U.S. patent application number 17/576970 was filed with the patent office on 2022-07-21 for loop-mediated isothermal amplification (lamp) analysis for pathogenic targets.
The applicant listed for this patent is Purdue Research Foundation, Raytheon BBN Technologies, Corp.. Invention is credited to Andres Dextre, Murali Kannan Maruthamuthu, Darby McChesney, Suraj Mohan, Ana Pascual-Garrigos, Jordan Seville, Mohit Verma, Jiangshan Wang.
Application Number | 20220228226 17/576970 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220228226 |
Kind Code |
A1 |
Seville; Jordan ; et
al. |
July 21, 2022 |
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ANALYSIS FOR
PATHOGENIC TARGETS
Abstract
The present disclosure is drawn to methods of preparing a saliva
sample for loop-mediated isothermal amplification (LAMP) detection
of a pathogen target. In some embodiments, such methods can include
providing an amount of saliva from a test subject, and diluting the
saliva in water to a degree that reduces a buffering capacity of
the saliva while maintaining a sufficient concentration to allow
for detection of the pathogen target.
Inventors: |
Seville; Jordan; (Delran,
NJ) ; McChesney; Darby; (Lawrenceville, NJ) ;
Wang; Jiangshan; (West Layfayette, IN) ;
Maruthamuthu; Murali Kannan; (Layfayette, IN) ;
Dextre; Andres; (West Lafayette, IN) ;
Pascual-Garrigos; Ana; (West Lafayette, IN) ; Mohan;
Suraj; (West Lafayette, IN) ; Verma; Mohit;
(West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation
Raytheon BBN Technologies, Corp. |
West Lafayette
Cambridge |
IN
IN |
US
US |
|
|
Appl. No.: |
17/576970 |
Filed: |
January 16, 2022 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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63138310 |
Jan 15, 2021 |
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63138312 |
Jan 15, 2021 |
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63138314 |
Jan 15, 2021 |
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63138316 |
Jan 15, 2021 |
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63138318 |
Jan 15, 2021 |
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63138320 |
Jan 15, 2021 |
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63138321 |
Jan 15, 2021 |
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63138323 |
Jan 15, 2021 |
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63138337 |
Jan 15, 2021 |
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63138341 |
Jan 15, 2021 |
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63148527 |
Feb 11, 2021 |
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International
Class: |
C12Q 1/6888 20060101
C12Q001/6888; C12Q 1/70 20060101 C12Q001/70; C12Q 1/6806 20060101
C12Q001/6806 |
Claims
1. A method of preparing a saliva sample for loop-mediated
isothermal amplification (LAMP) detection of a pathogen target,
comprising: providing an amount of saliva from a test subject; and
diluting the saliva in water to a degree that reduces a buffering
capacity of the saliva while maintaining a sufficient concentration
to allow for detection of the pathogen target.
2. The method of claim 1, further comprising: reducing a viscosity
of the saliva as compared to an original viscosity.
3. The method of claim 2, wherein the viscosity is reduced by one
or more of dilution, filtering, or combinations thereof.
4. The method of claim 2, wherein the viscosity is reduced using
filtering.
5. The method of claim 4, wherein the viscosity is reduced using a
10 micron filter.
6. The method of claim 2, wherein the viscosity is reduced to a
degree that increases flowability through a solid phase medium as
compared to an original viscosity.
7. The method of claim 2, wherein the viscosity is reduced to a
range of from about 1.0 cP to about 50 cP.
8. The method of claim 1, further comprising: filtering the saliva
sample to a degree that adjusts a saliva sample pH to a test sample
target range.
9. The method of claim 8, wherein the test sample target range is
from about 7.2 to about 8.6.
10. The method of claim 1, wherein the saliva is diluted in the
water to a saliva to water ratio of about 1:1 to about 1:20.
11. The method of claim 1, wherein the saliva is diluted in the
water to a degree that provides the sample with an optical density
at 600 nm (OD.sub.600) of less than 0.2.
12. The method of claim 1, wherein the water has a pH greater than
6.0 and is substantially free of contaminants.
13. The method of claim 1, wherein the saliva sample consists
essentially of saliva and water.
14. The method of claim 1, wherein the saliva has a volume of from
about 50 .mu.l to about 100 .mu.l.
15. The method of claim 14, wherein the saliva sample has a volume
of from about 100 .mu.l to about 1 ml.
16. The method of claim 1, wherein the saliva is collected using
sponge-based collection.
17. The method of claim 1, wherein the pathogen target comprises a
viral pathogen, a bacterial pathogen, a fungal pathogen, or a
protozoa pathogen.
18. The method of claim 1, wherein the pathogen target is a viral
target.
19. The method of claim 18, wherein the viral target comprises a
dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA
virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a
ds-DNA-RT virus.
20. The method of claim 18, wherein the viral target comprises
H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.
21. The method of claim 1, wherein the LAMP detection comprises
reverse transcription LAMP (RT-LAMP) detection.
22. A test sample composition for loop-mediated isothermal
amplification (LAMP) analysis, comprising: an amount of a test
subject's saliva that is sufficient to detect a pathogen target via
a LAMP analysis in combination with an amount of water that reduces
a buffering capacity of the saliva.
23. The composition of claim 22, wherein the composition has a
viscosity of from about 1.0 cP to about 50 cP.
24. The composition of claim 22, wherein the composition has a pH
of from about 7.2 to about 8.6.
25. The composition of claim 22, wherein the composition has a
saliva to water ratio of about 1:1 to about 1:20.
26. The composition of claim 22, wherein the composition has an
optical density at 600 nm (OD.sub.600) of less than 0.2.
27. The composition of claim 22, wherein the water has a pH greater
than 6.0 and is substantially free of contaminants.
28. The composition of claim 22, wherein the composition consists
essentially of saliva and water.
29. The composition of claim 22, wherein the saliva has a volume
ranging from about 50 .mu.1 to about 100 .mu.1.
30. The composition of claim 22, wherein the saliva sample has a
volume of from about 100 .mu.l to about 1 ml.
31. The composition of claim 22, wherein the pathogen target
comprises a viral pathogen, a bacterial pathogen, a fungal
pathogen, or a protozoa pathogen.
32. The composition of claim 22, wherein the pathogen target is a
viral target.
33. The composition of claim 32, wherein the viral target comprises
a dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand
ssRNA virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a
ds-DNA-RT virus.
34. The composition of claim 32, wherein the viral target comprises
H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.
35. The composition of claim 22, wherein the buffering capacity of
the composition is less than 5 mM.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 63/138,310 filed Jan. 15, 2021, U.S.
Provisional Patent Application Ser. No. 63/138,312 filed Jan. 15,
2021, U.S. Provisional Patent Application Ser. No. 63/138,314 filed
Jan. 15, 2021, U.S. Provisional Patent Application Ser. No.
63/138,316 filed Jan. 15, 2021, U.S. Provisional Patent Application
Ser. No. 63/138,318 filed Jan. 15, 2021, U.S. Provisional Patent
Application Ser. No. 63/138,320 filed Jan. 15, 2021, U.S.
Provisional Patent Application Ser. No. 63/138,321 filed Jan. 15,
2021, U.S. Provisional Patent Application Ser. No. 63/138,323 filed
Jan. 15, 2021, U.S. Provisional Patent Application Ser. No.
63/138,337 filed Jan. 15, 2021, U.S. Provisional Patent Application
Ser. No. 63/138,341 filed Jan. 15, 2021, U.S. Provisional Patent
Application Ser. No. 63/148,527 filed Feb. 11, 2021, the entire
contents of each of which are incorporated herein by reference.
BACKGROUND
[0002] Polymerase chain reaction (PCR) is a molecular biology
technique that allows amplification of nucleotides for various
analytical purposes. Quantitative PCR (qPCR) is an adaptation of
PCR which allows monitoring of the amplification of a targeted
nucleotide. Diagnostic qPCR has been applied to detect nucleotides
that are indicative of infectious diseases, cancer, and genetic
abnormalities. Reverse transcription PCR (RT-PCR) is an adaptation
of qPCR which allows detection of a target RNA nucleotides. Because
of this ability, RT-PCR is well-suited for detecting virus
pathogens. However, RT-PCR uses sizeable equipment which may not be
available in certain point of care settings. Additionally, RT-PCR
uses trained personnel, significant sample preparation, and time to
perform and obtain results.
[0003] By contrast, Loop-Mediated Isothermal Amplification (LAMP)
is a more simplistic approach to diagnostic identification of
target nucleotides. In particular, LAMP is a one-operation nucleic
acid amplification method to multiply specific nucleotide
sequences. In addition to use of an isothermal heating process,
LAMP can use a simple visual output test indicator, such as a color
change rather than a more complicated fluorescent indicator used by
PCR. Reverse-transcription LAMP (RT-LAMP) can be used like RT-PCR
in order to identify target nucleotides from RNA, and as such, can
be used in a diagnostic capacity to identify the presence or
absence of viral pathogens. Because LAMP is more simplistic, it can
be performed with less equipment and sample preparation and
therefore is more accessible for use in point of care settings,
such as clinics, emergency rooms, and even on a mobile basis.
