U.S. patent application number 16/987881 was filed with the patent office on 2021-06-03 for methods and compositions for detection of zika viral infections.
The applicant listed for this patent is Theranos IP Company, LLC. Invention is credited to Bernardo Araujo, Ushati Das Chakravarty, Jerzy Majka, Kristine Salazar, Jerald Sapida, Chandan Shee, Katrina Sullivan-Bibee.
Application Number | 20210164061 16/987881 |
Document ID | / |
Family ID | 1000005397740 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210164061 |
Kind Code |
A1 |
Shee; Chandan ; et
al. |
June 3, 2021 |
METHODS AND COMPOSITIONS FOR DETECTION OF ZIKA VIRAL INFECTIONS
Abstract
Applicant discloses herein kits for identifying the presence of
Zika virus in a sample. In embodiments, these kits comprise
reagents disclosed herein. Applicant further provides kits for use
in detecting ZIKV in a sample, the kits comprising reagents
disclosed herein. In embodiments, kits include primers directed to
Zika virus (ZIKV) nucleic acid sequences, the primers capable of
hybridizing to ZIKV nucleic acids and to copies of ZIKV nucleic
acids (including to cDNA copies of ZIKV nucleic acids). Applicant
discloses herein reagents for detecting Zika virus (ZIKV) in a
sample, the reagents including one or more nucleic acid primers
that are capable of hybridizing to a ZIKV nucleic acid (including
to cDNA copies of ZIKV nucleic acids).
Inventors: |
Shee; Chandan; (Newark,
CA) ; Majka; Jerzy; (Foster City, CA) ;
Araujo; Bernardo; (Mountain View, CA) ; Chakravarty;
Ushati Das; (Mountain View, CA) ; Sullivan-Bibee;
Katrina; (Palo Alto, CA) ; Sapida; Jerald;
(San Leandro, CA) ; Salazar; Kristine; (Fremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Theranos IP Company, LLC |
Healdsburg |
CA |
US |
|
|
Family ID: |
1000005397740 |
Appl. No.: |
16/987881 |
Filed: |
August 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16394679 |
Apr 25, 2019 |
10745766 |
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16987881 |
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15664807 |
Jul 31, 2017 |
10301689 |
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16394679 |
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62369179 |
Jul 31, 2016 |
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62369009 |
Jul 29, 2016 |
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62368961 |
Jul 29, 2016 |
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62368995 |
Jul 29, 2016 |
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62369006 |
Jul 29, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/701 20130101 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Claims
1-18. (canceled)
19. A method for detecting a virus, the method comprising:
providing a kit comprising: a sample process control reagent, a
virus positive control reagent, a nucleic acid primer probe
mixture, using said kit with a blood sample obtained from a
fingerstick to provide a processed sample; and using a nucleic acid
amplification process on the processed sample to determine a
presence of the virus in the processed sample.
20. The method of claim 19 wherein the kit further comprises a
buffer.
21. The method of claim 20, wherein the buffer is selected from
phosphate, tris(hydroxymethyl)aminomethane (TRIS), 3-(N-morpholino)
propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic
acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES),
N-(2-Acetamido)-iminodiacetic acid (ADA), and
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES).
22. The kit of claim 20, wherein the buffer is
tris(hydroxymethyl)aminomethane (TRIS).
23. The method of claim 19 further comprising a reporter
molecule.
24. The method of claim 19 wherein the kit comprises a nucleic acid
primer that comprises a reporter molecule.
25. The method of claim 23 wherein the reporter molecule comprises
a fluorescent moiety, and the nucleic acid primer further comprises
a quenching moiety effective to quench fluorescence from the
fluorescent moiety when the primer is not hybridized to a target
nucleic acid sequence.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Applications Nos.
62/368,961 filed Jul. 29, 2016, 62/368,995 filed Jul. 29, 2016,
62/369,006 filed Jul. 29, 2016, 62/369,179 filed Jul. 31, 2016, and
62/369,009 filed Jul. 29, 2016. All of the foregoing applications
and patents are incorporated herein by reference in their entirety
for all purposes.
BACKGROUND
[0002] A variety of methods for the amplification of nucleic acids
are known. For example, polymerase chain reaction ("PCR") (see,
e.g. U.S. Pat. No. 4,683,202) is a popular method for the
amplification of nucleic acids. PCR methods are in vitro methods
able to amplify a specific polynucleotide sequence. PCR can be used
to amplify specific polynucleotide sequences, including genomic
DNA, single-stranded cDNA, and mRNA among others. As described in
U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,800,159 (hereby
incorporated herein by reference), PCR typically comprises treating
separate complementary strands of a target nucleic acid with two
polynucleotide primers to form complementary primer extension
products on both strands that act as templates for synthesizing
copies of the desired nucleic acid sequences. By repeating the
separation and synthesis steps in an automated system, essentially
exponential duplication of the target sequences can be
achieved.
[0003] To successfully perform a PCR reaction, the reaction must be
performed at multiple different temperatures. This requires
hardware or other mechanisms for repeatedly changing the
temperature of the PCR reaction. In embodiments where the target
nucleic acid is RNA, reverse transcription PCR (rtPCR) may be
used.
[0004] Zika virus (ZIKV) is a member of the Flavivirus genus of
viruses (family Flaviviridae). Other members of the genus include
dengue virus (DENV), West Nile Virus (WNV), Japanese encephalitis
virus (JEV), yellow fever virus (YFV), and tick-borne encephalitic
virus (TBEV). Flaviviruses have a single-strand, positive-sense RNA
genome that serves both as a genome and messenger RNA. The RNA
genome is translated into a single polyprotein that is
proteolytically cleaved into three structural proteins (capsid,
prM, and envelope) and non-structural proteins NS1 to NS5. The
virion contains a nucleocapsid composed of the capsid protein (C)
and the RNA genome, surrounded by an icosahedral shell comprising
both the envelope (E) glycoprotein and membrane (M) protein or the
precursor membrane (prM) protein anchored in a lipid membrane.
[0005] Flaviviruses may be transmitted by the bite from an infected
arthropod (e.g., mosquito or tick) and may cause widespread
morbidity and mortality throughout the world. Most recently, the
Zika virus has spread rapidly across the Americas following its
introduction into Brazil in 2015. While Zika virus disease is
usually mild with non-specific symptoms, such as fever, rash,
conjunctivitis, and muscle and joint pain, there have been more
severe conditions linked to the Zika virus, such as congenital
microcephaly in newborns and Guillain-Barre syndrome (GBS) in
adults. Thus, it has become increasingly important to be able to
detect Zika virus infection in an individual.
[0006] Currently, Zika virus infection is diagnosed through
detection of the viral RNA and virus isolation from blood samples,
which can be time consuming. Diagnosis by serology can be difficult
as the Zika virus can cross-react with other flaviviruses, such as
DENV, WNV, and YFV. Thus, there is a need for better assays that
are capable of specifically detecting the Zika virus. Moreover,
there is a need for a rapid and simple test for detecting Zika
virus infection at or near the point of service.
SUMMARY
[0007] In one embodiment, Applicant discloses herein reagents,
methods, and kits for detecting Zika virus (ZIKV) in samples of
bodily fluid.
[0008] Applicant discloses herein reagents for use in PCR methods
for detecting Zika virus (ZIKV) in samples of bodily fluid. In
embodiments, provided herein is a method for detecting ZIKV in a
sample of bodily fluid, the method comprising: A) generating
multiple complementary DNA (cDNA) copies of at least portions of
ZIKV RNA, B) generating multiple copies of said cDNA copies of ZIKV
RNA by polymerase chain reaction (PCR) amplification. In
embodiments of the methods disclosed herein, generating multiple
complementary DNA (cDNA) copies of at least portions of ZIKV RNA
comprises using a reverse transcriptase to effect reverse
transcription of said at least portions of ZIKV RNA to provide said
cDNA copies of at least portions of ZIKV RNA. In embodiments of the
methods for detecting Zika virus (ZIKV) in samples of bodily fluid
disclosed herein, the PCR methods comprise reverse transcription
PCR (RT-PCR) methods. In embodiments, said cDNA copies comprise a
polynucleotide template for PCR amplification. In embodiments of
the methods disclosed herein, generating multiple complementary DNA
(cDNA) copies of at least portions of ZIKV RNA comprises PCR
amplification using a PCR reaction mixture that comprises a PCR
amplification reaction first primer and a PCR amplification
reaction second primer, wherein in the PCR amplification reaction
mixture, the PCR amplification reaction first primer anneals to the
polynucleotide template and the PCR second primer anneals to a
polynucleotide which is complementary to the polynucleotide
template, and wherein in the PCR amplification reaction mixture,
multiple copies of a PCR amplification reaction product are formed,
wherein the PCR amplification reaction product is a double-stranded
nucleic acid molecule comprising a first strand and a second
strand, and wherein a first strand of the PCR amplification
reaction product is a copy of the polynucleotide template. In
embodiments of the methods for detecting Zika virus (ZIKV) in
samples of bodily fluid disclosed herein, the PCR methods comprise
real-time PCR methods. In embodiments of the methods for detecting
Zika virus (ZIKV) in samples of bodily fluid disclosed herein, the
PCR methods comprise reverse transcription real-time PCR
methods.
[0009] In embodiments, a reagent for identifying the presence of
ZIKV in a sample comprises a nucleic acid that can serve as a
positive control for PCR nucleic amplification assays for
identifying the presence of ZIKV in a sample, and for detecting
ZIKV in a sample. In embodiments, a reagent for identifying the
presence of ZIKV in a sample comprises a nucleic acid that can
serve as a positive control for PCR nucleic amplification assays
for identifying the presence of ZIKV in a sample, and for detecting
ZIKV in a sample, and a buffer.
[0010] In embodiments, a reagent for identifying the presence of
ZIKV in a sample comprises a buffer and a variant of a nucleic acid
sequence that comprises a nucleic acid sequence found in, or is
complementary to a nucleic acid sequence found in, or is homologous
to a nucleic acid sequence found in, or complementary to a
homologous nucleic acid sequence found in, ZIKV. In embodiments,
such a nucleic acid sequence variant has at least about 95%
sequence identity to the nucleic acid sequence.
[0011] In embodiments, a reagent for identifying the presence of
ZIKV comprises a buffer selected from phosphate and
tris(hydroxymethyl)aminomethane (TRIS). In embodiments, a reagent
for identifying the presence of ZIKV comprises a TRIS buffer.
