of MRSA nucleic acid present in the
sample.
Example 7
Detecting the Presence or Absence of Streptococcus Pneumoniae in a
Human Sputum Sample
[0133] The presence or absence of Streptococcus pneumoniae in a
sputum sample collected from a human is detected using an enzymatic
amplification cascade. A Streptococcus pneumoniae nucleic acid
(GenBank.RTM. Accession No. NC_008533; GenBank.RTM. GI No.
116515308) was analyzed using the GenBank.RTM. genetic database and
CLC DNA Workbench software to identify a portion of target
Streptococcus pneumoniae nucleic acid that contains a pneumolysin
(ply) gene (GenBank.RTM. GI No. 116515308:c1722872-1721457) with a
cut site for the PstI restriction endonuclease, which cleaves at
the 6 bp nucleotide sequence 5'-CTGCAG-3'. A 40 nt probe nucleic
acid (5'-AACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGTCTATAT-3'; SEQ ID NO:16)
was designed from positions 1722127 to 1722166 of the selected
target nucleic acid.
[0134] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A human sputum sample to be
tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If Streptococcus pneumoniae is present
in the sample, at least a portion of the Streptococcus pneumoniae
nucleic acid hybridizes to the probe nucleic acid and thereby forms
a PstI cut site. PstI recognition restriction endonuclease, which
is present within the first well or which is added to the first
well, is allowed to cleave any formed recognition restriction
endonuclease cut sites by incubating the microtiter plate at
37.degree. C. for an appropriate length of time (e.g., 1 minute to
2 hours) for the cleavage reaction to proceed.
[0135] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by PstI, the reaction solution in the first well is
transferred to a second well containing reporter nucleic acid that
is immobilized to the surface of the second well and that has at
least one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0136] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of Streptococcus pneumoniae nucleic acid
present in the sample.
Example 8
Detecting the Presence or Absence of Streptococcus Pyogenes in a
Human Sputum or Blood Sample
[0137] The presence or absence of Streptococcus pyogenes in a
sputum or blood sample collected from a human is detected using an
enzymatic amplification cascade. A Streptococcus pyogenes nucleic
acid (GenBank.RTM. Accession No. NC_003485; GenBank.RTM. GI No.
19745201) was analyzed using the GenBank.RTM. genetic database and
CLC DNA Workbench software to identify a portion of target
Streptococcus pyogenes nucleic acid that contains an exotoxin type
A precursor (speA) gene (GenBank.RTM. GI No.
19745201:c332312-331557) with a cut site for the BstEII restriction
endonuclease, which cleaves at the 7 bp nucleotide sequence
5'-GGTGACC-3'. A 40 nt probe nucleic acid
(5'-ATATTTTCTTTATGAGGGTGACCCTGTTACTCACGAGAAT-3'; SEQ ID NO:17) was
designed from positions 331712 to 331751 of the selected target
nucleic acid.
[0138] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A sputum or blood sample to
be tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If Streptococcus pyogenes is present
in the sample, at least a portion of the Streptococcus pyogenes
nucleic acid hybridizes to the probe nucleic acid and thereby forms
a BstEII restriction endonuclease cut site. BstEII restriction
endonuclease, which is present within the first well or which is
added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0139] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by BstEII restriction endonuclease, the reaction solution in
the first well is transferred to a second well containing reporter
nucleic acid that is immobilized to the surface of the second well
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID
NO:13) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'; SEQ ID NO:14). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0140] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of Streptococcus pyogenes nucleic acid
present in the sample.
Example 9
Detecting the Presence or Absence of Mycobacterium Tuberculosis in
a Human Sputum Sample
[0141] The presence or absence of Mycobacterium tuberculosis in a
sputum sample collected from a human is detected using an enzymatic
amplification cascade. A Mycobacterium tuberculosis nucleic acid
(GenBank.RTM. Accession No. NC_000962; GenBank.RTM. GI No.
57116681) was analyzed using the GenBank.RTM. genetic database and
CLC DNA Workbench software to identify a portion of target
Mycobacterium tuberculosis nucleic acid that contains an RNA
polymerase subunit beta (rpoB) gene (GenBank.RTM. GI No.
57116681:759807-763325) with a cut site for the HincII restriction
endonuclease, which cleaves at the 6 bp nucleotide sequence
5'-GTTGAC-3'. A 40 nt probe nucleic acid
(5'-AACAACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGT-3'; SEQ ID NO:18) was
designed from positions 761115 to 761154 of the selected target
nucleic acid.