SUMMARY
[0004] The present disclosure is drawn to technology (e.g.,
compositions, methods, systems, and assemblies) for use in
detecting a target nucleotide using a LAMP analysis. In some
aspects, the target nucleotide can be known to reside in a pathogen
of interest. In cases where the pathogen is a virus, the LAMP
analysis can be an RT-LAMP analysis.
[0005] In some disclosure embodiments methods of preparing saliva
samples for loop-mediated isothermal amplification (LAMP) detection
of a pathogen target are provided. In one aspect, such a method can
include providing an amount of saliva from a test subject, and
diluting the saliva in water to a degree that reduces a buffering
capacity of the saliva while maintaining a sufficient concentration
to allow for detection of the pathogen target.
[0006] In one aspect, the method can include reducing a viscosity
of the saliva as compared to an original viscosity. In another
aspect, the viscosity can be reduced by one or more of dilution,
filtering, or combinations thereof. In another aspect, the
viscosity can be reduced using filtering. In a further aspect, the
viscosity can be reduced using a 10-micron filter. In yet another
aspect, the viscosity can be reduced to a degree that increases
flowability through a solid phase medium as compared to an original
viscosity. In yet another aspect, the viscosity can be reduced to a
range of from about 1.0 centipoise (cP) to about 50 cP.
[0007] In one aspect, such a method can include filtering the
saliva sample to a degree that adjusts a saliva sample pH to a test
sample target range. In another aspect, the test sample target
range can be from about 7.2 to about 8.6. In another aspect, the
water can have a pH greater than 6.0 and can be substantially free
of contaminants. In yet another aspect, the saliva sample can
consist essentially of saliva and water. In yet a further aspect,
the saliva can be collected using sponge-based collection.
[0008] In one aspect, the saliva can be diluted in the water to a
saliva to water ratio of about 1:1 to about 1:20. In another
aspect, the saliva can be diluted in the water to a degree that
provides the sample with an optical density at 600 nm (OD.sub.600)
of less than 0.2. In a further aspect, the saliva has a volume from
about 50 .mu.l to about 100 .mu.l. In yet another aspect, the
saliva sample has a volume ranging from about 100 .mu.l to about 1
ml.
[0009] In an additional aspect, the pathogen target can comprise a
viral pathogen, a bacterial pathogen, a fungal pathogen, or a
protozoa pathogen. In one aspect, the pathogen target can be a
viral target. In another aspect, the viral target can comprise a
dsDNA virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA
virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a
ds-DNA-RT virus. In yet another aspect, the viral target can be
H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.
[0010] In some aspects, the LAMP detection can comprise reverse
transcription LAMP (RT-LAMP) detection.
[0011] In other disclosure embodiments, test sample compositions
for LAMP analysis are disclosed and can include: an amount of a
test subject's saliva that is sufficient to detect a pathogen
target via a LAMP analysis in combination with an amount of water
that reduces a buffering capacity of the saliva.
[0012] In one aspect, the composition can have a viscosity of from
about 1.0 cP to about 50 cP. In another aspect, the composition can
have a pH of from about 7.2 to about 8.6. In another aspect, the
composition can have a saliva to water ratio of about 1:1 to about
1:20. In yet another aspect, the composition can have an optical
density at 600 nm (OD.sub.600) of less than 0.2. In another aspect,
the water can have a pH greater than 6.0 and can be substantially
free of contaminants. In one aspect, the composition can consist
essentially of saliva and water. In another aspect, the saliva can
have a volume ranging from about 50 .mu.l to about 100 .mu.L In yet
another aspect, the saliva sample can have a volume of from about
100 .mu.l to about 1 ml.
[0013] In one aspect, the pathogen target can comprise a viral
pathogen, a bacterial pathogen, a fungal pathogen, or a protozoa
pathogen. In another aspect, the pathogen target can be a viral
target. In another aspect, the viral target can comprise a dsDNA
virus, an ssDNA virus, a dsRNA virus, a positive-strand ssRNA
virus, a negative-strand ssRNA virus, an ssRNA-RT virus, or a
ds-DNA-RT virus. In yet another aspect, the viral target can
comprise H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2. In yet another
aspect, the buffering capacity of the composition can be less than
5 mM.
[0014] In yet other disclosure embodiments, compositions for LAMP
analysis on a solid phase medium can include one or more target
primers, a DNA polymerase, and a re-solubilization agent. In some
aspects, such a composition can be substantially free of non-pH
sensitive agents capable of discoloring the solid phase medium. In
one aspect, the composition can include an antioxidant. In another
aspect, the composition can be substantially free of volatile
agents. In yet another aspect, the composition can be substantially
free of hygroscopic agents. In one other aspect, the composition
can further include reverse transcriptase.
[0015] In one aspect, the hygroscopic agents can absorb more than
about 10 wt % when between about 40% and about 90% relative
humidity (RH) at 25.degree. C. In another aspect, the hygroscopic
agents can include glycerol, ethanol, methanol, calcium chloride,
potassium chloride, calcium sulfate, and combinations thereof.
[0016] In another aspect, the re-solubilization agent can be a
surfactant. In another aspect, the re-solubilization agent can
comprise bovine serum albumin (BSA), casein, polysorbate 20, or
combinations thereof.
[0017] In one aspect, the target primers can target a pathogen that
can comprise a viral pathogen, a bacterial pathogen, a fungal
pathogen, or a protozoa pathogen. In one aspect, the pathogen can
be a viral pathogen. In another aspect, the viral pathogen can
comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a
positive-strand ssRNA virus, a negative-strand ssRNA virus, an
ssRNA-RT virus, or a ds-DNA-RT virus. In another aspect, the viral
pathogen can comprise H1N1, H2N2, H3N2, H1N1pdm09, or
SARS-CoV-2.
[0018] In one aspect, the composition can further comprise a
non-discoloration additive. The non-discoloration additive can
comprise one or more of a sugar, a buffer, or combinations thereof.
In another aspect, the composition can further comprise an
indicator.
[0019] In other disclosure embodiments, a method for LAMP analysis
on a solid phase medium can include providing an assembly of a
solid phase medium and a composition as recited herein, depositing
a biological sample onto the solid phase medium, and heating the
assembly to an isothermal temperature sufficient to facilitate a
LAMP reaction. In one aspect, the biological sample can be one or
more of saliva, mucus, blood, urine, feces, sweat, exhaled breath
condensate, or combinations thereof. In another aspect, the
biological sample is saliva. In one aspect, the LAMP analysis can
be reverse transcriptase LAMP (RT-LAMP). In another aspect, the
method can further comprise detecting a viral pathogen.
[0020] In other disclosure embodiments, a system for performing the
LAMP analysis can comprise a composition as recited herein, and a
solid phase medium on to which the composition is deposited.
[0021] In yet further disclosure embodiments, compositions for
loop-mediated isothermal amplification (LAMP) analysis can utilize
a pH-dependent output signal that can include a pH sensitive dye,
and a plurality of non-interfering LAMP reagents. In one aspect,
the LAMP analysis can be RT-LAMP.
[0022] In one aspect, the pH sensitive dye can be at least one of
phenol red, phenolphthalein, azolitmin, bromothymol blue,
naphtholphthalein, cresol red, or combinations thereof In another
aspect, the plurality of non-interfering LAMP reagents can be
substantially free of volatile reagents, pH-interfering reagents,
magnesium-interfering reagents, or combinations thereof.
[0023] In one aspect, the plurality of non-interfering LAMP
reagents can be substantially free of magnesium, ammonium sulfate,
and ammonium carbonate. In aspect, the plurality of non-interfering
LAMP reagents can comprise DNA polymerase, reverse transcriptase,
target primers, or combinations thereof.
[0024] In another aspect, the composition can comprise an
antioxidant. In another aspect, the composition can further
comprise carrier RNA, carrier DNA, RNase inhibitors, DNase
inhibitors, guanidine hydrochloride, or combinations thereof. In
one aspect, the composition can further comprise a solid phase
medium.
[0025] In one aspect, the composition can comprise a
non-discoloration additive that can comprise a sugar, a buffer, a
blocking agent, or combinations thereof. In one aspect, the sugar
can comprise one or more of trehalose, glucose, sucrose, or
combinations thereof. In another aspect, the blocking agent can
comprise bovine serum albumin, casein, or combinations thereof.
[0026] In other disclosure embodiments, methods of performing a
LAMP analysis with a pH-dependent output signal are provided and
can include providing an assembly of a solid phase medium and a
composition as recited herein, depositing a biological sample onto
the solid phase medium, and heating the assembly to an isothermal
temperature sufficient to facilitate a LAMP reaction. In one
aspect, the LAMP analysis can be RT-LAMP. In one aspect, the
biological sample can be one or more of saliva, mucus, blood,
urine, feces, sweat, exhaled breath condensate, and combinations
thereof. In one aspect, the biological sample can be saliva. In
another aspect, the method can further comprise detecting a viral
pathogen.
[0027] In further disclosure embodiments, methods of maximizing
accuracy of an output signal in a pH-dependent LAMP analysis can
comprise providing a reagent mixture that minimizes non-LAMP
reaction produced discoloration from a signal output medium, and
performing the LAMP reaction. In one aspect, the method can
comprise controlling production of protons from a non-LAMP
reaction. In another aspect, the method can comprise controlling
oxidation from a non-LAMP reaction.