[0012] In embodiments, Applicant provides herein reagents for
identifying the presence of ZIKV in a sample. In embodiments, a
reagent for identifying the presence of ZIKV in a sample comprises
a nucleic acid sequence selected from the group SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:
6, and a buffer. In embodiments, the nucleic acid primer of a
reagent as disclosed herein comprises a nucleic acid sequence
selected from the group SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3.
[0013] In embodiments, a reagent for identifying the presence of
ZIKV in a sample comprises a buffer and a variant of a nucleic acid
sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the
variant has at least about 95% sequence identity to the nucleic
acid sequence. In embodiments, the nucleic acid primer of a reagent
as disclosed herein comprises a variant of a nucleic acid sequence
selected from the group SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3, wherein the variant has at least about 95% sequence identity to
the nucleic acid sequence.
[0014] In embodiments, a reagent for identifying the presence of
ZIKV comprises a buffer selected from phosphate and
tris(hydroxymethyl)aminomethane (TRIS). In embodiments, a reagent
for identifying the presence of ZIKV comprises a TRIS buffer.
[0015] In embodiments of reagents for identifying the presence of
ZIKV as disclosed herein, the primer comprises a reporter molecule.
In embodiments wherein the primer comprises a reporter molecule,
the reporter molecule may comprise a fluorescent moiety. In
embodiments wherein the primer comprises a fluorescent moiety, the
nucleic acid primer may further comprise a quenching moiety
effective to quench fluorescence from the fluorescent moiety when
the primer is not hybridized to a target nucleic acid sequence.
[0016] Applicant discloses herein kits for detecting Zika virus
(ZIKV) in samples of bodily fluid. Kits for detecting Zika virus
(ZIKV) in samples include reagents as disclosed herein. Kits for
detecting Zika virus (ZIKV) in samples include nucleic acids as
disclosed herein. Kits for detecting Zika virus (ZIKV) in samples
include nucleic acid primers as disclosed herein. Kits for
detecting Zika virus (ZIKV) in samples include nucleic acids
comprising reporter molecules as disclosed herein. Kits for
detecting Zika virus (ZIKV) in samples include nucleic acid primers
comprising reporter molecules as disclosed herein.
[0017] The assays and methods disclosed herein may be performed on
a device, or on a system, for processing a sample. The assays and
methods disclosed herein can be readily incorporated into and used
in an automated assay device, and in an automated assay system. The
assays and methods disclosed herein can be readily incorporated
into and used in an automated sample analysis device. For example,
devices and systems as disclosed herein may include a communication
assembly for transmitting or receiving a protocol based on the
analyte to be detected (e.g., ZIKV) or based on other analytes to
be detected by the device or system.
[0018] Methods and compositions disclosed herein provide rapid
assays which require only small amounts of sample, such as only
small amounts of blood. Device and systems disclosed herein are
configured to perform such rapid assays which require only small
amounts of sample, such as only small amounts of blood.
Accordingly, the methods, compositions, devices, and systems
provide rapid tests, which require only small biological samples,
and thus provide advantages over other methods, reagents, kits,
assays, devices, and systems.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a perspective image of a rRT-PCR Reagent kit
and related articles, as disclosed herein.
[0020] FIG. 2 is a plot of fluorescence (a measure of the numbers
of cDNA copies of the target nucleic acid sequence) versus time (as
cycles) showing the results of rRT-PCR amplification of Zika
virus.
[0021] FIG. 3 is a graph showing the copies resulting from rRT-PCR
amplification of Zika virus.
DETAILED DESCRIPTION
[0022] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. It may be noted that, as used in the specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a material" may include mixtures
of materials, reference to "a compound" may include multiple
compounds, and the like. References cited herein are hereby
incorporated by reference in their entirety, except to the extent
that they conflict with teachings explicitly set forth in this
specification.
[0023] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0024] As used herein, a "sample" may be, but is not limited to,
blood, cerebrospinal fluid, bile, plasma, serum, saliva, sputum,
mucus, and urine, or any portion thereof. The sample may be of any
suitable size or volume. In some embodiments of the assays and
methods disclosed herein, measurements may be made using a small
volume of the sample, or no more than a small volume portion of the
sample, where a small volume comprises no more than about 5 mL; or
comprises no more than about 3 mL; or comprises no more than about
2 mL; or comprises no more than about 1 mL; or comprises no more
than about 500 .mu.L; or comprises no more than about 250 .mu.L; or
comprises no more than about 100 .mu.L; or comprises no more than
about 75 .mu.L; or comprises no more than about 50 .mu.L; or
comprises no more than about 35 .mu.L; or comprises no more than
about 25 .mu.L; or comprises no more than about 20 .mu.L; or
comprises no more than about 15 .mu.L; or comprises no more than
about 10 .mu.L; or comprises no more than about 8 .mu.L; or
comprises no more than about 6 .mu.L; or comprises no more than
about 5 .mu.L; or comprises no more than about 4 .mu.L; or
comprises no more than about 3 .mu.L; or comprises no more than
about 2 .mu.L; or comprises no more than about 1 .mu.L; or
comprises no more than about 0.8 .mu.L; or comprises no more than
about 0.5 .mu.L; or comprises no more than about 0.3 .mu.L; or
comprises no more than about 0.2 .mu.L; or comprises no more than
about 0.1 .mu.L; or comprises no more than about 0.05 .mu.L; or
comprises no more than about 0.01 .mu.L.
[0025] As used herein, the term "analyte" refers to a molecule of
interest that is detected or to be detected in an analytical
procedure.
[0026] As used in the description herein and throughout the claims
that follow, the meaning of "or" includes both the conjunctive and
disjunctive unless the context expressly dictates otherwise. Thus,
the term "or" includes "and/or" unless the context expressly
dictates otherwise.
[0027] The term "moiety" as used herein refers to any particular
composition of matter, e.g., a molecular fragment, an intact
molecule, or a mixture of materials.
[0028] As used herein, "nucleic acid" includes both DNA and RNA,
including DNA and RNA containing non-standard nucleotides. A
"nucleic acid" contains at least one polynucleotide (a "nucleic
acid strand"). A "nucleic acid" may be single-stranded or
double-stranded. This, the term "nucleic acid" refers to
nucleotides and nucleosides which make up, for example,
deoxyribonucleic acid (DNA) macromolecules and ribonucleic acid
(RNA) macromolecules. Nucleic acids may be identified by the base
attached to the sugar (e.g., deoxyribose or ribose); as used
herein, the abbreviations for these bases (shown in Table 1) are
used to represent nucleic acids in sequence listings identifying
and describing their structures (either upper-case or lower-case
may be used).
TABLE-US-00001 TABLE 1 Base (in Nucleic Acid) Letter Code Adenine A
Thymine T Guanine G Cytosine C Uracil U
[0029] RNA molecules found in nature consist of sequences of A, G,
C, and U, while DNA molecules found in nature consist of A, G, C,
and T; that is, where a RNA sequence that is complementary to a
nucleic acid target sequence includes U, a DNA sequence that is
complementary to that nucleic acid target sequence includes T in
place of U.
[0030] As used herein, the term "polynucleotide" is used to refer
to a polymeric chain containing two or more nucleotides.
"Polynucleotides" include primers, oligonucleotides, nucleic acid
strands, etc. A polynucleotide may contain standard or non-standard
nucleotides. Typically, a polynucleotide contains a 5' phosphate at
one terminus ("5' terminus") and a 3' hydroxyl group at the other
terminus ("3' terminus) of the chain. The most 5' nucleotide of a
polynucleotide may be referred to herein as the "5' terminal
nucleotide" of the polynucleotide. The most 3' nucleotide of a
polynucleotide may be referred to herein as the "3' terminal
nucleotide" of the polynucleotide.
[0031] The term "downstream" as used herein in the context of a
polynucleotide containing a 5' terminal nucleotide and a 3'
terminal nucleotide refers to a position in the polynucleotide
which is closer to the 3' terminal nucleotide than a reference
position in the polynucleotide. For example, in a primer having the
sequence: 5' ATAAGC 3', the "G" is downstream from the "T" and all
of the "A"s.
[0032] The term "upstream" as used herein in the context of a
polynucleotide containing a 5' terminal nucleotide and a 3'
terminal nucleotide, refers to a position in the polynucleotide
which is closer to the 5' terminal nucleotide than a reference
position in the polynucleotide. For example, in a primer having the
sequence: 5' TAGC 3', the "T" is upstream from the "G", the "C",
and the "A".
[0033] As used herein, a nucleic acid molecule which is described
as containing the "sequence" of a template or other nucleic acid
may also be considered to contain the template or other nucleic
acid itself (e.g. a molecule which is described as containing the
sequence of a template may also be described as containing the
template), unless the context clearly dictates otherwise.
[0034] As used herein "cDNA" refers to DNA molecules
("complementary DNA") produced by reverse transcription of an RNA
molecule. Such reverse transcription produces a DNA molecule having
a nucleotide sequence that is the same as the nucleotide sequence
of that RNA molecule, with the exception that where the RNA
molecule has a uracil moiety (U) the DNA molecule has instead a
thymine (T). A cDNA produced by reverse transcription of an RNA
molecule is complementary to the complement of that RNA
molecule.
[0035] As used herein, a "target" nucleic acid or molecule refers
to a nucleic acid of interest. A target nucleic acid/molecule may
be of any type, including single-stranded or double stranded DNA or
RNA (e.g. mRNA). In some instances, a target nucleic acid may be a
nucleic acid which may directly function as a double-stranded
nucleic acid template in a method provided herein (e.g. a
double-stranded DNA molecule), or it may be a nucleic acid which
requires further processing or conversion to function as a
double-stranded nucleic acid template in a method provided herein
(e.g. mRNA).
[0036] As used herein, "complementary" sequences refer to two
nucleotide sequences which, when aligned anti-parallel to each
other, contain multiple individual nucleotide bases which pair with
each other. It is not necessary for every nucleotide base in two
sequences to pair with each other for sequences to be considered
"complementary". Sequences may be considered complementary, for
example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two
sequences pair with each other. In addition, sequences may still be
considered "complementary" when the total lengths of the two
sequences are significantly different from each other. For example,
a primer of 15 nucleotides may be considered "complementary" to a
longer polynucleotide containing hundreds of nucleotides if
multiple individual nucleotide bases of the primer pair with
nucleotide bases in the longer polynucleotide when the primer is
aligned anti-parallel to a particular region of the longer
polynucleotide.