[0142] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A sputum sample to be tested
is obtained, and the nucleic acid of that sample is incubated in
the first well. If Mycobacterium tuberculosis is present in the
sample, at least a portion of the Mycobacterium tuberculosis
nucleic acid hybridizes to the probe nucleic acid and thereby forms
a HincII restriction endonuclease cut site. HincII restriction
endonuclease, which is present within the first well or which is
added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0143] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by HincII restriction endonuclease, the reaction solution in
the first well is transferred to a second well containing reporter
nucleic acid that is immobilized to the surface of the second well
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID
NO:13) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'; SEQ ID NO:14). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0144] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of Mycobacterium tuberculosis nucleic acid
present in the sample.
Example 10
Detecting the Presence or Absence of Influenza A Virus in a Human
Nasal Swab Sample
[0145] The presence or absence of influenza A virus in a nasal swab
sample collected from a human is detected using an enzymatic
amplification cascade. An influenza A virus nucleic acid
(GenBank.RTM. Accession No. NC_002016; GenBank.RTM. GI No. 8486122)
was analyzed using the GenBank.RTM. genetic database and CLC DNA
Workbench software to identify a portion of target influenza A
virus nucleic acid that contains a matrix protein 1 (M1) gene
(GenBank.RTM. GI No. 8486122:26-784) with a cut site for the PstI
restriction endonuclease, which cleaves at the 6 bp nucleotide
sequence 5'-CTGCAG-3'. A 40 nt probe nucleic acid
(5'-ACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTG-3'; SEQ ID NO:19) was
designed from positions 225 to 264 of the selected target nucleic
acid.
[0146] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A nasal swab sample to be
tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If influenza A virus is present in the
sample, at least a portion of the influenza A virus nucleic acid
hybridizes to the probe nucleic acid and thereby forms a PstI
restriction endonuclease cut site. PstI restriction endonuclease,
which is present within the first well or which is added to the
first well, is allowed to cleave any formed recognition restriction
endonuclease cut sites by incubating the microtiter plate at
37.degree. C. for an appropriate length of time (e.g., 1 minute to
2 hours) for the cleavage reaction to proceed.
[0147] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by PstI restriction endonuclease, the reaction solution in
the first well is transferred to a second well containing reporter
nucleic acid that is immobilized to the surface of the second well
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID
NO:13) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'; SEQ ID NO:14). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0148] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of influenza A virus nucleic acid present
in the sample.
Example 11
Detecting the Presence or Absence of Adenovirus 4 in a Human Nasal
Swab Sample
[0149] The presence or absence of adenovirus 4 in a nasal swab
sample collected from a human is detected using an enzymatic
amplification cascade. An adenovirus 4 nucleic acid (GenBank.RTM.
Accession No. NC_003266; GenBank.RTM. GI No. 51527264) was analyzed
using the GenBank.RTM. genetic database and CLC DNA Workbench
software to identify a portion of target adenovirus 4 nucleic acid
that contains a glycoprotein 12 (gp 12) gene (GenBank.RTM. GI No.
51527264:26686-31469) with a cut site for the BglII restriction
endonuclease, which cleaves at the 6 bp nucleotide sequence
5'-AGATCT-3'. A 40 nt probe nucleic acid
(5'-CCAACTCGCCGGATCGGGAAGATCTTCCTTCACGCCTCGT-3'; SEQ ID NO:20) was
designed from positions 26776 to 26815 of the selected target
nucleic acid.
[0150] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A nasal swab sample to be
tested is obtained, and the nucleic acid from that sample is
incubated in the first well. If adenovirus 4 is present in the
sample, at least a portion of the Adenovirus 4 nucleic acid
hybridizes to the probe nucleic acid and thereby forms a BglII
restriction endonuclease cut site. BglII restriction endonuclease,
which is present within the first well or which is added to the
first well, is allowed to cleave any formed recognition restriction
endonuclease cut sites by incubating the microtiter plate at
37.degree. C. for an appropriate length of time (e.g., 1 minute to
2 hours) for the cleavage reaction to proceed.
[0151] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by BglII restriction endonuclease, the reaction solution in
the first well is transferred to a second well containing reporter
nucleic acid that is immobilized to the surface of the second well
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID
NO:13) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'; SEQ ID NO:14). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0152] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of adenovirus 4 nucleic acid present in
the sample.