[0028] In other disclosure embodiments, a method of maximizing
accuracy of an output signal in a pH-dependent LAMP analysis can
comprise substantially eliminating non-LAMP reaction produced
discoloration from a signal output medium.
[0029] In other disclosure embodiments, methods of maximizing a
limit of detection (LOD) in a pH-dependent LAMP analysis can
include substantially eliminating non-LAMP reaction produced
discoloration from a signal output medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Features and advantages of the disclosure will be apparent
from the detailed description which follows, taken in conjunction
with the accompanying drawings, which together illustrate, by way
of example, features of the disclosure; and, wherein:
[0031] FIG. 1 depicts a method of preparing a saliva sample for
loop-mediated isothermal amplification (LAMP) detection of a
pathogen target in accordance with an example embodiment;
[0032] FIG. 2 depicts a method for LAMP analysis in accordance with
an example embodiment;
[0033] FIG. 3 depicts a method of maximizing accuracy of an output
signal in a pH-dependent LAMP analysis in accordance with an
example embodiment;
[0034] FIG. 4 illustrates that loop-mediate isothermal
amplification (LAMP) can be obtained in a saliva sample in
accordance with an example embodiment;
[0035] FIG. 5A illustrates a sponge-based collection device in
accordance with an example embodiment;
[0036] FIG. 5B illustrates a passive drool collection device in
accordance with an example embodiment;
[0037] FIG. 6A illustrates the limit of detection for various
concentrations of sample and various collection devices in
accordance with an example embodiment;
[0038] FIG. 6B illustrates the effect of RNase inhibitors for
various concentrations of template on the RT-LAMP colorimetric
response in accordance with an example embodiment;
[0039] FIG. 6C illustrates the effect of the saliva processing
technique on the colorimetric LoD in accordance with an example
embodiment;
[0040] FIG. 6D illustrates the effect of carrier DNA concentration
on colorimetric RT-LAMP response in accordance with an example
embodiment;
[0041] FIG. 6E illustrates the effect of Guanidine HCl on RT-LAMP
colorimetric response in accordance with an example embodiment;
[0042] FIG. 6F illustrates the effect of UDG on end-point RT-LAMP
colorimetric response response in accordance with an example
embodiment;
[0043] FIG. 6G illustrates the effect of saliva processing on
colorimetric response in accordance with an example embodiment;
[0044] FIG. 7 is a chart illustrating the stability of frozen
saliva samples in accordance with an example embodiment;
[0045] FIG. 8 illustrates the limit of detection of fresh saliva in
accordance with an example embodiment;
[0046] FIG. 9 illustrates the limit of detection in a bovine nasal
swab in accordance with an example embodiment;
[0047] FIG. 10 illustrates the limit of detection on paper in
accordance with an example embodiment;
[0048] FIG. 11 illustrates the colorimetric transition for phenol
red in accordance with an example embodiment;
[0049] FIG. 12 illustrates buffer used for the paper-based assay in
accordance with an example embodiment;
[0050] FIG. 13A illustrates paper LAMP validation in accordance
with an example embodiment;
[0051] FIG. 13B illustrates paper LAMP validation in accordance
with an example embodiment;
[0052] FIG. 14A illustrates low template concentration LAMP on
paper at a 0-minute time point in accordance with an example
embodiment;
[0053] FIG. 14B illustrates low template concentration LAMP on
paper at a 60-minute time point in accordance with an example
embodiment;
[0054] FIG. 15 illustrates whole untreated saliva with heat
inactivated SARS-CoV-2 virus in accordance with an example
embodiment;
[0055] FIG. 16 illustrates a comparison of colorimetric and
fluorometric RT-LAMP responses in accordance with an example
embodiment;
[0056] FIG. 17A illustrates use of calmagite as a LAMP colorimetric
indicator in accordance with an example embodiment;
[0057] FIG. 17B illustrates use of EBT as a LAMP indicator in
accordance with an example embodiment;
[0058] FIG. 17C illustrates LAMP on chromatography paper using EBT
as a colorimetric reporter in accordance with an example
embodiment;
[0059] FIG. 17D illustrates colorimetric response of LAMP on
various papers using EBT as an indicator in accordance with an
example embodiment;
[0060] FIG. 17E illustrates LAMP detection on biodyne A amphoteric
paper using EBT as a colorimetric indicator in accordance with an
example embodiment;
[0061] FIG. 17F illustrates the effect of crystal violet
concentration on LAMP colorimetric response in accordance with an
example embodiment;
[0062] FIG. 17G illustrates colorimetric LAMP using crystal violet
at various concentration on paper in accordance with an example
embodiment;
[0063] FIG. 17H illustrates pH indicators as colorimetric reporters
for RT-LAMP in accordance with an example embodiment;
[0064] FIG. 17I illustrates the effect of cresol red concentration
on colorimetric response of LAMP reaction in accordance with an
example embodiment;
[0065] FIG. 17J the effect of concentration of various pH
indicators on colorimetric response for RT-LAMP reaction in
accordance with an example embodiment;
[0066] FIG. 17K gel electrophoresis scans of RT-LAMP products using
pH indicators in accordance with an example embodiment;
[0067] FIG. 17L the effect of initial pH on RT-LAMP colorimetric
response using Phenol red in accordance with an example
embodiment;
[0068] FIG. 18 illustrates the color stability of the drying
process in accordance with an example embodiment;
[0069] FIG. 19A illustrates the effect of elimination of single
reactant on initial color of paper after drying in accordance with
an example embodiment; and
[0070] FIG. 19B illustrates the effect of trehalose and Tween 20 on
RT-LAMP colorimetric response in accordance with an example
embodiment.
[0071] Reference will now be made to the exemplary embodiments
illustrated, and specific language will be used herein to describe
the same. It will nevertheless be understood that no limitation of
the scope of the technology is thereby intended.
DESCRIPTION OF EMBODIMENTS
[0072] Before invention embodiments are described, it is to be
understood that this disclosure is not limited to the particular
structures, process steps, or materials disclosed herein, but is
extended to equivalents thereof as would be recognized by those
ordinarily skilled in the relevant arts. It should also be
understood that terminology employed herein is used for the purpose
of describing particular examples or embodiments only and is not
intended to be limiting. The same reference numerals in different
drawings represent the same element. Numbers provided in flow
charts and processes are provided for clarity in illustrating steps
and operations and do not necessarily indicate a particular order
or sequence.
[0073] Furthermore, the described features, structures, or
characteristics can be combined in any suitable manner in one or
more embodiments. In the following description, numerous specific
details are provided, such as examples of compositions, dosage
forms, treatments, etc., to provide a thorough understanding of
various invention embodiments. One skilled in the relevant art will
recognize, however, that such detailed embodiments do not limit the
overall inventive concepts articulated herein, but are merely
representative thereof.
Definitions
[0074] It should be noted that as used herein, the singular forms
"a," "an," and, "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to "an
excipient" includes reference to one or more of such excipients,
and reference to "the carrier" includes reference to one or more of
such carriers.
[0075] As used herein, the terms "formulation" and "composition"
are used interchangeably and refer to a mixture of two or more
compounds, elements, or molecules. In some aspects, the terms
"formulation" and "composition" may be used to refer to a mixture
of one or more active agents with a carrier or other
excipients.
[0076] As used herein, the term "soluble" is a measure or
characteristic of a substance or agent with regards to its ability
to dissolve in a given solvent. The solubility of a substance or
agent in a particular component of the composition refers to the
amount of the substance or agent dissolved to form a visibly clear
solution at a specified temperature such as about 25.degree. C. or
about 37.degree. C.
[0077] As used herein, the term "lipophilic," refers to compounds
that are not freely soluble in water. Conversely, the term
"hydrophilic" refers to compounds that are soluble in water.
[0078] As used herein, a "subject" refers to an animal. In one
aspect the animal may be a mammal. In another aspect, the mammal
may be a human.
[0079] As used herein, "non-liquid" when used to refer to the state
of a composition disclosed herein refers to the physical state of
the composition as being a semi-solid or solid.
[0080] As used herein, "solid" and "semi-solid" refers to the
physical state of a composition that supports its own weight at
standard temperature and pressure and has adequate viscosity or
structure to not freely flow. Semi-solid materials may conform to
the shape of a container under applied pressure.
[0081] As used herein, a "solid phase medium," "solid phase base"
"solid phase substrate" "solid phase test substrate" "solid phase
testing substrate," and the like refer to a non-liquid medium,
device, system, or environment. In some aspects, the non-liquid
medium may be substantially free of liquid or entirely free of
liquid. In one example, the non-liquid medium can comprise or be a
porous material or a material with a porous surface. In another
example, the non-liquid medium can comprise or be a fibrous
material or a material with a fibrous surface. In yet another
example, the non-liquid medium can be a paper.
[0082] As used herein, a "non-discoloration additive" refers to an
additive that minimizes or prevents a color change in the color of
the solid phase medium from an original or starting color to a
different color for reasons other than nucleotide amplification
from a LAMP reaction taking place thereon or therein. For example,
in one embodiment, such a color change can be minimized or reduced
as compared to a color change that would take place without the
non-discoloration additive present.