[0037] "Identical" or "identity," as used herein in the context of
two or more polypeptide or polynucleotide sequences, can mean that
the sequences have a specified percentage of residues that are the
same over a specified region. The percentage can be calculated by
optimally aligning the two sequences, comparing the two sequences
over the specified region, determining the number of positions at
which the identical residue occurs in both sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the specified region,
and multiplying the result by 100 to yield the percentage of
sequence identity. In cases where the two sequences are of
different lengths or the alignment produces one or more staggered
ends and the specified region of comparison includes only a single
sequence, the residues of the single sequence are included in the
denominator but not the numerator of the calculation.
[0038] "Homology" or "homologous" as used herein in the context of
two or more polypeptide or polynucleotide sequences, can mean that
the sequences have a specified percentage of residues that are
either i) the same, or ii) conservative substitutions of the same
residue, over a specified region. Conservative substitutions
include substitutions of one amino acid by an amino acid of the
same group, and include substitutions of one amino acid by an amino
acid as an exemplary or as a preferred substitution as known in the
art. In determining homology of two sequences, identical residues
and homologous residues are given equal weight. The percentage can
be calculated by optimally aligning the two sequences, comparing
the two sequences over the specified region, determining the number
of positions at which either identical or homologous residues occur
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the specified region, and multiplying the result by
100 to yield the percentage of sequence homology. In cases where
the two sequences are of different lengths or the alignment
produces one or more staggered ends and the specified region of
comparison includes only a single sequence, the residues of the
single sequence are included in the denominator but not the
numerator of the calculation.
[0039] As used herein, in the context of two or more polymeric
molecules (e.g. nucleic acids, proteins), "corresponds to",
"corresponding to", and the like refers to polymeric molecules or
portions thereof which have the same or similar sequence of
component elements (e.g. nucleotides, amino acids). For example, if
a first nucleic acid is described as containing a region which
"corresponds to" the sequence of a second nucleic acid, the
relevant region of the first nucleic acid has a nucleotide sequence
which is the same or similar to the sequence of the second nucleic
acid.
[0040] The term "primer" as used herein refers to a polynucleotide,
whether occurring naturally as in a purified restriction digest or
produced synthetically, which is capable of acting as a point of
initiation of synthesis when placed under conditions in which
synthesis of a primer extension product which is complementary to a
nucleic acid strand is induced, i.e., in the presence of
nucleotides and an inducing agent such as DNA polymerase and at a
suitable temperature and pH. The primer is preferably single
stranded for maximum efficiency in amplification, but may
alternatively be double stranded. If double stranded, the primer is
first treated to separate its strands before being used to prepare
extension products. Preferably, the primer is a
poly-deoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and use of
the method. For example, for diagnostics applications, depending on
the complexity of the target sequence, the polynucleotide primer
typically contains about 10-30 or more nucleotides, or about 15-25
or more nucleotides, although it may contain fewer nucleotides. For
other applications, the polynucleotide primer is typically shorter,
e.g., 7-15 nucleotides. Such short primer molecules generally
require cooler temperatures to form sufficiently stable hybrid
complexes with template.
[0041] A primer may include a marker moiety, such as a fluorescent
moiety, or a quencher moiety (e.g., for quenching fluorescence by
fluorescence (or Forster) resonance energy transfer FRET)), or
combinations thereof. Fluorescein dyes (e.g., FAM.TM.) are suitable
fluorescent moieties ("dye"). Other suitable dyes include VIC.RTM.,
ROX.TM., SYBR.RTM. Green, JOE, TAMRA.TM., and NED.TM. dyes, all of
which are commercially available and may be linked to nucleic acid
molecules. Dabcyl (4-((4-(dimethylamino)phenyl)azo)benzoic Acid) is
an example of a suitable quencher which is commercially available
and may be linked to nucleic acid molecules.
[0042] As used herein, when a first polynucleotide is described as
"annealed", "annealing" or the like to a second polynucleotide, the
entirety of the first polynucleotide or any portion thereof may
anneal to the second polynucleotide, and vice versa.
[0043] The "Tm" indicates the annealing temperature for a
particular primer set; a primer set may have a different Tm than
other primer sets, or may have the same Tm as another primer set.
In many cases, Tm is typically between about 45.degree. C. to about
80.degree. C., or between about 50.degree. C. to about 75.degree.
C.
[0044] As used herein, "reverse transcriptase" (RT) refers to an
enzyme which can be used to produce a DNA molecule that is
complementary to a RNA molecule. The act of producing such a DNA
molecule from an RNA template is termed "reverse
transcription".
[0045] As used herein, polymerase chain reaction, abbreviated by
the acronym "PCR", refers to any of the nucleic acid amplification
methods in which a target nucleic acid (typically a double-stranded
deoxyribonucleic acid) is exposed to a thermostable DNA polymerase
during multiple thermal cycles, and in which multiple copies of the
target nucleic acid (typically including copies of nucleic acid
sequences disposed between a first target region on one strand of a
double-stranded target nucleic acid and a second target region on
the complementary strand of a double-stranded target nucleic acid)
are produced, amplifying the target nucleic acid. Thermal cycles
typically include low temperature portions (typically at
temperatures between about 40.degree. C. and about 59.degree. C.,
or between about 45.degree. C. and about 55.degree. C.),
intermediate temperature portions (typically at temperatures
between about 60.degree. C. and about 74.degree. C.), and higher
temperature portions (typically at temperatures between about
75.degree. C. and about 99.degree. C., or between about 80.degree.
C. and about 95.degree. C.). For example, some PCR reactions
include a) incubation of a mixture including target molecules and
primers at high temperature (e.g., about 90.degree. C. to about
95.degree. C.) to denature the target DNA; b) cooling the mixture
to an intermediate temperature (e.g., about 50.degree. C. to about
60.degree. C.) to allow annealing between the primers and target
DNA; and c) in the presence of DNA polymerase, generating
extensions of the primers (e.g., by action of the polymerase at,
e.g. temperatures of about 65.degree. C. to about 75.degree. C.);
and repeating this cycle of steps a), b), and c). Steps a), b), and
c) together may be termed a "thermal cycle".
[0046] Amplification occurs with each thermal cycle, and, following
multiple cycles, significant amplification of the target nucleic
acid molecule produces large numbers of DNA copies of the target
sequence. PCR requirements include a DNA polymerase (e.g., a
thermostable DNA polymerase), deoxynucleotides, and appropriate
buffer solutions. Where a target nucleic acid is an RNA target,
reverse transcriptase (RT) may be used to produce a DNA copy of the
RNA, and PCR applied to the DNA copies.
[0047] As used herein, reverse transcription PCR (RT-PCR) refers to
methods for amplifying RNA targets, in which copy DNA molecules
(cDNAs) are produced from RNA target polynucleotides by application
of reverse transcriptase, and PCR is applied to the cDNA copies to
amplify the cDNA copies for detection and/or amplification of the
target polynucleotide. RT-PCR requirements include a reverse
transcriptase, a DNA polymerase (e.g., a thermostable DNA
polymerase), deoxynucleotides (typically as deoxynucleotide
tri-phosphates ("dNTPs") such as dATP, dTTP, dGTP, and dCTP), and
appropriate buffer solutions.
[0048] As used herein, "real-time PCR" refers to PCR amplification
methods in which the progress, or extent, of target amplification
is monitored during the course of the assay (e.g., at each thermal
cycle). Progress of the amplification reactions may be monitored,
for example, detecting the amount of fluorescence or absorbance of
reporter molecules. Suitable reporter molecules include
intercalating dyes (which are detectable when bound to
double-stranded DNA, or to the minor groove of DNA, such as
ethidium bromide and SYBR Green dye); fluorogenic probes, such as
self-quenching dyes, or dye pairs (the pairs including a dye and a
quencher) attached to primers (which fluoresce when the primer is
bound to target, but do not produce significant fluorescence when
not hybridized to target nucleic acid molecules); and other
reporter molecules.
[0049] As used herein, "rRT-PCR" refers to reverse-transcription
real-time PCR. rRT-PCR is real-time PCR applied to RNA targets,
using reverse-transcription PCR to amplify nucleic acids based on
RNA target molecules, and monitoring the amplification using
real-time PCR methods. As used herein, "rRT-PCR" refers to
reverse-transcription real-time PCR. rRT-PCR is real-time PCR
applied to RNA targets, using reverse-transcription PCR to amplify
nucleic acids based on RNA target molecules, and monitoring the
amplification using real-time PCR methods. Reverse-transcription
PCR methods provide the DNA substrate required for PCR by
contacting a sample, under the appropriate conditions, with a
reverse transcriptase and producing cDNA copies of RNA molecules in
the sample.
[0050] The terms "polypeptide" and "protein" may be used
interchangeably to refer to molecules comprised of amino acids
linked by peptide bonds. Individual amino acids may be termed
"residues" of a polypeptide or protein. The amino acid sequences of
polypeptides disclosed herein may be identified by SEQ ID NO:
presented as a string of letters, where the letters have the
following meanings:
TABLE-US-00002 TABLE 1B AminoAcid 3-Letter Code 1-Letter Code
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D
Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0051] Amino acid sequence of polypeptides, including enzymes, may
include variants of a parent sequence with substitutions,
insertions and deletions as compared to the sequence of the parent
polypeptide. Amino acid variants of parent polypeptides may be
suitable for the same or similar use as the parent polypeptide. For
example, amino acid variants of parent polypeptides having amino
acid sequences that are 95% or greater identical or similar to the
amion acid sequence of the parent polypeptide may be suitable for
the same or similar use as the parent polypeptide.
[0052] A composition may include a buffer. Buffers include, without
limitation, phosphate, citrate, ammonium, acetate, carbonate,
tris(hydroxymethyl)aminomethane (TRIS), 3-(N-morpholino)
propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic
acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES),
N-(2-Acetamido)-iminodiacetic acid (ADA),
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES),
N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), cholamine
chloride, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic
acid (TES), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid
(HEPES), acetamidoglycine, tricine
(N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), glycinamide,
and bicine (2-(Bis(2-hydroxyethyl)amino)acetic acid) buffers.
Buffers include other organic acid buffers in addition to the
phosphate, citrate, ammonium, acetate, and carbonate buffers
explicitly mentioned herein.