Example 12
Detecting the Presence or Absence of Respiratory Syncytial Virus in
a Human Nasal Swab Sample
[0153] The presence or absence of respiratory syncytial virus in a
nasal swab sample collected from a human is detected using an
enzymatic amplification cascade. A Respiratory syncytial virus
nucleic acid (GenBank.RTM. Accession No. NC_001803; GenBank.RTM. GI
No. 9629367) was analyzed using the GenBank.RTM. genetic database
and CLC DNA Workbench software to identify a portion of target
respiratory syncytial virus nucleic acid that contains a matrix
protein 2 (M2) gene (GenBank.RTM. GI No. 9629367:7567-8527) with a
cut site for the EcoRV restriction endonuclease, which cleaves at
the 6 bp nucleotide sequence 5'-GATATC-3'. A 40 nt probe nucleic
acid (5'-CCATAAAAACCACATTGGATATCCACAAGAGCATAACCAT-3'; SEQ ID NO:21)
was designed from positions 8054 to 8093 of the selected target
nucleic acid.
[0154] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A nasal swab sample to be
tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If respiratory syncytial virus is
present in the sample, at least a portion of the respiratory
syncytial virus nucleic acid hybridizes to the probe nucleic acid
and thereby forms a EcoRV restriction endonuclease cut site. EcoRV
restriction endonuclease, which is present within the first well or
which is added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0155] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by EcoRV restriction endonuclease, the reaction solution in
the first well is transferred to a second well containing reporter
nucleic acid that is immobilized to the surface of the second well
and that has at least one double-stranded portion having an
amplifying restriction endonuclease NcoI cut site. The reporter
nucleic acid can be a double-stranded nucleic acid having a first
strand (e.g., 5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID
NO:13) and a second strand (e.g.,
5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3'; SEQ ID NO:14). The
reporter nucleic acid also has a fluorescent label. In some cases,
first signal expansion nucleic acid and second signal expansion
nucleic acid are used prior to the reporter nucleic acid step to
increase the level of target nucleic acid detection. The first
signal expansion nucleic acid and second signal expansion nucleic
acid can include labels, in which case they can be used together
with reporter nucleic acid or in place of reporter nucleic
acid.
[0156] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of Respiratory syncytial virus nucleic
acid present in the sample.
Example 13
Detecting the Presence or Absence of Neisseria Gonorrhoeae in a
Human Endocervical or Urethral Swab Sample
[0157] The presence or absence of Neisseria gonorrhoeae in a
endocervical or urethral swab sample collected from a human is
detected using an enzymatic amplification cascade. A N. gonorrhoeae
target nucleic acid (GenBank.RTM. Accession No. NC_002946;
GenBank.RTM. GI No. 59800473:1572106-1573584) was analyzed using
the GenBank.RTM. genetic database and CLC DNA Workbench software to
identify a portion of target N. gonorrhoeae nucleic acid that
contains orf1 gene (gene accession number NC_002946.2) with a cut
site for the EcoRI restriction endonuclease, which cleaves at the 6
bp nucleotide sequence 5'-GAATTC-3'. A 40 nt probe nucleic acid
(5'-CGCCGTCGAAGACGAAGAATTCGGGTTCGGGGCCGAAATA-3'; SEQ ID NO:22) was
designed from positions 1573069 to 1573109 of the selected target
gene.
[0158] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. An endocervical or urethral
swab sample to be tested is obtained, and the nucleic acid of that
sample is incubated in the first well. If N. gonorrhoeae is present
in the sample, at least a portion of the N. gonorrhoeae nucleic
acid hybridizes to the probe nucleic acid and thereby forms a EcoRI
cut site. EcoRI, which is present within the first well or which is
added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0159] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by EcoRI, the reaction solution in the first well is
transferred to a second well containing reporter nucleic acid that
is immobilized to the surface of the second well and that has at
least one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0160] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of N. gonorrhoeae nucleic acid present in
the sample.
Example 14
Detecting the Presence or Absence of Chlamydia Trachomatis in a
Human Endocervical or Urethral Swab Sample
[0161] The presence or absence of Chlamydia trachomatis in an
endocervical and urethral swab sample collected from a human is
detected using an enzymatic amplification cascade. A C. trachomatis
target nucleic acid (GenBank.RTM. Accession No. CP000052;
GenBank.RTM. GI No. 76168153) was analyzed using the GenBank.RTM.
genetic database and CLC DNA Workbench software to identify a
portion of target C. trachomatis nucleic acid that contains plasmid
pCTA (gene accession number CP000052.1) with a cut site for the
BamHI restriction endonuclease, which cleaves at the 6 bp
nucleotide sequence 5'-GGATCC-3'. A 40 nt probe nucleic acid
(5'-ATTTCGTCTAACTTACGGATCCCTTGTACAATCAATTTAC-3'; SEQ ID NO:23) was
designed from positions 628 to 667 of the selected target nucleic
acid.