[0083] As used herein, "non-LAMP reaction produced discoloration"
refers to any discoloration (e.g., change in color from an original
color to another color) of the solid phase medium which is not the
result of a nucleotide amplification from a LAMP reaction. In some
examples, non-LAMP reaction produced discoloration can refer to
discoloration of the solid phase medium resulting from one or more
of: a volatile agent, a magnesium-interfering agent, an oxidizing
agent, a pH change resulting from causes other than amplification
from a LAMP reaction, drying, or combinations thereof.
[0084] As used herein, a "volatile agent" refers to an agent that
includes a composition that has a high vapor pressure or a low
boiling point. In one example, ammonium sulfate can be a volatile
agent because the ammonia can volatilize and leave behind sulfuric
acid. In one example, a composition, component, or element, can
have a high vapor pressure when the composition is in a gas phase
at a temperature of more than about 30.degree. C. In one example, a
composition can have a low boiling point when the composition forms
is in a gas phase at a temperature of less than about 80.degree.
C.
[0085] As used herein, a "pH-interfering reagent" is a reagent that
can affect the pH of a reaction, system, or environment for reasons
other than amplification from a LAMP reaction. In one example, the
ammonium ion can volatilize from ammonium sulfate, and the sulfate
ion can react to form sulfuric acid and affect the pH of the
reaction in the absence of amplification from the LAMP
reaction.
[0086] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. Patent law and can mean "includes," "including," and the like,
and are generally interpreted to be open ended terms. The terms
"consisting of" or "consists of" are closed terms, and include only
the components, structures, steps, or the like specifically listed
in conjunction with such terms, as well as that which is in
accordance with U.S. Patent law. "Consisting essentially of" or
"consists essentially of" have the meaning generally ascribed to
them by U.S. Patent law. In particular, such terms are generally
closed terms, with the exception of allowing inclusion of
additional items, materials, components, steps, or elements, that
do not materially affect the basic and novel characteristics or
function of the item(s) used in connection therewith. For example,
trace elements present in a composition, but not affecting the
compositions nature or characteristics would be permissible if
present under the "consisting essentially of" language, even though
not expressly recited in a list of items following such
terminology. When using an open ended term, like "comprising" or
"including," in the written description it is understood that
direct support should be afforded also to "consisting essentially
of" language as well as "consisting of" language as if stated
explicitly and vice versa.
[0087] The terms "first," "second," "third," "fourth," and the like
in the description and in the claims, if any, are used for
distinguishing between similar elements and not necessarily for
describing a particular sequential or chronological order. It is to
be understood that any terms so used are interchangeable under
appropriate circumstances such that the embodiments described
herein are, for example, capable of operation in sequences other
than those illustrated or otherwise described herein. Similarly, if
a method is described herein as comprising a series of steps, the
order of such steps as presented herein is not necessarily the only
order in which such steps may be performed, and certain of the
stated steps may possibly be omitted and/or certain other steps not
described herein may possibly be added to the method.
[0088] As used herein, comparative terms such as "increased,"
"decreased," "better," "worse," "higher," "lower," "enhanced,"
"maximized," "minimized," and the like refer to a property of a
device, component, composition, or activity that is measurably
different from other devices, components, compositions or
activities that are in a surrounding or adjacent area, that are
similarly situated, that are in a single device or composition or
in multiple comparable devices or compositions, that are in a group
or class, that are in multiple groups or classes, or as compared to
the known state of the art.
[0089] The term "coupled," as used herein, is defined as directly
or indirectly connected in a chemical, mechanical, electrical or
nonelectrical manner. Objects described herein as being "adjacent
to" each other may be in physical contact with each other, in close
proximity to each other, or in the same general region or area as
each other, as appropriate for the context in which the phrase is
used. Occurrences of the phrase "in one embodiment," or "in one
aspect," herein do not necessarily all refer to the same embodiment
or aspect.
[0090] As used herein, the term "substantially" refers to the
complete or nearly complete extent or degree of an action,
characteristic, property, state, structure, item, or result. For
example, an object that is "substantially" enclosed would mean that
the object is either completely enclosed or nearly completely
enclosed. The exact allowable degree of deviation from absolute
completeness may in some cases depend on the specific context.
However, generally speaking the nearness of completion will be so
as to have the same overall result as if absolute and total
completion were obtained. The use of "substantially" is equally
applicable when used in a negative connotation to refer to the
complete or near complete lack of an action, characteristic,
property, state, structure, item, or result. For example, a
composition that is "substantially free of" particles would either
completely lack particles, or so nearly completely lack particles
that the effect would be the same as if it completely lacked
particles. In other words, a composition that is "substantially
free of" an ingredient or element may still actually contain such
item as long as there is no measurable effect thereof.
[0091] As used herein, the term "about" is used to provide
flexibility to a numerical range endpoint by providing that a given
value may be "a little above" or "a little below" the endpoint.
Unless otherwise stated, use of the term "about" in accordance with
a specific number or numerical range should also be understood to
provide support for such numerical terms or range without the term
"about". For example, for the sake of convenience and brevity, a
numerical range of "about 50 angstroms to about 80 angstroms"
should also be understood to provide support for the range of "50
angstroms to 80 angstroms." Furthermore, it is to be understood
that in this specification support for actual numerical values is
provided even when the term "about" is used therewith. For example,
the recitation of "about" 30 should be construed as not only
providing support for values a little above and a little below 30,
but also for the actual numerical value of 30 as well.
[0092] As used herein, a plurality of items, structural elements,
compositional elements, and/or materials may be presented in a
common list for convenience. However, these lists should be
construed as though each member of the list is individually
identified as a separate and unique member. Thus, no individual
member of such list should be construed as a de facto equivalent of
any other member of the same list solely based on their
presentation in a common group without indications to the
contrary.
[0093] Concentrations, amounts, levels and other numerical data may
be expressed or presented herein in a range format. It is to be
understood that such a range format is used merely for convenience
and brevity and thus should be interpreted flexibly to include not
only the numerical values explicitly recited as the limits of the
range, but also to include all the individual numerical values or
sub-ranges or decimal units encompassed within that range as if
each numerical value and sub-range is explicitly recited. As an
illustration, a numerical range of "about 1 to about 5" should be
interpreted to include not only the explicitly recited values of
about 1 to about 5, but also include individual values and
sub-ranges within the indicated range. Thus, included in this
numerical range are individual values such as 2, 3, and 4 and
sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well
as 1, 2, 3, 4, and 5, individually. This same principle applies to
ranges reciting only one numerical value as a minimum or a maximum.
Furthermore, such an interpretation should apply regardless of the
breadth of the range or the characteristics being described.
[0094] Reference throughout this specification to "an example"
means that a particular feature, structure, or characteristic
described in connection with the example is included in at least
one embodiment. Thus, appearances of the phrases "in an example" in
various places throughout this specification are not necessarily
all referring to the same embodiment.
EMBODIMENTS
[0095] Many molecular tests for pathogens (e.g., severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus
responsible for COVID-19) can be limited to the laboratory and thus
have significant lag times (>24 hours) to provide a result,
preventing their adoption in point-of-care settings. Despite
several attempts at developing a point-of-care test for SARS-CoV-2,
some limitations remain: i) scalability (the demand for testing is
in the order of millions per week, but manufacturing new tests at
that scale is difficult), ii) sample processing (many tests still
use an extraction operation when using saliva), and iii)
readability (molecular tests often use fluorescence and thus, a
fluorescence reader to report the results).
[0096] The current testing methods can be overcome by using a
point-of-care test using paper-based devices and
reverse-transcription loop-mediated isothermal amplification
(RT-LAMP) that report a color change in the presence of a pathogen
(e.g., SARS-CoV-2) within 60 minutes using diluted saliva (e.g., 5%
v/v in water) as the sample. RT-LAMP is a nucleic acid
amplification technique conducted at a constant temperature with
adequate diagnostic performance especially during the acute phase
of infection. Since RT-LAMP can be conducted at a constant
temperature, expensive thermal cycling equipment is not used.
Additionally, existing colorimetric reporters for LAMP products do
not use fluorescence readers. Consequently, this test is suitable
for use in point-of-care settings and is amenable to rapid
development and scale-up, making it appropriate for use in public
health emergencies.
[0097] RT-LAMP can be implemented on microfluidic paper-based
analytical devices (.mu.PADs) to detect various pathogens (e.g.,
SARS-CoV-2) where image analysis can be performed using a portable
electronic device to distinguish between positive and negative
responses. In one example, a high-contrast RT-LAMP reaction on
paper can provide a color change that can be visible to the naked
eye. In addition, instead of using wax-printing--which would have
precise alignment of printed areas and dispensing of
reagents--polystyrene spacers can be used for preventing crosstalk
between samples. The polystyrene spacers can be amenable to
roll-to-roll fabrication for scale up of production.
[0098] Nucleic-acid-based COVID-19 diagnosis methods use
pre-processing to provide results. As disclosed herein, on-paper
colorimetric detection of SARS-CoV-2 can be performed with minimal
pre-processing. The device can have a sensitivity and specificity
that can detect SARS-CoV-2 on paper without pre-amplification.
Other assays conducted in solution may not be as scalable during
manufacturing as paper-based assays. Additionally, the assay
disclosed herein uses a dilution operation that can be completed in
seconds, whereas other assays use various operations such as
treatment with protease, heat-inactivation, and/or RNA extraction
to detect SARS-CoV-2 (operations completed in at least 10 minutes
and using additional equipment).