[0053] In embodiments of the compositions disclosed herein,
including embodiments of the aqueous compositions and embodiments
of the buffered aqueous compositions, the composition may comprise
albumin, gelatin, cytochrome C, an immunoglobulin, an amino acid,
agar, glycerol, ethylene glycol, a protease inhibitor, an
antimicrobial agent, a metal chelating agent, a monosaccharide, a
disaccharide, a polysaccharide, a reducing agent, a chelating
agent, or combinations thereof.
[0054] An article of manufacture may comprise a container; and a
composition contained within the container, wherein the composition
comprises a nucleic acid molecule (such as, e.g., a primer directed
to a target related to ZIKA). An article of manufacture may
comprise a container; and a composition contained within the
container, wherein the composition comprises a nucleic acid
molecule (such as, e.g., a primer directed to a target related to
ZIKA). An article of manufacture may comprise a container; and a
composition contained within the container, wherein the composition
comprises a nucleic acid molecule (such as, e.g., a primer directed
to a target related to ZIKA). The containers may be formed from a
variety of materials such as glass or plastic, and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). The article of manufacture may
further comprise a label or package insert on or associated with
the container indicating that the composition may be used to detect
the presence of a nucleic acid molecule (such as, e.g., a primer
directed to a target related to ZIKA) in a sample.
[0055] Description and disclosure of examples of reagents, kits,
assays, methods, kits, devices, and systems which may use, or be
used with, the reagents, kits, methods, devices, and systems
disclosed herein may be found, for example, in U.S. Pat. Nos.
8,088,593; 8,380,541; 8,435,738; 8,475,739; 8,840,838; 9,250,229;
U.S. Pub. No. 2014/0057255; U.S. Pub. No. 2013/0078624; WO
2013/052318; WO 2014/015199; U.S. patent application Ser. No.
14/183,503, filed Feb. 18, 2014; U.S. patent application Ser. No.
13/933,035, filed Jul. 1, 2013; U.S. patent application Ser. No.
13/769,820, filed Feb. 18, 2013; U.S. patent application Ser. No.
14/183,503, filed Feb. 18, 2014; Patent application Ser. No.
14/214,850, filed Mar. 15, 2014; International Patent Application
PCT/US2014/030034, filed Mar. 15, 2014; International Patent
Application PCT/US2014/056151, filed Sep. 17, 2014; U.S. patent
application Ser. No. 13/769,798, filed Feb. 18, 2013; U.S. patent
application Ser. No. 13/769,779, filed Feb. 18, 2013; U.S. patent
application Ser. No. 13/244,947 filed Sep. 26, 2011;
PCT/US2012/57155, filed Sep. 25, 2012; U.S. application Ser. No.
13/244,946, filed Sep. 26, 2011; U.S. patent application Ser. No.
13/244,949, filed Sep. 26, 2011; and U.S. application Ser. No.
13/945,202, filed Jul. 18, 2013, the disclosures of which patents
and patent applications are all hereby incorporated by reference in
their entireties.
[0056] In embodiments of such methods, the nucleic acid target
molecules amplified by the PCR amplification method comprise DNA.
In embodiments of such methods, the nucleic acid target molecules
amplified by the PCR amplification method comprise RNA. In
embodiments, the nucleic acid may include uracil, and in
embodiments may include dideoxyuracil (e.g., may include
dideoxyuracil in place of a thymine during amplification). In
embodiments of such methods, said second nucleic acid amplification
method comprises an isothermal nucleic amplification method.
[0057] In embodiments of the methods disclosed herein, primers used
in the amplification methods are directed to a single target
nucleic acid sequence, and its complement. In embodiments of the
methods disclosed herein, primers used in the amplification methods
are directed to a plurality of target nucleic acid sequences, and
complements thereof.
[0058] In embodiments, the target molecule is a RNA target, and
cDNA molecules are generated from RNA target molecules by reverse
transcription. These cDNA molecules provide substrate molecules for
PCR amplification.
[0059] Optionally, the PCR amplification reaction first primer is
at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50,
or 60 and no more than 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50,
60, 70, 80, 90, or 100 nucleotides in length, and wherein when the
PCR amplification reaction first primer is annealed to the
polynucleotide template, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
nucleotides of the PCR amplification reaction first primer are
mis-matched according to Watson-Crick base-pairing rules with
corresponding nucleotides on the polynucleotide template.
Optionally, the PCR amplification reaction second primer is at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or
60 and no more than 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60,
70, 80, 90, or 100 nucleotides in length, and wherein when the PCR
amplification reaction second primer is annealed to the
polynucleotide which is complementary to the polynucleotide
template, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of
the PCR amplification reaction second primer are mis-matched
according to Watson-Crick base-pairing rules with corresponding
nucleotides on the polynucleotide which is complementary to the
polynucleotide template.
[0060] In embodiments, PCR methods for amplifying a polynucleotide
target molecule include: A) generating multiple copies of a
polynucleotide template in a polymerase chain reaction (PCR)
amplification reaction mixture, wherein the PCR amplification
reaction mixture comprises a first PCR amplification reaction
primer and a second PCR amplification reaction primer, wherein in
the PCR amplification reaction mixture, the first PCR amplification
reaction primer anneals to the polynucleotide template and the
second PCR amplification reaction primer anneals to a
polynucleotide which is complementary to the polynucleotide
template, and wherein in the PCR amplification reaction mixture,
multiple copies of a PCR amplification reaction product are formed,
wherein the PCR amplification reaction product is a double-stranded
nucleic acid molecule comprising a first strand and a second
strand, and wherein a first strand of the PCR amplification
reaction product is a copy of the polynucleotide template.
[0061] Applicant discloses herein methods for detecting Zika virus
(ZIKV) in samples of bodily fluid. In embodiments, provided herein
is a method for detecting ZIKV in a sample of bodily fluid, the
method comprising: A) generating multiple complementary DNA (cDNA)
copies of at least portions of ZIKV RNA, B) generating multiple
copies of said cDNA copies of ZIKV RNA by polymerase chain reaction
(PCR) amplification. In embodiments of the methods disclosed
herein, generating multiple complementary DNA (cDNA) copies of at
least portions of ZIKV RNA comprises using a reverse transcriptase
to effect reverse transcription of said at least portions of ZIKV
RNA to provide said cDNA copies of at least portions of ZIKV RNA.
In embodiments of the methods for detecting Zika virus (ZIKV) in
samples of bodily fluid disclosed herein, the PCR methods comprise
reverse transcription PCR (RT-PCR) methods. In embodiments, said
cDNA copies comprise a polynucleotide template for PCR
amplification. In embodiments of the methods disclosed herein,
generating multiple complementary DNA (cDNA) copies of at least
portions of ZIKV RNA comprises PCR amplification using a PCR
reaction mixture that comprises a PCR amplification reaction first
primer and a PCR amplification reaction second primer, wherein in
the PCR amplification reaction mixture, the PCR amplification
reaction first primer anneals to the polynucleotide template and
the PCR second primer anneals to a polynucleotide which is
complementary to the polynucleotide template, and wherein in the
PCR amplification reaction mixture, multiple copies of a PCR
amplification reaction product are formed, wherein the PCR
amplification reaction product is a double-stranded nucleic acid
molecule comprising a first strand and a second strand, and wherein
a first strand of the PCR amplification reaction product is a copy
of the polynucleotide template. In embodiments of the methods for
detecting Zika virus (ZIKV) in samples of bodily fluid disclosed
herein, the PCR methods comprise real-time PCR methods. In
embodiments of the methods for detecting Zika virus (ZIKV) in
samples of bodily fluid disclosed herein, the PCR methods comprise
reverse transcription real-time PCR methods.
[0062] The assays and methods disclosed herein may be performed on
a device, or on a system, for processing a sample. The assays and
methods disclosed herein can be readily incorporated into and used
in device for processing a sample, or a system for processing a
sample, which may be an automated assay device, or may be an
automated assay system. Such a device, and such a system, may be
useful for the practice of the methods disclosed herein. For
example, a device may be useful for receiving a sample. A device
may be useful for preparing, or for processing a sample. A device
may be useful for performing an assay on a sample. A device may be
useful for obtaining data from a sample. A device may be useful for
transmitting data obtained from a sample. A device may be useful
for disposing of a sample following processing or assaying of a
sample.
[0063] A device may be part of a system, a component of which may
be an automatic assay device. A device may be an automatic assay
device. An automatic assay device may be configured to facilitate
collection of a sample, prepare a sample for a clinical test, or
effect a chemical reaction with one or more reagents or other
chemical or physical processing, as disclosed herein. An automatic
assay device may be configured to obtain data from a sample. An
automatic assay device may be configured to transmit data obtained
from a sample. An automatic assay device may be configured to
analyze data from a sample. An automatic assay device may be
configured to communicate with another device, or a laboratory, or
an individual affiliated with a laboratory, to analyze data
obtained from a sample.
[0064] A sample may be, for example, a blood sample (e.g., a sample
obtained from a fingerstick, or from venipuncture, or an arterial
blood sample), a urine sample, a biopsy sample, a tissue slice,
stool sample, or other biological sample; a water sample, a soil
sample, a food sample, an air sample; or other sample. A blood
sample may comprise, e.g., whole blood, plasma, or serum. An
automatic assay device may receive a sample from the subject
through a housing of the device. The sample collection may occur at
a sample collection site, or elsewhere. The sample may be provided
to the device at a sample collection site.
[0065] Accordingly, Applicants disclose devices configured to
measure ZIKV in a sample of blood according to a method disclosed
herein. Devices configured to measure ZIKV in a sample of blood
according to a method disclosed herein may be configured to
determine ZIKV from a sample of blood that comprises no more than
about 1000 .mu.L of blood, or no more than about 500 .mu.L of
blood, no more than about 250 .mu.L of blood, or no more than about
150 .mu.L of blood, or no more than about 100 .mu.L of blood, or no
more than about 50 .mu.L of blood, or, in embodiments, wherein said
sample of blood comprises no more than about 25 .mu.L of blood, or
wherein said sample of blood comprises no more than about 10 .mu.L
of blood, or wherein said sample of blood comprises less than about
10 .mu.L of blood. Such devices may be configured to measure ZIKV
in a sample of blood in less than about one hour, or, in
embodiments, in less than about 40 minutes, or in less than about
30 minutes.