[0162] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. An endocervical and urethral
swab sample to be tested is obtained, and the nucleic acid of that
sample is incubated in the first well. If C. trachomatis is present
in the sample, at least a portion of the C. trachomatis nucleic
acid hybridizes to the probe nucleic acid and thereby forms a BamHI
cut site. BamHI, which is present within the first well or which is
added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0163] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by, the reaction solution in the first well is transferred
to a second well containing reporter nucleic acid that is
immobilized to the surface of the second well and that has at least
one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0164] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of C. trachomatis nucleic acid present in
the sample.
Example 15
Detecting the Presence or Absence of Treponema Pallidum in a Human
Lesion Swab Sample
[0165] The presence or absence of Treponema pallidum in a lesion
swab sample collected from a human is detected using an enzymatic
amplification cascade. A T. pallidum target nucleic acid
(GenBank.RTM. Accession No. NC_010741; GenBank.RTM. GI No.
189025236:115803-118796) was analyzed using the GenBank.RTM.
genetic database and CLC DNA Workbench software to identify a
portion of target T. pallidum nucleic acid that contains polA gene
(gene accession number NC_010741.1) with a cut site for the BamHI
restriction endonuclease, which cleaves at the 6 bp nucleotide
sequence 5'-GGATCC-3'. A 40 nt probe nucleic acid
(5'-TTGCAGCTTGGTTGCTGGATCCCGATCGCGGTACATACGG-3'; SEQ ID NO:24) was
designed from positions 117359 to 117398 of the selected target
nucleic acid.
[0166] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A lesion swab sample to be
tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If T. pallidum is present in the
sample, at least a portion of the T. pallidum nucleic acid
hybridizes to the probe nucleic acid and thereby forms a BamHI cut
site. BamHI, which is present within the first well or which is
added to the first well, is allowed to cleave any formed
recognition restriction endonuclease cut sites by incubating the
microtiter plate at 37.degree. C. for an appropriate length of time
(e.g., 1 minute to 2 hours) for the cleavage reaction to
proceed.
[0167] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by BamHI, the reaction solution in the first well is
transferred to a second well containing reporter nucleic acid that
is immobilized to the surface of the second well and that has at
least one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0168] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of T. pallidum nucleic acid present in the
sample.
Example 16
Detecting the Presence or Absence of Herpes Simplex Virus 2 (HSV-2)
in a Human Herpes Sore Sample
[0169] The presence or absence of HSV-2 in a herpes sore swab
sample collected from a human is detected using an enzymatic
amplification cascade. A HSV-2 target nucleic acid (GenBank.RTM.
Accession No. X01712.1; GenBank.RTM. GI No. 59898) was analyzed
using the GenBank.RTM. genetic database and CLC DNA Workbench
software to identify a portion of target HSV-2 nucleic acid that
contains thymidine kinase gene (gene accession number X01712
J02225) with a cut site for the BamHI restriction endonuclease,
which cleaves at the 6 bp nucleotide sequence 5'-GGATCC-3'. A 40 nt
probe nucleic acid (5'-CCTCCGAAGCCCGCGGGGATCCGGAGCTGCCCACGCTGCT-3';
SEQ ID NO:25) was designed from positions 442 to 481 of the
selected target nucleic acid.
[0170] Once designed and obtained by standard oligonucleotide
synthesis methods, probe nucleic acid is conjugated to an
amplifying restriction endonuclease and immobilized to the surface
of a first well of a microtiter plate. A herpes sore swab sample to
be tested is obtained, and the nucleic acid of that sample is
incubated in the first well. If HSV-2 is present in the sample, at
least a portion of the HSV-2 nucleic acid hybridizes to the probe
nucleic acid and thereby forms a BamHI cut site. BamHI, which is
present within the first well or which is added to the first well,
is allowed to cleave any formed recognition restriction
endonuclease cut sites by incubating the microtiter plate at
37.degree. C. for an appropriate length of time (e.g., 1 minute to
2 hours) for the cleavage reaction to proceed.