Sample Collection and Properties for LAMP Analysis
[0099] Saliva has various physical, chemical, and antibacterial
properties that can cause difficulty in the context of a LAMP
reaction. For example, in one physical property, saliva can dilute
and remove organic acids from dental plaque that can interfere with
a LAMP reaction. Some of the chemical properties--electrolytes and
buffering molecules that minimize changes in pH--can also interfere
with a LAMP reaction. The antibacterial agents in saliva, e.g.,
mucins, amylases, lysozyme, and peroxidase enzyme, also present
challenges. For example, peroxidase enzyme can form free radical
compounds in bacterial cells that can cause them to undergo
apoptosis-like death. However, such a reaction can also provide an
unstable redox environment that can complicate a LAMP reaction.
[0100] With the above-described background in mind, as depicted in
the flowchart in FIG. 1, a method 100 of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target is provided. Depending on the final signal output
selected to show a test result, for example an optically detected
pH-based color change, it can be desirable to ensure that the
saliva in the sample does not skew the overall sample pH
significantly away from neutral. A variety of techniques and
processes can be implemented to check or otherwise limit the
buffering capacity or influence of the saliva in the sample. An
excessive amount of buffering capacity can prevent the fluctuations
in pH used to detect a pH-based color change.
[0101] One way of reducing the buffering capacity of saliva can
include dilution. In one embodiment, such a method can comprise
providing an amount of saliva from a test subject, as shown in
block 110 and diluting the saliva in water to a degree that reduces
a buffering capacity of the saliva while maintaining a sufficient
concentration to allow for detection of the pathogen target, as
shown in block 120.
[0102] The proteins present in saliva offer another challenge. For
example, an excessively viscous sample can be difficult to test on
a solid-based or solid-phase medium. A slow flow rate in a
solid-based medium can increase the reaction time, decrease
uniformity of spreading, increase variability in results, and
increase invalidity of results. For example, when a viscous form of
saliva does not spread evenly throughout a solid-based medium, a
color-based indication can be difficult to read. The decrease in
uniform spreading can also increase the variability of results by
adding uncertainty to the reading of results. Different technicians
may interpret the results differently. In some cases, the results
may be impracticable to read due to ambiguous or absent color
changes. Therefore, controlling the viscosity of the saliva can
prevent various complications that may occur.
[0103] Therefore, in another embodiment, the method can further
comprise reducing a viscosity of the saliva as compared to an
original viscosity. In one aspect, the saliva can be reduced by one
or more of dilution, filtering, the like, or combinations thereof
In one aspect, when the viscosity of the saliva is reduced using
dilution, the saliva can be diluted in water to a saliva to water
ratio of from about 1:1 to about 1:20. In another aspect, the
viscosity of the saliva can be diluted in water to a saliva to
water ratio of about 1:1, 1:2, 1:4, 1:8, 1:10, 1:12, 1:14, 1:16,
1:18, or 1:20. In one aspect, the saliva can be diluted in the
water to a degree that provides the sample with an optical density
at 600 nm (OD.sub.600) of less than about 0.2. In one aspect, the
saliva can have a volume range of from about 50 .mu.l to about 100
.mu.l. In another aspect, the saliva sample can have a volume
ranging from about 100 .mu.l to about 1 ml.
[0104] In some cases, diluting the saliva sample can reduce the
impact of the effects arising from the buffering capacity and
viscosity of the saliva. Another way of reducing the impact of the
viscosity of the saliva can include filtering. In another aspect,
the viscosity can be reduced using a filter having a rating between
about 2 microns and 50 microns. In one example, the filter rating
can be one or more of 2 microns, 5 microns, 8 microns, 10 microns,
15 microns, 20 microns, 25 microns, 40 microns, or 50 microns. In
one aspect, the filter rating can be an absolute micron rating in
which the filter can remove at least about 98.7% of a specific
particle size. Filtering the saliva, rather than dilution alone,
can also remove saliva proteins (e.g., mucins, amylases, lysozyme,
and peroxidase enzyme) that may interfere with a LAMP reaction.
[0105] Through a combination of dilution, filtering, or both, the
viscosity can be controlled to fall within a specific range. In yet
another aspect, the viscosity can be reduced to a degree that
increases flowability through a solid phase medium as compared to
an original viscosity. In one example, a viscosity of saliva can
have a range of from about 1 centipoise (cP) and to about 100 cP
before dilution or filtering. In one example, the viscosity of the
saliva can be reduced to a range of from about 1.0 cP to about 50
cP after dilution or filtering. In another example, the viscosity
of the saliva can be reduced to a range of from about 1.0 cP to
about 10 cP after dilution or filtering.
[0106] Filtering the saliva can also adjust the pH range to a
desirable level. For example, some pH indicators may display a
color change within a specific pH range (e.g., 7.2 to about 8.6).
Therefore, the saliva can be filtered to a test sample target range
depending on the type of pH indicator to be used. However,
maintaining the test sample target range within physiological
conditions can increase the uniformity of the LAMP reaction
results. In one aspect, the saliva can be filtered to a degree that
adjusts the saliva sample pH to a test sample target range. In one
example, the test sample target range can include a pH range
between about 7.2 to about 8.6. In another example, the test sample
target range can include a pH range between about 7.6 to about
8.2.
[0107] Adjusting the test sample target range to a desired level
may not be sufficient to detect a pH change (or other colorimetric
indication) in a LAMP reaction. In another example, the saliva can
be diluted with water to a degree that the buffering capacity of
the composition is reduced relative to the buffering capacity
before the dilution with water to allow a pH indication to be
detected. In one example, buffering capacity can be defined as the
ability of a solution (e.g., the saliva, the water, or the saliva
diluted with water) to resist changes in pH when acids or bases are
added. In one example, the buffering capacity can be defined as the
amount of strong acid or strong base, grams equivalents, that is to
be added to 1 liter of the solution to change the pH by one unit.
In one aspect, the buffering capacity of the saliva can be between
0.03 mg/ml to about 0.30 mg/ml before dilution with water, and the
buffering capacity of the saliva diluted water can be between about
0.003 mg/ml to about 0.03 mg/ml after dilution with water. In
another example, the buffering capacity of the saliva diluted water
can be less than about 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM.
[0108] The water used to dilute the sample should be free of any
contaminants or properties that might interfere with the LAMP
reaction. For example, a pH that is too acidic can prevent the LAMP
reaction from being detected if the pH prevents a pH-based
indication change. In one aspect, the saliva can be diluted in
water, wherein the water can have a pH greater than about 6.0. In
another aspect, the water can have a pH less than about 8.0. In one
example, the water can be molecular grade water that is
substantially free of contaminants, such as RNase and DNase. RNase
can degrade the RNA in the saliva that is to be detected, and DNase
can degrade the DNA formed during the LAMP reaction. In another
example, the saliva sample can consist essentially of saliva and
water.
[0109] Minimizing the presence of undesirable saliva proteins can
be accomplished using a specific saliva collection method. In one
aspect, the saliva can be collected using one or more of a
sponge-based collection method or a passive drool collection
method. Collecting saliva using a sponge-based collection method
may provide the benefit of inherently filtering mucins and high
molecular weight proteins out of the saliva as they will not be
absorbed by the sponge, which can reduce the viscosity of the
saliva and increase the rapidity, uniformity, and reliability of
the saliva when used on a solid-based medium. When the saliva is
collected using a drooling method, the unfiltered saliva can have a
greater viscosity, and therefore a reduced absorption, and
distribution on a solid-based medium. As a result, in some
embodiments when the saliva is collected via drooling, it can be
subsequently filtered in order to remove mucins and other debris
and reduce its viscosity.
[0110] A selected pathogen target can be detected from the saliva.
In one aspect, the pathogen target can be one or more of a viral
pathogen, a bacterial pathogen, a fungal pathogen, a protozoa
pathogen, the like, or combinations thereof. The pathogen target in
saliva can be detected when the nucleic acid from the pathogen
target can be released from a cell wall, a cell membrane, a protein
coat, or the like.
[0111] More specifically, in one aspect, the pathogen target can be
a viral target. In some aspects, the viral target can be H1N1,
H2N2, H3N2, H1N1pdm09, severe acute respiratory syndrome
coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome
(MERS), influenza, the like, or combinations thereof.
[0112] The viral target can be selected from a number of different
viral species. In one example, the viral target can be human
coronavirus 229E, human coronavirus OC43, human coronavirus HKU1,
human coronavirus NL63, MERS-coronavirus, human respirovirus 1,
human rubulavirus 2, human respirovirus 3, human rubulavirus 4,
human enterovirus, human respiratory virus, rhinovirus A,
rhinovirus B, rhinovirus C, or combinations thereof.
[0113] The viral target can also be a form of influenza. In one
aspect, influenza can be any of Influenza A, Influenza B, Influenza
C, or Influenza D. In one aspect, the viral target can be a virus
chosen from the order Nidovirale. In one aspect, the viral target
can be chosen from the alpha, beta, gamma or delta genera of the
Nidovirale order.
[0114] There are various families of viruses that may be detected.