[0066] In some embodiments, an automatic assay device may be
configured to accept or hold a cartridge. In some embodiments, an
automatic assay device may comprise a cartridge. The cartridge may
be removable from the automatic assay device. In some embodiments,
a sample may be provided to the cartridge of the automatic assay
device. Alternatively, a sample may be provided to another portion
of an automatic assay device. The cartridge and/or device may
comprise a sample collection unit that may be configured to accept
a sample.
[0067] A cartridge may include a sample, and may include reagents
for use in processing or testing a sample, disposables for use in
processing or testing a sample, or other materials. Following
placement of a cartridge on, or insertion of a cartridge into, an
automatic assay device, one or more components of the cartridge may
be brought into fluid communication with other components of the
automatic assay device. For example, if a sample is collected at a
cartridge, the sample may be transferred to other portions of the
automatic assay device. Similarly, if one or more reagents are
provided on a cartridge, the reagents may be transferred to other
portions of the automatic assay device, or other components of the
automatic assay device may be brought to the reagents. In some
embodiments, the reagents or components of a cartridge may remain
on-board the cartridge. In some embodiments, no fluidics are
included that require tubing or that require maintenance (e.g.,
manual or automated maintenance).
[0068] A sample or reagent may be transferred to a device, such as
an automatic assay device. A sample or reagent may be transferred
within a device. Such transfer of sample or reagent may be
accomplished without providing a continuous fluid pathway from
cartridge to device. Such transfer of sample or reagent may be
accomplished without providing a continuous fluid pathway within a
device. In embodiments, such transfer of sample or reagent may be
accomplished by a sample handling system (e.g., a pipette); for
example, a sample, reagent, or aliquot thereof may be aspirated
into an open-tipped transfer component, such as a pipette tip,
which may be operably connected to a sample handling system which
transfers the tip, with the sample, reagent, or aliquot thereof
contained within the tip, to a location on or within the automatic
assay device. The sample, reagent, or aliquot thereof can be
deposited at a location on or within the automatic assay device.
Sample and reagent, or multiple reagents, may be mixed using a
sample handling system in a similar manner. One or more components
of the cartridge may be transferred in an automated fashion to
other portions of the automatic assay device, and vice versa.
[0069] A device, such as an automatic assay device, may have a
fluid handling system. The fluid may be a sample, a reagent,
diluent, wash, dye, or any other fluid that may be used by the
device, and may include, but not limited to, homogenous fluids,
different liquids, emulsions, suspensions, and other fluids. A
fluid handling system, including without limitation a pipette, may
also be used to transport vessels (with or without fluid contained
therein) around the device. The fluid handling system may dispense
or aspirate a fluid. The sample may include one or more particulate
or solid matter floating within a fluid.
[0070] In embodiments, a fluid handling system may comprise a
pipette, pipette tip, syringe, capillary, or other component. The
fluid handling system may include one or more fluidically isolated
or hydraulically independent units. For example, the fluid handling
system may include one, two, or more pipette tips. The pipette tips
may be configured to accept and confine a fluid. The tips may be
fluidically isolated from or hydraulically independent of one
another. The fluid contained within each tip may be fluidically
isolated or hydraulically independent from one fluids in other tips
and from other fluids within the device. The fluidically isolated
or hydraulically independent units may be movable relative to other
portions of the device and/or one another. The fluidically isolated
or hydraulically independent units may be individually movable. A
fluid handling system may comprise one or more base or support. A
base or support may support one or more pipette or pipette units. A
base or support may connect one or more pipettes of the fluid
handling system to one another.
[0071] An automatic assay device may be configured to perform
processing steps or actions on a sample obtained from a subject.
Sample processing may include sample preparation, including, e.g.,
sample dilution, division of a sample into aliquots, extraction,
contact with a reagent, filtration, separation, centrifugation, or
other preparatory or processing action or step. An automatic assay
device may be configured to perform one or more sample preparation
action or step on the sample. Optionally, a sample may be prepared
for a chemical reaction and/or physical processing step. A sample
preparation action or step may include one or more of the
following: centrifugation, separation, filtration, dilution,
enriching, purification, precipitation, incubation, pipetting,
transport, chromatography, cell lysis, cytometry, pulverization,
grinding, activation, ultrasonication, micro column processing,
processing with magnetic beads, processing with nanoparticles, or
other sample preparation action or steps. For example, sample
preparation may include one or more step to separate blood into
serum and/or particulate fractions, or to separate any other sample
into various components. Sample preparation may include one or more
step to dilute and/or concentrate a sample, such as a blood sample,
or other biological samples. Sample preparation may include adding
an anti-coagulant or other ingredients to a sample. Sample
preparation may also include purification of a sample. In
embodiments, all sample processing, preparation, or assay actions
or steps are performed by a single device. In embodiments, all
sample processing, preparation, or assay actions or steps are
performed within a housing of a single device. In embodiments, most
sample processing, preparation, or assay actions or steps are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, many sample processing,
preparation, or assay actions or steps are performed by a single
device, and may be performed within a housing of a single device.
In embodiments, sample processing, preparation, or assay actions or
steps may be performed by more than one device.
[0072] An automatic assay device may be configured to run one or
more assay on a sample, and to obtain data from the sample. An
assay may include one or more physical or chemical treatments, and
may include running one or more chemical or physical reactions. An
automatic assay device may be configured to perform one, two or
more assays on a small sample of bodily fluid. One or more chemical
reaction may take place on a sample having a volume, as described
elsewhere herein. For example one or more chemical reaction may
take place in a pill having less than femtoliter volumes. In an
instance, the sample collection unit is configured to receive a
volume of the bodily fluid sample equivalent to a single drop or
less of blood or interstitial fluid. In embodiments, the volume of
a sample may be a small volume, where a small volume may be a
volume that is less than about 1000 .mu.L, or less than about 500
.mu.L, or less than about 250 .mu.L, or less than about 150 .mu.L,
or less than about 100 .mu.L, or less than about 75 .mu.L, or less
than about 50 .mu.L, or less than about 40 .mu.L, or less than
about 20 .mu.L, or less than about 10 .mu.L, or other small volume.
In embodiments, all sample assay actions or steps are performed on
a single sample. In embodiments, all sample assay actions or steps
are performed by a single device. In embodiments, all sample assay
actions or steps are performed within a housing of a single device.
In embodiments, most sample assay actions or steps are performed by
a single device, and may be performed within a housing of a single
device. In embodiments, many sample assay actions or steps are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, sample processing, preparation,
or assay actions or steps may be performed by more than one
device.
[0073] An automatic assay device may be configured to perform a
plurality of assays on a sample. In embodiments, an automatic assay
device may be configured to perform a plurality of assays on a
single sample. In embodiments, an automatic assay device may be
configured to perform a plurality of assays on a single sample,
where the sample is a small sample. For example, a small sample may
have a sample volume that is a small volume of less than about 1000
.mu.L, or less than about 500 .mu.L, or less than about 250 .mu.L,
or less than about 150 .mu.L, or less than about 100 .mu.L, or less
than about 75 .mu.L, or less than about 50 .mu.L, or less than
about 40 .mu.L, or less than about 20 .mu.L, or less than about 10
.mu.L, or other small volume. An automatic assay device may be
capable of performing multiplexed assays on a single sample. A
plurality of assays may be run simultaneously; may be run
sequentially; or some assays may be run simultaneously while others
are run sequentially. One or more control assays and/or calibrators
(e.g., including a configuration with a control of a calibrator for
the assay/tests) can also be incorporated into the device; control
assays and assay on calibrators may be performed simultaneously
with assays performed on a sample, or may be performed before or
after assays performed on a sample, or any combination thereof. In
embodiments, all sample assay actions or steps are performed by a
single device. In embodiments, all of a plurality of assay actions
or steps are performed within a housing of a single device. In
embodiments, most sample assay actions or steps, of a plurality of
assays, are performed by a single device, and may be performed
within a housing of a single device. In embodiments, many sample
assay actions or steps, of a plurality of assays, are performed by
a single device, and may be performed within a housing of a single
device. In embodiments, sample processing, preparation, or assay
actions or steps may be performed by more than one device.
[0074] In embodiments, all of a plurality of assays may be
performed in a short time period. In embodiments, such a short time
period comprises less than about three hours, or less than about
two hours, or less than about one hour, or less than about 40
minutes, or less than about 30 minutes, or less than about 25
minutes, or less than about 20 minutes, or less than about 15
minutes, or less than about 10 minutes, or less than about 5
minutes, or less than about 4 minutes, or less than about 3
minutes, or less than about 2 minutes, or less than about 1 minute,
or other short time period.
[0075] An automatic assay device may perform nucleic acid assays,
including isothermal nucleic acid assays (e.g., assays for
detecting and measuring nucleic acid targets in a sample, including
DNA and RNA targets). In embodiments, an automatic assay device may
perform nucleic acid assays as disclosed in U.S. patent application
Ser. No. 14/183,503, filed Feb. 18, 2014; U.S. patent application
Ser. No. 14/214,850, filed Mar. 15, 2014; International Patent
Application PCT/US2014/030034, filed Mar. 15, 2014; and in
International Patent Application PCT/US2014/056151, filed Sep. 17,
2014. An automatic assay device may perform antibody assays,
including enzyme-linked immunosorbent assays (ELISA), and other
assays for detecting and measuring the amounts of proteins
(including antibodies), peptides, and small molecules in samples.
An automatic assay device may perform general chemistry assays,
including electrolyte assays (e.g., assays for detecting and
measuring the amounts of electrolytes such as sodium and potassium
in a sample).
[0076] An automatic assay device may be configured to detect one or
more signals relating to the sample. An automatic assay device may
be configured to identify one or more properties of the sample. For
instance, the automatic assay device may be configured to detect
the presence or concentration of one analyte or a plurality of
analytes or a disease condition in the sample (e.g., in or through
a bodily fluid, secretion, tissue, or other sample). Alternatively,
the automatic assay device may be configured to detect a signal or
signals that may be analyzed to detect the presence or
concentration of one or more analytes (which may be indicative of a
disease condition) or a disease condition in the sample. The
signals may be analyzed on board the device, or at another
location. Running a clinical test may or may not include any
analysis or comparison of data collected.