[0171] After cleavage of the probe nucleic acid:target nucleic acid
hybrid by BamHI, the reaction solution in the first well is
transferred to a second well containing reporter nucleic acid that
is immobilized to the surface of the second well and that has at
least one double-stranded portion having an amplifying restriction
endonuclease NcoI cut site. The reporter nucleic acid can be a
double-stranded nucleic acid having a first strand (e.g.,
5'-CATTGCTAGTTGTTTCCATGGGGTAGTGCGAAATGC-3'; SEQ ID NO:13) and a
second strand (e.g., 5'-GCATTTCGCACTACCCCATGGAAACAACTAGCAATG-3';
SEQ ID NO:14). The reporter nucleic acid also has a fluorescent
label. In some cases, first signal expansion nucleic acid and
second signal expansion nucleic acid are used prior to the reporter
nucleic acid step to increase the level of target nucleic acid
detection. The first signal expansion nucleic acid and second
signal expansion nucleic acid can include labels, in which case
they can be used together with reporter nucleic acid or in place of
reporter nucleic acid.
[0172] After transferring the reaction mixture to the second
chamber, the amplifying restriction endonucleases of the released
portions of probe nucleic acid contact reporter nucleic acid, and
the microtiter plate is incubated at an appropriate temperature
(e.g., at 37.degree. C.) for an appropriate length of time (e.g., 1
minute to 2 hours) for the cleavage reaction to proceed. The
amplifying restriction endonucleases cleave reporter nucleic acid
at the double-stranded amplifying restriction endonuclease cut site
to form at least two portions. The reaction solution of the second
well is transferred to a third well for fluorescence detection
using a fluorescent microtiter plate reader. Fluorescence in the
third well is indicative of HSV-2 nucleic acid present in the
sample.
Other Embodiments
[0173] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
25140DNAStaphylococcus aureus 1tgatctagcg aaagcaagat atcacaaaat
cgtcattatg 40240DNAStaphylococcus aureus 2attggcaaat ccggtactgc
agaactcaaa atgaaacaag 40340DNAStreptococcus pneumoniae 3aacagagagg
aatttctgca gagcgtcctt tggtctatat 40440DNAStreptococcus pyogenes
4atattttctt tatgagggtg accctgttac tcacgagaat 40540DNAMycobacterium
tuberculosis 5aacaacccgc tgtcggggtt gacccacaag cgccgactgt
40640DNAInfluenza A virus 6accgtgccca gtgagcgagg actgcagcgt
agacgctttg 40740DNAInfluenza B virus 7aatgagaaga tgtgtaagct
ttcatgaagc atttgaaata 40840DNAAdenovirus 4 (E) 8ccaactcgcc
ggatcgggaa gatcttcctt cacgcctcgt 40940DNARespiratory syncytial
virus 9ccataaaaac cacattggat atccacaaga gcataaccat
401030DNAArtificial Sequencethiol and biotin containing nucleic
acid 10ggtagtgcga aatgccattg ctagttgttt 301121DNAArtificial
Sequencepartial complement of SEQ ID NO10 11aaacaactag caatggcatt t
211240DNAStaphylococcus aureus 12tgatctagcg aaagcaagat atcacaaaat
cgtcattatg 401336DNAArtificial Sequencenucleic acid containing
cleavage site 13cattgctagt tgtttccatg gggtagtgcg aaatgc
361436DNAArtificial Sequencepartial complement of SEQ ID NO13
14gcatttcgca ctaccccatg gaaacaacta gcaatg 361540DNAStaphylococcus
aureus 15attggcaaat ccggtactgc agaactcaaa atgaaacaag
401640DNAStreptococcus pneumoniae 16aacagagagg aatttctgca
gagcgtcctt tggtctatat 401740DNAStreptococcus pyogenes 17atattttctt
tatgagggtg accctgttac tcacgagaat 401840DNAMycobacterium
tuberculosis 18aacaacccgc tgtcggggtt gacccacaag cgccgactgt
401940DNAInfluenza A virus 19accgtgccca gtgagcgagg actgcagcgt
agacgctttg 402040DNAAdenovirus 4 20ccaactcgcc ggatcgggaa gatcttcctt
cacgcctcgt 402140DNARespiratory syncytial virus 21ccataaaaac
cacattggat atccacaaga gcataaccat 402240DNANeisseria gonorrhoeae
22cgccgtcgaa gacgaagaat tcgggttcgg ggccgaaata 402340DNAChlamydia
trachomatis 23atttcgtcta acttacggat cccttgtaca atcaatttac
402440DNATreponema pallidum 24ttgcagcttg gttgctggat cccgatcgcg
gtacatacgg 402540DNAHerpes simplex virus 2 (HSV-2) 25cctccgaagc
ccgcggggat ccggagctgc ccacgctgct 40
* * * * *