In one aspect, the viral target can be a DNA virus selected from
the group of families including: Adenoviridae, Papovaviridae,
Parvoviridae, Herpesviridae, Poxviridae, Anelloviridae,
Pleolipoviridae, the like, and combinations thereof. In another
aspect, the viral target can be an RNA virus selected from the
group of families including: Reoviridae, Picornaviridae,
Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae,
Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae,
Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, the like
and combinations thereof. In another aspect, the viral target can
be a reverse transcribing virus selected from the group of families
including: Retroviridae, Caulimoviridae, Hepadnaviridae, the like,
and combinations thereof.
[0115] More generally, the viral target can be a virus categorized
by the Baltimore classification. In one aspect, the viral target
can be an RNA virus (e.g., Influenza A, Zika, Hepatitis C). In one
aspect, the viral target can be a DNA virus (e.g., Epstein Barr,
Smallpox). In one aspect, the viral target can be a positive sense
RNA virus (e.g., Hepatitis A, rubella). In one aspect, the viral
target can be a negative sense RNA virus (e.g., Ebola, measles,
mumps). In another aspect, the viral target can be a dsDNA virus
(e.g., chickenpox, herpes), an ssDNA virus, a dsRNA virus (e.g., a
rotavirus), a positive-strand ssRNA virus, a negative-strand ssRNA
virus, an ssRNA-RT virus (e.g., retroviruses), or a ds-DNA-RT virus
(e.g., Hepatitis B).
[0116] Besides viral targets, in another aspect, the pathogen
target can be a bacterial target. In some examples, the bacterial
target can be selected from a genus including: Bacillus,
Bartonella, Bordetella, Borrelia, Brucella, Campylobacter,
Chlamydia, Chlamydophila, Clostridium, Corynebacterium,
Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter,
Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma,
Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,
Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio,
Yersinia, the like, and combinations thereof. In another example,
the bacterial target can be selected from a species including:
Actinomyces israelii, Bacillus anthracis, Bordetella pertussis, B.
abortus, B. canis, B. melitensis, B. suis, Corynebacterium
diphtherias, E. coli, Enterotoxigenic E. coli, Enteropathogenic E.
coli, Enteroinvasive E. coli, Haemophilus influenzae, Helicobacter
pylori, Klebsiella pneumoniae, Legionella pneumophila, M.
tuberculosis, Mycoplasma pneumoniae, N. meningitidis, S. typhi, S.
sonnei, S. dysenteriae, Streptococcus pneumoniae, Streptococcus
pyogenes, Streptococcus viridans, Vibrio cholerae, Yersinia pestis,
the like, and combinations thereof In another aspect, the pathogen
target can be selected from the species including: Chlamydia
pneumoniae, Pneumocystis jirovecii, Candida albicans, Pseudomonas
aeruginosa, Staphylococcus epidermis, Streptococcus salivarius, the
like, and combinations thereof.
[0117] The pathogen target can also include various types of
fungus. In one aspect, the pathogen target can be a fungal target.
In some examples, the fungal target can be selected from a genus
including: Aspergillus, Histoplasma, Pneumocystis, Stachybotrys,
the like, and combinations thereof. In another aspect, the pathogen
target can be a protist target. In some examples, the protist
target can be selected from a genus including: plasmodium,
trypanosomes, the like, and combinations thereof.
[0118] When the pathogen target in saliva includes RNA, the RNA can
be reverse transcribed. Therefore, in another aspect, the LAMP
detection can be reverse transcription LAMP (RT-LAMP). In this
example, cDNA can be generated from a target RNA with a reverse
transcriptase enzyme. The cDNA can be amplified to a detectable
amount. When the pathogefn target can be detected directly from
DNA, then LAMP can be used to amplify the DNA to a detectable
amount without reversed transcribing the RNA to DNA.
[0119] In another aspect, the specific target nucleotide sequences
to be detected can be target nucleotides corresponding to human
biomarkers. Any disease that has a target nucleotide corresponding
to a human biomarker for a disease can be detected. Various types
of diseases can be detected including one or more of: breast
cancer, pancreatic cancer, colorectal cancer, ovarian cancer,
gastrointestinal cancer, cervix cancer, lung cancer, bladder
cancer, many types of carcinomas, salivary gland cancer, kidney
cancer, liver cancer, lymphoma, leukemia, melanoma, prostate
cancer, thyroid cancer, stomach cancer, the like, or combinations
thereof. For example, biomarkers for various types of diseases can
be detected by detecting target nucleotides corresponding to one or
more of: alpha fetoprotein, CA15-3 and CA27-29, CA19-9, C!-125,
calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99MIC 2,
CD117, chromogranin, chromosomes 3, 7, 17, and 9p21, cytokeratin,
cesmin, epithelial membrane antigen, factor VIII, CD31 FL1, glial
fibrillary acidic protein, gross cystic disease fluid protein,
hPG80, HMB-45, human chorionic gonadotropin, immunoglobulin,
inhibin, keratin, lymphocyte marker, MART-1, Myo D1,
muscle-specific actin, neurofilament, neuron-specific enolase,
placental alkaline phosphatase, prostate-specific antigen, PTPRC,
S100 protein, smooth muscle action, synaptophysin, thymidine
kinase, thyroglobulin, thyroid transcription factor-1, tumor M2-PK,
vimentin, the like, or combinations thereof.
[0120] In another embodiment, a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis can comprise
an amount of a test subject's saliva that is sufficient to detect a
pathogen target via a LAMP analysis in combination with an amount
of water that reduces a buffering capacity of the saliva. In one
aspect, the viscosity of the composition can be from about 1.0 cP
to about 50 cP. In another aspect, the pH of the composition can be
from about 7.2 to about 8.6. Selecting a viscosity and pH within
these respective ranges can enhance the change in pH and therefore
the color change resulting from a pH-based indicator.
[0121] The saliva can be diluted with water to place the viscosity
and pH within the ranges enumerated above. In one aspect, the
saliva can be combined with the amount of water in a saliva to
water ratio of from about 1:1 to about 1:20. In another aspect, the
saliva to water ratio can be about 1:1, 1:2, 1:4, 1: 6, 1:8, 1:10,
1:12, 1:14, 1:16, 1:18, or 1:20. In another aspect, the saliva can
be combined with the amount of water to a degree that provides the
sample with an optical density at 600 nm (OD.sub.600) of less than
0.2.
[0122] In order to ensure that the amount of saliva contains a
detectable amount of virus, the amount of collected saliva can be
higher than a threshold amount. In one aspect, the saliva can have
a volume ranging from about 50 .mu.l to about 100 .mu.l. In another
aspect, the saliva sample can have a volume ranging from about 100
.mu.l to about 1 ml.
[0123] The saliva can also have various chemical properties (e.g.,
pH and buffering capacity) that can facilitate the LAMP reaction.
In one aspect, the water can have a pH greater than about 6.0 and
can be substantially free of contaminants such as RNase and DNase.
In another aspect, the water can have a pH less than about 8.0 and
can be substantially free of contaminants. In another aspect, the
composition can consist essentially of the saliva and the water. In
one aspect, the buffering capacity of the composition can be
between about 0.003 mg/ml to about 0.03 mg/ml. In another example,
the buffering capacity of the composition can be less than about 5
mM, 4 mM, 3 mM, 2 mM, or 1 mM.
[0124] As disclosed in the preceding, the pathogen target can
comprise a viral pathogen, a bacterial pathogen, a fungal pathogen,
or a protozoa pathogen. The pathogen target can be a viral target.
Based on the Baltimore classification of viruses, in another
aspect, the viral target can comprise a dsDNA virus, an ssDNA
virus, a dsRNA virus, a positive-strand ssRNA virus, a
negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT
virus. In another aspect, the viral target can comprise H1N1, H2N2,
H3N2, H1N1pdm09, or SARS-CoV-2.
Reagent Compositions
[0125] A variety of reagents can be used in a LAMP analysis
depending on the testing medium, readout type, and overall
environment of the designed system. Further, reaction components
such as primers and enzymes can be selected in view of the specific
target nucleotide sequences to be detected, organisms to be
identified, etc. Additionally, the specifics of the test
environment, such as liquid environment, anhydrous environment,
housing, substrates, etc. can be taken into account as well as
other needs such as storage on stability when selecting specific
reagents to be involved in the reaction underlying the LAMP
analysis.
[0126] In one embodiment, a composition for loop-mediated
isothermal amplification (LAMP) analysis on a solid phase medium
can comprise one or more target primers, a DNA polymerase, and a
re-solubilization agent. In one aspect, the composition can be
substantially free of non-pH sensitive agents capable of
discoloring the solid phase medium.
[0127] When conducting LAMP analysis on a solid phase medium, the
concentration of reagents can be increased when compared to a LAMP
analysis in a liquid phase medium. In one aspect, the concentration
of DNA polymerase when used on the solid phase medium can be at
least twice the concentration of DNA polymerase when used with a
liquid medium. In another aspect, the concentration of DNA
polymerase when used on the solid phase medium can be at least
three times the concentration of DNA polymerase when used with a
liquid medium. In one example, the concentration of DNA polymerase
can be from about 300 U/mL to about 1000 U/mL when used on the
solid-phase medium. In another example, the concentration of DNA
polymerase can be from about 600 U/mL to about 1000 U/mL when used
on the solid phase medium. In yet another example the concentration
of DNA polymerase can be from about 620 U/m to about 680 U/mL when
used on the solid phase medium.