[0077] A chemical reaction or other processing step may be
performed, with or without the sample. Examples of steps, tests, or
assays that may be prepared or run by the device may include, but
are not limited to immunoassay, nucleic acid assay, receptor-based
assay, cytometric assay, colorimetric assay, enzymatic assay,
electrophoretic assay, electrochemical assay, spectroscopic assay,
chromatographic assay, microscopic assay, topographic assay,
calorimetric assay, turbidmetric assay, agglutination assay,
radioisotope assay, viscometric assay, coagulation assay, clotting
time assay, protein synthesis assay, histological assay, culture
assay, osmolarity assay, and/or other types of assays,
centrifugation, separation, filtration, dilution, enriching,
purification, precipitation, pulverization, incubation, pipetting,
transport, cell lysis, or other sample preparation action or steps,
or combinations thereof. Steps, tests, or assays that may be
prepared or run by the device may include imaging, including
microscopy, cytometry, and other techniques preparing or utilizing
images. Steps, tests, or assays that may be prepared or run by the
device may further include an assessment of histology, morphology,
kinematics, dynamics, and/or state of a sample, which may include
such assessment for cells.
[0078] A device may be capable of performing all on-board steps
(e.g., steps or actions performed by a single device) in a short
amount of time. A device may be capable of performing all on-board
steps on a single sample in a short amount of time. For example,
from sample collection from a subject to transmitting data and/or
to analysis may take about 3 hours or less, 2 hours or less, 1 hour
or less, 50 minutes or less, 45 minutes or less, 40 minutes or
less, 30 minutes or less, 20 minutes or less, 15 minutes or less,
10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes
or less, 2 minutes or less, or 1 minute or less. The amount of time
from accepting a sample within the device to transmitting data
and/or to analysis from the device regarding such a sample may
depend on the type or number of steps, tests, or assays performed
on the sample. The amount of time from accepting a sample within
the device to transmitting data and/or to analysis from the device
regarding such a sample may take about 3 hours or less, 2 hours or
less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40
minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes
or less, 10 minutes or less, 5 minutes or less, 4 minutes or less,
3 minutes or less, 2 minutes or less, or 1 minute or less.
[0079] A device may be configured to prepare a sample for disposal,
or to dispose of a sample, such as a biological sample, following
processing or assaying of a sample.
[0080] In embodiments, an automatic assay device may be configured
to transmit data obtained from a sample. In embodiments, an
automatic assay device may be configured to communicate over a
network. An automatic assay device may include a communication
module that may interface with the network. An automatic assay
device may be connected to the network via a wired connection or
wirelessly. The network may be a local area network (LAN) or a wide
area network (WAN) such as the Internet. In some embodiments, the
network may be a personal area network. The network may include the
cloud. The automatic assay device may be connected to the network
without requiring an intermediary device, or an intermediary device
may be required to connect an automatic assay device to a network.
An automatic assay device may communicate over a network with
another device, which may be any type of networked device,
including but not limited to a personal computer, server computer,
or laptop computer; personal digital assistants (PDAs) such as a
Windows CE device; phones such as cellular phones, smartphones
(e.g., iPhone, Android, Blackberry, etc.), or location-aware
portable phones (such as GPS); a roaming device, such as a
network-connected roaming device; a wireless device such as a
wireless email device or other device capable of communicating
wireless with a computer network; or any other type of network
device that may communicate possibly over a network and handle
electronic transactions. Such communication may include providing
data to a cloud computing infrastructure or any other type of data
storage infrastructure which may be accessed by other devices.
[0081] An automatic assay device may provide data regarding a
sample to, e.g., a health care professional, a health care
professional location, such as a laboratory, or an affiliate
thereof. One or more of a laboratory, health care professional, or
subject may have a network device able to receive or access data
provided by the automatic assay device. An automatic assay device
may be configured to provide data regarding a sample to a database.
An automatic assay device may be configured to provide data
regarding a sample to an electronic medical records system, to a
laboratory information system, to a laboratory automation system,
or other system or software. An automatic assay device may provide
data in the form of a report.
[0082] A laboratory, device, or other entity or software may
perform analysis on data regarding a sample in real-time. A
software system may perform chemical analysis and/or pathological
analysis, or these could be distributed amongst combinations of
lab, clinical, and specialty or expert personnel. Analysis may
include qualitative and/or quantitative evaluation of a sample.
Data analysis may include a subsequent qualitative and/or
quantitative evaluation of a sample. Optionally, a report may be
generated based on raw data, pre-processed data, or analyzed data.
Such a report may be prepared so as to maintain confidentiality of
the data obtained from the sample, the identity and other
information regarding the subject from whom a sample was obtained,
analysis of the data, and other confidential information. The
report and/or the data may be transmitted to a health care
professional. Data obtained by an automatic assay device, or
analysis of such data, or reports, may be provided to a database,
an electronic medical records system, to a laboratory information
system (LIS), to a laboratory automation system (LAS), or other
system or software.
[0083] Reagents
[0084] Applicant discloses reagents herein, where the reagents are
suitable for identifying the presence of ZIKA virus in a sample,
and are suitable for detecting ZIKA virus in a sample. Methods for
identifying the presence of ZIKA virus in a sample, and for
detecting ZIKA virus in a sample, include PCR nucleic acid
amplification methods. In embodiments, the PCR nucleic acid
amplification methods may be reverse transcription PCR methods. In
embodiments, the PCR nucleic acid amplification methods may be
real-time PCR methods. In embodiments, the PCR nucleic acid
amplification methods may be real-time reverse transcription PCR
methods.
[0085] A reagent for identifying the presence of ZIKA virus in a
sample, the reagent comprising a nucleic acid primer comprising a
nucleic acid sequence selected from the group SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6,
and a buffer. In embodiments, the nucleic acid primer comprises a
nucleic acid sequence selected from the group SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
[0086] A reagent for identifying the presence of ZIKA virus in a
sample, the reagent comprising a variant of a nucleic acid primer
comprising a nucleic acid sequence selected from the group SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and
SEQ ID NO: 6, and a buffer, wherein the variant has at least about
95% sequence identity to the nucleic acid sequence. In embodiments,
the a variant of a nucleic acid primer comprises a variant of a
nucleic acid sequence selected from the group SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein the variant has at
least about 95% sequence identity to the nucleic acid sequence.
[0087] The reagent for identifying the presence of a ZIKA virus in
a sample, wherein the buffer is selected from phosphate and TRIS.
In embodiments, the buffer is TRIS.
[0088] The reagent comprising a primer and a buffer, wherein the
primer comprises a reporter molecule.
[0089] The reagent comprising a primer and a buffer, wherein the
primer comprises a reporter molecule, and wherein the reporter
molecule comprises a fluorescent moiety, and the nucleic acid
primer further comprises a quenching moiety effective to quench
fluorescence from the fluorescent moiety when the primer is not
hybridized to a target nucleic acid sequence.
[0090] Kits
[0091] A kit for identifying the presence of ZIKA virus in a
sample, comprising a comprises a reagent including a nucleic acid
primer, the primer comprising a nucleic acid sequence selected from
the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, and SEQ ID NO: 6, and a buffer.
[0092] In embodiments, a kit may comprise a reagent including a
nucleic acid primer, the primer comprising a nucleic acid sequence
selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
and SEQ ID NO: 4.
[0093] In embodiments, a kit for identifying the presence of ZIKA
virus in a sample may include a reagent comprising a nucleic acid
primer, the primer comprising a variant of a nucleic acid sequence
selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and a buffer, wherein
the variant has at least about 95% sequence identity to the nucleic
acid sequence.
[0094] In embodiments, a kit for identifying the presence of ZIKA
virus in a sample may include a reagent comprising a nucleic acid
primer, the nucleic acid primer comprising a variant of a nucleic
acid sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, and SEQ ID NO: 4, and a buffer, wherein the variant
has at least about 95% sequence identity to the nucleic acid
sequence.
[0095] In embodiments, a kit for identifying the presence of ZIKA
virus in a sample may include a reagent including a nucleic acid
primer, wherein the nucleic acid primer comprises a reporter
molecule.
[0096] In embodiments, a kit for identifying the presence of ZIKA
virus in a sample may include a reagent including a primer with a
reporter molecule, wherein the reporter molecule comprises a
fluorescent moiety, and the nucleic acid primer further comprises a
quenching moiety effective to quench fluorescence from the
fluorescent moiety when the primer is not hybridized to a target
nucleic acid sequence.
[0097] In embodiments, a kit for identifying the presence of ZIKA
virus in a sample may include a reagent comprising a primer with a
reporter molecule, wherein the reporter molecule comprises a
fluorescent moiety, and the nucleic acid primer further comprises a
quenching moiety effective to quench fluorescence from the
fluorescent moiety when the primer is not hybridized to a target
nucleic acid sequence.
[0098] In the following, abbreviations and acronyms have their
standard meanings. For example, "mM" means millimolar; "uL" and
".mu.L" mean microliter; "PFU/uL" means plaque forming units per
uL; "mg/ml" means milligram per milliliter; "BSA" means bovine
serum albumin; "cp/uL" means copies per microliter; and "1E4" means
1.times.10.sup.4, the "E" indicating the exponent to the power of
ten.
[0099] A kit for performing rRT-PCR for the detection of ZIKV
contains primers and buffers. In embodiments, a kit includes the
primers, probes, and positive and sample processing controls for
use in PCR assays. In such an embodiment, the user needs to
purchase commercially available enzymes, negative control and
buffer.
[0100] In a first embodiments, a kit for detection of Zika contains
primers and buffers and lacks enzymes, negative control, and
buffer. Such a first embodiment of a kit for performing rRT-PCR for
the detection of ZIKV may contain:
[0101] A) Sample processing control--MS2 bacteriophage supplied at
100.times. (200 PFU/uL), used at 1.times. concentration (2 PFU/uL).
Formulation buffer for MS2: 10 mM Tris pH 7.5, 1 mM MgCl.sub.2, 100
mM NaCl, 0.1% gelatin, 2 mg/ml BSA. Volume=80 uL.
[0102] B) Zika virus positive control--Synthetic RNA target
designed via in vitro transcription of a g-block, supplied at 1E4
cp/uL, used at a final concentration of 4E3 cp/uL. Formulation
buffer for positive control: 10 mM Tris pH 8.0, 0.1 mM EDTA (IDTE,
IDT Cat No. 11-05-01-09), 1 unit/.mu.L RNase inhibitor (RI, NEB Cat
No. M0314S). Volume=250 uL per tube, 4 tubes provided.