[0128] When the LAMP analysis involves reverse transcriptase LAMP
(RT-LAMP), the composition can further comprise reverse
transcriptase. The reverse transcriptase can aid in the detection
of RNA-based viruses. In one aspect, the concentration of reverse
transcriptase when used on the solid phase medium can be at least
twice the concentration of reverse transcriptase when used with a
liquid medium. In another aspect, the concentration of reverse
transcriptase can be least three times the concentration of reverse
transcriptase when used with a liquid medium. In one example, the
concentration of reverse transcriptase can be from about 200 U/mL
to about 600 U/mL when used on the solid-phase medium. In another
example, the concentration of reverse transcriptase can be from
about 250 U/mL to about 500 U/mL when used on the solid phase
medium. In yet another example the concentration of reverse
transcriptase can be from about 290 U/mL to about 310 U/mL when
used on the solid phase medium.
[0129] Besides the target primers, DNA polymerase, and reverse
transcriptase, the composition can comprise a re-solubilization
agent. A re-solubilization agent can aid in the re-hydration of the
LAMP reagents on the solid-based medium when a saliva sample is
deposited on the solid-based medium. In one aspect, the
re-solubilization agent can be a surfactant. For example, the
re-solubilization agent can comprise bovine serum albumin (BSA),
casein, polysorbate 20, the like, or combinations thereof. BSA and
casein can facilitate re-solubilization of the DNA polymerase,
reverse transcriptase, and other related enzymes when the dried
reagents are rehydrated. Polysorbate 20 is a surfactant that can
also aid in the re-solubilization of dried reagents. In one
example, the concentration of the re-solubilization agent can be
from about 0.05 wt % to about 5 wt % when used on the solid-phase
medium. In another example, the concentration of the
re-solubilization agent can be from about 0.5 wt % to about 3 wt %.
In yet another example, the concentration of the re-solubilization
agent can be from about 0.5 wt % to about 1.5 wt %.
[0130] The composition can further comprise an agent that can speed
up the reaction, increase sensitivity, or a combination thereof In
one example, BSA can be included to speed up the reaction and
increase sensitivity. However, the inclusion of BSA can also
introduce pH variations that can interfere with the readability of
the results. Therefore, in some examples, the re-solubilization
agent can include casein, polysorbate 20, the like, or combinations
thereof.
[0131] Volatile agents can interfere with the LAMP reaction. For
example, a volatile compound can ionize to a plurality of ions, and
one of the ions can have a low boiling point. When the ion with the
low boiling point evaporates, the remaining ion can further react.
Some of the further reactions can include redox reactions,
acid-base reactions, or other reactions that can affect the
interpretation of a pH-based signal. In one aspect, the composition
can be substantially free of volatile agents. In one example, the
removal of volatile agents can increase the color contrast and
decrease the reaction time of the solid-based medium when compared
to the color contrast and reaction time when volatile agents are
included. In one aspect, the composition can contain less than one
or more of: 1.0 wt %, 0.5 wt %, 0.1 wt %, or 0.01 wt % of the
volatile agents.
[0132] Volatile agents can cause instability in the solid-based
medium. In some examples, a LAMP reaction that contains a volatile
compound, such as ammonium sulfate, can cause instability in the
solid-based medium when the ammonium ions partially convert the
ammonium sulfate to ammonium which can volatilize and leave behind
sulfate. The sulfate can become sulfuric acid and reduce the pH
which can affect the reading of the pH-based indicator (e.g.,
changing the phenol red indicator from red to yellow even when the
LAMP reaction does not occur). Replacing ammonium sulfate with
betaine can prevent the non-LAMP reaction-based discoloration and
stabilize the solid-based medium by preventing discoloration under
storage.
[0133] As such, reducing the presence of volatile agents in the
composition can reduce the degree of interference with the LAMP
reaction and its reading via the pH-based indicator. In one aspect,
the LAMP composition can comprise a non-volatile agent including a
quaternary ammonium of low molecular weight of neutral charge, or
an amide compound of low molecular weight of neutral charge, the
like, or combinations thereof. In one example, the non-volatile
agent can include, but is not limited to, N-Formylurea, Urea,
L-Asparagine, Trimethylglycine (Betaine),
3-(Cyclohexylamino)-1-propanesulphonic acid (CAPS),
3-(1-Pyridinio)-1-propanesulfonate (NDSB-201), N-Methylurea,
Acetamide, Propionamide, Isobutyramide, Piracetam,
1,3-Dimethylurea, 1,1-Dimethylurea, Glycolamide, 2-Chloroacetamide,
Succinimide, 2-Imidazolidone, Choline chloride, Acetylcholine
chloride, Bethanechol chloride, L-Carnitine inner salt,
O-Acetyl-L-carnitine hydrochloride,
4-(Cyclohexylamino)-1-butanesulfonic acid (CABS),
Dimethylethylammoniumpropane sulfonate (NDSB-195),
3-(1-Methylpiperidinium)-1-propane sulfonate (NDSB-221),
3-(Benzyldimethylammonio)propanesulfonate (NDSB-256), and
Dimethyl-2-hydroxyethylammonium-l-propane sulfonate (NDSB-211), the
like, or combinations thereof.
[0134] In one example, the concentration of the non-volatile agent
including a quaternary ammonium of low molecular weight of neutral
charge, or the amide compound of low molecular weight of neutral
charge can be from about 1 mM to about 200 mM when used on the
solid-phase medium. In another example, the concentration of the
non-volatile agent can be from about 10 mM to about 50 mM when used
on the solid-phase medium. In yet another example, the
concentration of the non-volatile agent can be from about 15 mM to
about 25 mM when used on the solid-phase medium.
[0135] In addition to volatile agents, hygroscopic agents can
interfere with the LAMP reaction. A hygroscopic agent can retain an
excessive amount of water and destabilize the reagents in the
solid-based medium by slowing down or preventing drying. In one
aspect, the composition can be substantially free of hygroscopic
agents. In some examples, a LAMP reaction that contains a
hygroscopic agent, such as glycerol can contribute to the
instability of reagents in the solid-based medium because the
hygroscopic agent attract can attract water. In one example, a
hygroscopic agent can absorb more than about 10 wt % when between
about 40% and about 90% relative humidity (RH) at 25.degree. C. In
one example, a hygroscopic agent can include, but is not limited
to, one or more of glycerol, ethanol, methanol, calcium chloride,
potassium chloride, calcium sulfate, the like, or combinations
thereof. In one aspect, the composition can contain less than one
or more of: 1.0 wt %, 0.5 wt %, 0.1 wt %, or 0.01 wt % of the
hygroscopic agents.
[0136] Some additional agents can be included to prevent carryover
contamination of previous LAMP reactions, primer dimerization,
non-specific amplification, or a combination thereof. Carryover
contamination can be prevented by including deoxyuridine
triphosphate (dUTP), uracil DNA glycosylase (UDG), or a combination
thereof in the LAMP reaction. These agents can catalyze the release
of free uracil from single-stranded or double stranded DNA
containing uracil.
[0137] It has been discovered that some pH-based indicators with an
antioxidant effect, such as phenol red can have increased contrast
and uniformity in comparison to other pH-based indicators with a
reduced degree of antioxidant activity. In one aspect, the
composition can further comprise an antioxidant. In one example,
the concentration of the antioxidant can be from about 0.1 mM to
about 1 mM when used on the solid-phase medium. In another example,
the concentration of the antioxidant can be from about 0.2 mM to
about 0.8 mM when used on the solid-phase medium. In yet another
example, the concentration of the antioxidant can be from about 0.2
mM to about 0.3 mM when used on the solid-phase medium. The
antioxidant can stabilize the reagents on the solid-based medium by
preventing oxidization-reduction reactions.
[0138] Various antioxidants can be used including, but not limited
to: N-acetyl-cysteine, hydroxytyrosol (HXT), superoxide dismutase
(SOD), catalase, Vitamin A, Vitamin C, Vitamin E, coenzyme Q10,
manganese, iodide, melatonin, alpha-carotene, astaxanthin,
beta-carotene, canthaxanthin, cryptoxanthin, lutein, lycopene,
zeaxanthin, apigenin, luteolin, tangeritin, isorhamnetin,
kaempferol, myricetin, proanthocyanidins, quercetin, eriodyctiol,
hesperetin, naringenin, catechin, gallocatechin, epicatechin,
epigallocatechin, theaflavin, thearubigins, daidzein, genistein,
glycitein, resveratrol, pterostilbene, cyanidin, delphinidin,
malvidin, pelargonidin, peonidin, petunidin, chicoric acid,
cholorogenic acid, cinnamic acid, ellagic acid, ellagitannins,
gallic acid, gallotannins, rosmarinic acid, salicylic acid,
curcumin, flavonolignans, xanthones, eugenol, capsaicin, bilirubin,
citric acid, oxalic acid, phytic acid, R-alpha-Lipoic acid, the
like, or combinations thereof.