[0103] C) Reagent A (Primer-probe mix)--comprises Zika primers
(forward and reverse), MS2 primers (forward and reverse), Zika
probe (FAM fluorophore, Dabcyl quencher), MS2 probe (SIMA-HEX
fluorophore, Dabcyl quencher). Formulation buffer for primers and
probes: 10 mM Tris pH 8.0, 0.1 mM EDTA (IDTE, IDT Cat No.
11-05-01-09) Volume=54 uL per tube, 4 tubes provided.
[0104] Reagent A includes Zika primers (forward and reverse) and
Zika probe:
[0105] RLX 4877 GACATGGCTTCGGACAG (SEQ ID NO: 1)
[0106] RLX4878 ATATTGAGTGTCTGATTGCTTG (SEQ ID NO: 2)
[0107] RLX4879 FAM TGCCCAACACAAGGTGAAGCC Dabcyl (SEQID NO: 3)
[0108] Reagent A includes MS2 probe (SIMA-HEX fluorophore, Dabcyl
quencher):
TABLE-US-00003 (SEQ ID NO: 4) RLX 4943 AACGAGTCATATGAATTTAGGC (SEQ
ID NO: 5) RLX 4944 GCAGCCCGATCTATTTTATTAT (SEQ ID NO: 6) RLX 5156
HEX AGGGAACGGAGTGTTTACAGTTCC Dabcyl
[0109] This kit works with venous serum, venous plasma, venous
whole blood and capillary whole blood matrices. This kit has been
tested and found to be suitable for use in PCR assays. This kit is
suitable for use on the following RT-PCR platforms: Roche
LightCycler.RTM. 480 Instrument II; Bio Rad CFX96 Touch.TM. Real
Time PCR Detection System; and ABI 7500 Fast Real Time RT PCR.
[0110] A suitable thermo-cycling program used for PCR for such a
kit is illustrated in the following table:
TABLE-US-00004 Temp Time Cycles 50.degree. C. 30 min 1 95.degree.
C. 15 min 1 95.degree. C. 15 sec 45 60.degree. C. 1 min*
[0111] Fluorescence is measured after each of the 45 cycles, as
indicated by the asterisk (which indicates when fluorescence
measurements are made).
[0112] In embodiments, an RT-PCR kit may include further
components, including, for example, enzymes and additional controls
(e.g., negative controls, sample processing controls, in addition
to positive controls as provided in the kit embodiments discussed
above).
[0113] In a second embodiment, an RT-PCR kit may comprise the
following basic components: Enzymes--reverse transcriptase (RT) and
DNA polymerase; Reaction buffer--containing MgCl.sub.2 and dNTPs
besides salt, buffer; Primers--DNA oligonucleotides; Probes--DNA
oligonucleotides with 5'-fluorophore and 3'-quencher; and
Controls--positive, negative, sample processing.
[0114] For example, such a second embodiment of a kit for
performing rRT-PCR for the detection of ZIKV may contain:
[0115] Enzymes: reverse transcriptase; for example the following
reverse transcriptase has the following amino acid sequence (SEQ ID
NO: 7):
TABLE-US-00005 MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI
IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVL
DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF
NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGN
LGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQ
LREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQ
ALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPRRRPVAYLSKKL
DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPD
RWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDIL
AEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWA
KALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYR
RRGLLTSKGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGN
RMADQAARKAAITETPDTSTLL.
[0116] A suitable DNA Taq polymerase for use in a second embodiment
of a kit as disclosed herein has the following amino acid sequence
(SEQ ID NO: 8):
TABLE-US-00006 MGHHHHHHHHHHSSGHIEGRASSSGHENLYFQSMGMRGMLPLFEPKGRVL
LVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVV
FDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPG
YEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITP
AWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLE
ALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPD
RERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKE
PMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGL
GLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFAN
LWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAE
EIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTS
AAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFN
QTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIE
LRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTI
NFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGR
RRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAM
VKLEPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAV
PLEVEVGIGEDWLSAKE.
[0117] A suitable formulation buffer for use in a second embodiment
of a kit as disclosed herein may be: 20 mM Tris pH 7.5, 300 mM KCl,
2 mM DTT, 0.1 mM EDTA, 50% Glycerol, 0.2% Triton X-100.
[0118] Thermal cycling parameters for PCR assays using reagents of
a second embodiment of a kit disclosed herein are presented in the
following table. It will be understood that the number of cycles
can be adjusted as desired; for example, the number of cycles may
be decreased from 60 to 45-50.
TABLE-US-00007 PCR cycle Temp Time Cycles 50.degree. C. 10 min
1.times. 95.degree. C. 1 min 1.times. 95.degree. C. 15 sec
60.times. 64.degree. C. 1 min*
[0119] The asterisk indicates that fluorescence may be measured
after this step.
[0120] A reagent for use in a second embodiment of a kit having
features as disclosed herein may be termed a "mastermix" and may
have a composition as described in the following table:
TABLE-US-00008 # of # of reactions reactions Units [Stock] [Final]
1 60 water 7.01 420.6 NEB 5X HF X 5 1 5 300 Phusion Buffer dNTP mM
10 0.2 0.5 30 BSA X 100 3.5 0.88 52.5 MS2 Forward uM 150 1 0.17 10
primer MS2 Reverse uM 150 1 0.17 10 primer MS2 probe uM 25 0.3 0.3
18 Zika Forward uM 150 1 0.17 10 primer Zika Reverse uM 150 1 0.17
10 primer Zika probe uM 25 0.3 0.3 18 RT RDP205 mg/ 0.1 0.0005 0.13
7.5 mL His-tagged Taq mg/ 0.25 0.002 0.2 12 (RDP282B) mL template
10 600 total 25 1500 Total MM (no template) 15 900
[0121] The buffer indicated in the table above is Phusion.RTM. HF
Buffer (NEB, catalog # B0518S) used after five-fold dilution.
[0122] A second embodiment of a kit having features as disclosed
herein may include dNTPs, which may be provided in solution, or may
be provided in dry (powdered) form.
[0123] A second embodiment of a kit having features as disclosed
herein may include BSA, for example, BSA for use following 100-fold
dilution. BSA may be provided in, and/or may be diluted in, a
formulation buffer, such as the following formulation buffer: 20 mM
KPO.sub.4 pH 7.0, 50 mM NaCl, 0.1 mM EDTA, 5% Glycerol.
[0124] Kits as disclosed herein may be stored at -20.degree. C. In
embodiments, kits as disclosed herein may be stored at other
temperatures, such as, e.g., at 0-4.degree. C.; and at room
temperature.
Example
[0125] Thermal cycling protocols for polymerase chain reactions for
nucleic acid amplification may include, for example: about 5 to 20
minutes at "low temperature" such as a temperature of between about
45.degree. C. and about 55.degree. C., followed by about 1-15
minute at "high temperature" such as a temperature of between about
80.degree. C. to about 95.degree. C., followed by about 20 to 100
thermal cycles, where each thermal cycle consists of about 10 to
120 seconds at "high temperature" then about 1 to 15 minutes at
"low temperature". In some PCR methods, an "intermediate
temperature" of between about 60.degree. C. and about 74.degree. C.
may be applied, e.g., between applications of "low temperature" and
applications of "high temperature".
[0126] FIG. 1 shows components of a kit having features disclosed
herein (e.g., a Zika RT-PCR kit). The box with labeling on the
outside is shown in a closed configuration (on the right). The box
with reagent vials is shown in an open configuration (central box
in the figure) with the lid open and showing reagent vials in place
in receptacles within the box. The contents of the reagent vials
provide an integrated set of reagents to perform the assay. The kit
may also include capillary sample collection devices and a shipment
container. Shown in FIG. 1B are an open shipment container for
transporting the samples stably, with each sample held in a
Nanotainer.TM. (the left-most box in the figure), and three sample
collection devices (shown in the center foreground of the
figure).
[0127] FIG. 2 provides results of a RT-PCR assays performed using
reagents and using automated sample analysis devices and systems as
disclosed herein.
[0128] The primers used in the nucleic acid amplification Zika
assay were designed from a consensus of a multi-sequence alignment
of all Zika strains deposited in GenBank. The gene target we
selected is a 100-base pair region within the highly conserved
polyprotein gene.
[0129] FIG. 3 presents inflection time measurements (average of
four replicates) showing the average inflection time on the
vertical axis plotted against the number of copies of the nucleic
acid target sequences in the sample that was analyzed.
[0130] An example of a suitable thermal cycling protocol useful for
the polymerase chain reaction portion of the rRT-PCR amplification
is: 10 minutes at 50.degree. C., followed by 1 minute at 95.degree.
C., followed by 60 thermal cycles; each thermal cycle consisted of
15 seconds at 95.degree. C. then 1 minute at 64.degree. C.
[0131] For further example, the following thermal cycling protocol
was used for the polymerase chain reaction portion of the rRT-PCR
amplification: 30 minutes at 50.degree. C., followed by 15 minutes
at 95.degree. C., followed by 45 thermal cycles; each thermal cycle
consisted of 15 seconds at 95.degree. C. then 1 minute at
60.degree. C. Following the last thermal cycle (after the final 1
minute at 60.degree. C.), fluorescence measurements may be
taken.
[0132] Reagents suitable for use in PCR amplification methods
include:
TABLE-US-00009 TABLE 2 RT-PCR formulation # of # of RT-PCR
reactions reactions Master Mix Units [Stock] [Final] 1 60 water
7.62 457.00 NEB 5X HF X 5 1 5.00 300.00 Phusion Buffer dNTP mM 10
0.2 0.50 30.00 BSA X 100 3.5 0.88 52.50 Zika F RLX4877 uM 150 1
0.17 10.00 Zika R RLX4878 uM 150 1 0.17 10.00 Zika probe uM 25 0.3
0.30 18.00 RLX4879 RT RDP205 mg/mL 0.1 0.0005 0.13 7.50 His-tagged
Taq mg/mL 0.25 0.0025 0.25 15.00 (RDP282) template 10 600 total 25
1500 Total MM 15.00 900.00 (no template)
[0133] 5.times. Phusion buffer (HF & CG, ("high fidelity" and
CG-rich) from New England BioLabs (NEB) contains 7.5 mM MgCl2 (1.5
mM at 1.times. (i.e., no) dilution).
[0134] Primer sequences used to identify ZIKV in a sample were:
TABLE-US-00010 TABLE 3 RLX 4877 GACATGGCTTCGGACAG SEQ ID NO: 1
RLX4878 ATATTGAGTGTCTGATTGCTTG SEQ ID NO: 2 RLX4879 FAM
TGCCCAACACAAGGTGAAGCC SEQ ID NO: 3 Dabcyl
[0135] Primer sequences used to identify MS2 in a sample were:
TABLE-US-00011 TABLE 4 RLX 4943 AACGAGTCATATGAATTTAGGC SEQ ID NO: 4
RLX 4944 GCAGCCCGATCTATTTTATTAT SEQ ID NO: 5 RLX 5156 HEX
AGGGAACGGAGTGTTTACAGTTCC SEQ ID NO: 6 Dabcyl
[0136] As shown, FIG. 2 shows the results of a RT-PCR amplification
of Zika virus, as relative fluorescence units (RFU) plotted against
cycles (thermal cycles).
[0137] FIG. 2 is a plot of fluorescence (a measure of the numbers
of cDNA copies of the target nucleic acid sequence) versus time (as
cycles) showing the results of rRT-PCR amplification of Zika
virus.
[0138] The average Cq was calculated from four Cq measurements for
different numbers of copies per reaction. These results are
presented in the following table (Table 5):
TABLE-US-00012 TABLE 5 T276A1 c/rxn in neg. In-house RT-PCR
formulation sample prep Cq1 Cq2 Cq3 Cq4 Cq(avg) Stdev 4,000 30.72
30.25 30.19 30.39 30.39 0.24 100 36.31 35.95 34.97 35.74 35.74 0.57
50 36.29 36.32 37.14 36.10 36.46 0.46 25 38.15 36.77 38.65 37.68
37.81 0.80 10 39.37 40.13 38.69 38.50 39.17 0.74 7.5 39.44 60.00
39.29 40.16 44.72 10.19 5 40.80 40.35 40.25 60.00 45.35 9.77 0
60.00 60.00 60.00 60.00 60.00 0.00
[0139] FIG. 3 is a graph showing the average numbers of copies
resulting from rRT-PCR amplification of Zika virus as a function of
copies per reaction.
[0140] As shown, FIG. 3 shows average Cq versus copies per
reaction.
[0141] While the invention has been described and illustrated with
reference to certain particular embodiments thereof, those skilled
in the art will appreciate that various adaptations, changes,
modifications, substitutions, deletions, or additions of procedures
and protocols may be made without departing from the spirit and
scope of the invention.
[0142] The publications discussed or cited herein are provided
solely for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed. All publications and patent
applications mentioned herein are incorporated herein by reference
to disclose and describe the structures and/or methods in
connection with which the publications and/or patent applications
are cited.
Sequence CWU 1
1
8117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gacatggctt cggacag 17222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2atattgagtg tctgattgct tg 22321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 3tgcccaacac aaggtgaagc c
21422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4aacgagtcat atgaatttag gc 22522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5gcagcccgat ctattttatt at 22624DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 6agggaacgga gtgtttacag ttcc
247672PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu
His Glu Thr Ser Lys1 5 10 15Glu Pro Asp Val Ser Leu Gly Ser Thr Trp
Leu Ser Asp Phe Pro Gln 20 25 30Ala Trp Ala Glu Thr Gly Gly Met Gly
Leu Ala Val Arg Gln Ala Pro 35 40 45Leu Ile Ile Pro Leu Lys Ala Thr
Ser Thr Pro Val Ser Ile Lys Gln 50 55 60Tyr Pro Met Ser Gln Glu Ala
Arg Leu Gly Ile Lys Pro His Ile Gln65 70 75 80Arg Leu Leu Asp Gln
Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn 85 90 95Thr Pro Leu Leu
Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro 100 105 110Val Gln
Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro 115 120
125Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Pro Pro Pro Ser His
130 135 140Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys
Leu Arg145 150 155 160Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe
Glu Trp Arg Asp Pro 165 170 175Glu Met Gly Ile Ser Gly Gln Leu Thr
Trp Thr Arg Leu Pro Gln Gly 180 185 190Phe Lys Asn Ser Pro Thr Leu
Phe Asn Glu Ala Leu His Arg Asp Leu 195 200 205Ala Asp Phe Arg Ile
Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val 210 215 220Asp Asp Leu
Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly225 230 235
240Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu
Gly Tyr 260 265 270Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala
Arg Lys Glu Thr 275 280 285Val Met Gly Gln Pro Thr Pro Lys Thr Pro
Arg Gln Leu Arg Glu Phe 290 295 300Leu Gly Thr Ala Gly Phe Cys Arg
Leu Trp Ile Pro Gly Phe Ala Glu305 310 315 320Met Ala Ala Pro Leu
Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn 325 330 335Trp Gly Pro
Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu 340 345 350Leu
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu 355 360
365Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380Lys Leu Gly Pro Arg Arg Arg Pro Val Ala Tyr Leu Ser Lys
Lys Leu385 390 395 400Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu
Arg Met Val Ala Ala 405 410 415Ile Ala Val Leu Thr Lys Asp Ala Gly
Lys Leu Thr Met Gly Gln Pro 420 425 430Leu Val Ile Leu Ala Pro His
Ala Val Glu Ala Leu Val Lys Gln Pro 435 440 445Pro Asp Arg Trp Leu
Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu 450 455 460Leu Leu Asp
Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn465 470 475
480Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu
Thr Asp 500 505 510Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr
Asp Gly Ser Ser 515 520 525Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly
Ala Ala Val Thr Thr Glu 530 535 540Thr Glu Val Ile Trp Ala Lys Ala
Leu Pro Ala Gly Thr Ser Ala Gln545 550 555 560Arg Ala Glu Leu Ile
Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly 565 570 575Lys Lys Leu
Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala 580 585 590His
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Lys 595 600
605Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly
His Gln625 630 635 640Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg
Met Ala Asp Gln Ala 645 650 655Ala Arg Lys Ala Ala Ile Thr Glu Thr
Pro Asp Thr Ser Thr Leu Leu 660 665 6708867PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Met Gly His His His His His His His His His His Ser Ser Gly His1 5
10 15Ile Glu Gly Arg Ala Ser Ser Ser Gly His Glu Asn Leu Tyr Phe
Gln 20 25 30Ser Met Gly Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys
Gly Arg 35 40 45Val Leu Leu Val Asp Gly His His Leu Ala Tyr Arg Thr
Phe His Ala 50 55 60Leu Lys Gly Leu Thr Thr Ser Arg Gly Glu Pro Val
Gln Ala Val Tyr65 70 75 80Gly Phe Ala Lys Ser Leu Leu Lys Ala Leu
Lys Glu Asp Gly Asp Ala 85 90 95Val Ile Val Val Phe Asp Ala Lys Ala
Pro Ser Phe Arg His Glu Ala 100 105 110Tyr Gly Gly Tyr Lys Ala Gly
Arg Ala Pro Thr Pro Glu Asp Phe Pro 115 120 125Arg Gln Leu Ala Leu
Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala 130 135 140Arg Leu Glu
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu145 150 155
160Ala Lys Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala
165 170 175Asp Lys Asp Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val
Leu His 180 185 190Pro Glu Gly Tyr Leu Ile Thr Pro Ala Trp Leu Trp
Glu Lys Tyr Gly 195 200 205Leu Arg Pro Asp Gln Trp Ala Asp Tyr Arg
Ala Leu Thr Gly Asp Glu 210 215 220Ser Asp Asn Leu Pro Gly Val Lys
Gly Ile Gly Glu Lys Thr Ala Arg225 230 235 240Lys Leu Leu Glu Glu
Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu 245 250 255Asp Arg Leu
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp 260 265 270Asp
Leu Lys Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro 275 280
285Leu Glu Val Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu
290 295 300Arg Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His
Glu Phe305 310 315 320Gly Leu Leu Glu Ser Pro Lys Ala Leu Glu Glu
Ala Pro Trp Pro Pro 325 330 335Pro Glu Gly Ala Phe Val Gly Phe Val
Leu Ser Arg Lys Glu Pro Met 340 345 350Trp Ala Asp Leu Leu Ala Leu
Ala Ala Ala Arg Gly Gly Arg Val His 355 360 365Arg Ala Pro Glu Pro
Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg 370 375 380Gly Leu Leu
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu385 390 395
400Gly Leu Pro Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp
405 410 415Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly
Gly Glu 420 425 430Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser
Glu Arg Leu Phe 435 440 445Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu
Glu Arg Leu Leu Trp Leu 450 455 460Tyr Arg Glu Val Glu Arg Pro Leu
Ser Ala Val Leu Ala His Met Glu465 470 475 480Ala Thr Gly Val Arg
Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu 485 490 495Glu Val Ala
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu 500 505 510Ala
Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val 515 520
525Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr
530 535 540Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg
Glu Ala545 550 555 560His Pro Ile Val Glu Lys Ile Leu Gln Tyr Arg
Glu Leu Thr Lys Leu 565 570 575Lys Ser Thr Tyr Ile Asp Pro Leu Pro
Asp Leu Ile His Pro Arg Thr 580 585 590Gly Arg Leu His Thr Arg Phe
Asn Gln Thr Ala Thr Ala Thr Gly Arg 595 600 605Leu Ser Ser Ser Asp
Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro 610 615 620Leu Gly Gln
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu625 630 635
640Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His
645 650 655Leu Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly
Arg Asp 660 665 670Ile His Thr Glu Thr Ala Ser Trp Met Phe Gly Val
Pro Arg Glu Ala 675 680 685Val Asp Pro Leu Met Arg Arg Ala Ala Lys
Thr Ile Asn Phe Gly Val 690 695 700Leu Tyr Gly Met Ser Ala His Arg
Leu Ser Gln Glu Leu Ala Ile Pro705 710 715 720Tyr Glu Glu Ala Gln
Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro 725 730 735Lys Val Arg
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg 740 745 750Gly
Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu 755 760
765Glu Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe
770 775 780Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu
Ala Met785 790 795 800Val Lys Leu Phe Pro Arg Leu Glu Glu Met Gly
Ala Arg Met Leu Leu 805 810 815Gln Val His Asp Glu Leu Val Leu Glu
Ala Pro Lys Glu Arg Ala Glu 820 825 830Ala Val Ala Arg Leu Ala Lys
Glu Val Met Glu Gly Val Tyr Pro Leu 835 840 845Ala Val Pro Leu Glu
Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser 850 855 860Ala Lys
Glu865
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