[0139] Although pH-based indicators have been discussed thus far,
other indicators can also be used. In one aspect, the composition
can further comprise an indicator. In one example, the indicator
can be a pH-based indicator, such as phenol red, when used with a
solid-based medium. Phenol red has antioxidant properties that some
other dyes do not have. The phenol red molecule is a conjugated
bond system that might also contribute antioxidant properties. In
one example, the concentration of the indicator can be from about
0.1 mM to about 1 mM when used on the solid-phase medium. In
another example, the concentration of the indicator can be from
about 0.2 mM to about 0.8 mM when used on the solid-phase medium.
In yet another example, the concentration of the indicator can be
from about 0.2 mM to about 0.3 mM when used on the solid-phase
medium.
[0140] Some other indicators can also provide an adequate
colorimetric signal. In another example, the indicator can be one
or more of a (i) magnesium colorimetric indicator, (ii) a pH
colorimetric indicator, or (iii) a DNA intercalating colorimetric
indicator. When the indicator is a magnesium colorimetric
indicator, the concentration of magnesium should be monitored to
maintain the magnesium within a range of from about 0.01 mM to
about 2 mM. Also, the concentration of magnesium should be
monitored to prevent interference with DNA polymerase. Magnesium--a
cofactor of DNA polymerase, can interfere with DNA polymerase when
the magnesium concentration is outside a target range.
[0141] The LAMP reaction can also use various types of target
primers. Some target primers can include about 4 or 6 primers that
can target 6 or 8 regions within a genome, respectively. In one
aspect, the concentration of the target primers can have a
concentration from about 0.05 .mu.M to about 5 .mu.M when used on
the solid-phase medium. In another example, the concentration of
the target primers can be from about 0.1 .mu.M to about 3 .mu.M
when used on the solid-phase medium. In yet another example, the
concentration of the target primers can be from about 0.2 .mu.M to
about 1.6 .mu.M when used on the solid-phase medium.
[0142] The target primers can be selected to target the genomes of
various pathogens. In one aspect, the target primers can target a
pathogen that can comprise a viral pathogen, a bacterial pathogen,
a fungal pathogen, or a protozoa pathogen. In another aspect, the
pathogen target can be a viral target. In another aspect, the viral
target can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a
positive-strand ssRNA virus, a negative-strand ssRNA virus, an
ssRNA-RT virus, or a ds-DNA-RT virus. In another aspect, the viral
target can comprise H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2. In
sum, the target primers can target nearly any pathogen target, in
particular, those target pathogens as disclosed herein.
[0143] When the solid-based medium includes an excessive amount of
volatile agents, oxidizing agents, pH-interfering agents,
magnesium-interfering agents, the like, or combinations thereof,
then the color of the solid-based medium can be affected in the
absence of amplification from the LAMP reaction. To address this
issue, in another aspect, the composition can comprise a
non-discoloration additive. In one aspect, the concentration of the
non-discoloration additive can be from about 0.01 mM to about 1 M
when used on the solid-phase medium. In another example, the
concentration of the non-discoloration additive can be from about
10 mM to about 500 mM when used on the solid-phase medium. In yet
another example, the concentration of the non-discoloration
additive can be from about 200 mM to about 400 mM when used on the
solid-phase medium.
[0144] There are various non-discoloration additives that can
preserve the color of the solid-based medium in the absence of
LAMP-reaction produced amplification and potentially increase the
contrast when LAMP-reaction produced amplification occurs. In one
example, the non-discoloration additive can comprise one or more of
a sugar, a buffer, the like, or combinations thereof.
[0145] In one example, a non-discoloration additive such as sugar
can stabilize the solid-based medium and prevent discoloration
under long-term storage conditions. For example, trehalose can
preserve the stability of enzymes under freeze-drying conditions or
when dried at ambient temperatures. In one aspect, the sugar can
comprise one or more of: glucose, sucrose, trehalose, dextran, the
like, or combinations thereof In one aspect, the concentration of
the sugar can be from about 0.01 mM to about 1 M when used on the
solid-phase medium. In another example, the concentration of the
sugar can be from about 10 mM to about 500 mM when used on the
solid-phase medium. In yet another example, the concentration of
the sugar can be from about 200 mM to about 400 mM when used on the
solid-phase medium.
[0146] The LAMP reaction can also include other reagents. In one
aspect, the composition can comprise one or more of an enzyme, a
nucleic acid, or combinations thereof. In one example, the enzyme
can be an RNase inhibitor or a DNase inhibitor. Inclusion of an
RNase inhibitor can slow the degradation of an RNA target to allow
for an increased limit of detection. Inclusion of a DNase inhibitor
can slow the degradation of a DNA target to also allow for an
increased limit of detection. In one aspect, the composition can
comprise carrier DNA or carrier RNA. The carrier DNA or carrier RNA
can provide decoy substrate that sequesters the activity of DNase
or RNase, respectively. In another example, a selected amount of
guanidine hydrochloride can stimulate the denaturing and exposing
of RNA molecules which can further stabilize the LAMP reaction.
[0147] In one aspect, the concentration of the RNase or DNase
inhibitor can be from about 0.01 .mu.L per mL of saliva sample to
about 5 .mu.L per mL of saliva sample when used on the solid-phase
medium. In another example, the concentration of the RNase or DNase
inhibitor can be from about 0.1 .mu.L per mL of saliva sample to
about 1 .mu.L per mL of saliva sample when used on the solid-phase
medium. In yet another example, the concentration of the RNase or
DNase inhibitor can be from about 0.5 .mu.L per mL of saliva sample
to about 1.5 .mu.L per mL of saliva sample when used on the
solid-phase medium.
[0148] In one aspect, the concentration of the carrier RNA or
carrier DNA can be from about 0.01 ng/.mu.L to about 10 ng/.mu.L
when used on the solid-phase medium. In another example, the
concentration of the carrier RNA or carrier DNA can be from about
0.1 ng/.mu.L to about 1 ng/.mu.L when used on the solid-phase
medium. In yet another example, the concentration of the carrier
RNA or carrier DNA can be from about 0.2 ng/.mu.L to about 0.4
ng/.mu.L when used on the solid-phase medium.
[0149] In addition to the foregoing, a number of other agents or
ingredients can be used in a composition that is suitable for
carrying out a LAMP reaction as recited herein. For example, in
another aspect, the composition can further comprise a tonicity
agent, a pH adjuster, a preservative, water, the like, or
combinations thereof. Moreover, these ingredients/agents can be
used to provide the composition with a range of specifically
desired properties. In one aspect, the tonicity of the composition
can be from about 250 to about 350 milliosmoles/liter (mOsm/L). In
another aspect, the tonicity of the composition can be from about
270 to about 330 mOsm/L. Tonicity agents can be present in the
composition in various amounts. In one aspect, the tonicity agent
can have a concentration in the composition of from about 0.1 wt %,
about 0.5 wt %, or about 1 wt % to about, 2 wt %, about 5 wt %, or
about 10 wt %.
[0150] Although the composition should be substantially free of
pH-interfering reagents, pH adjusters can be used to select an
initial pH of the composition before a LAMP reaction. Furthermore,
pH adjusters can also be used when the effects of the pH adjusters
can be compensated for when interpreting results from the LAMP
reaction. Non-limiting examples of pH adjusters can include a
number of acids, bases, and combinations thereof, such as
hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide,
potassium hydroxide, calcium hydroxide, and the like. The pH
adjusters can be used to provide an appropriate pH for the
composition. In one aspect, the pH can be from about 5.5 to about
8.5. In one aspect, the pH can be from about 5.8 to about 7.8. In
another aspect, the pH can be from about 6.5 to about 7.8. In yet
other examples, the pH can be from about 7.0 to about 7.6. pH
adjusters can be present in the composition in various amounts. In
one aspect, the pH adjuster can have a concentration in the
composition of from about 0.01 wt %, about 0.05 wt %, about 0.1 wt
%, or about 0.5 wt % to about 1 wt %, about 2 wt %, about 5 wt %,
or about 10 wt %.
[0151] The shelf life of the composition can be enhanced by using
preservatives. Non-limiting examples of preservatives can include
benzalkoniurn chloride (BAK), cetrimonium, sodium perborate,
ethylenediaminetetraaceticacid (EDTA) and its various salt forms,
chlorobutanol, and the like. Preservatives can be present in the
composition in various amounts. In one aspect, the preservative can
have a concentration in the composition of from about 0.001 wt %,
about 0.005 wt %, about 0.01 wt %, or about 0.05 wt % to about 0.1
wt %, about 0.25 wt %, about 0.5 wt %, or about 1 wt %.
[0152] In another embodiment, as depicted in FIG. 2, a method 200
for LAMP analysis on a solid phase medium can comprise providing an
assembly of a solid phase medium and a reaction composition in
combination therewith, such as any of the ingredients or
compositions as recited herein, as shown in block 210. In one
aspect, the method can comprise depositing a biological sample onto
the solid phase medium, as shown in block 220. In another aspect,
the method can comprise heating the assembly to an isothermal
temperature sufficient to facilitate a LAMP reaction, as shown in
block 230.
[0153] In one aspect, the biological sample can be one or more of
saliva, mucus, blood, urine, feces, sweat, exhaled breath
condensate, the like, or combinations thereof. In another aspect,
the biological sample can be saliva. In one aspect, the method can
comprise detecting a viral pathogen. In one aspect, the viral
pathogen can be a pathogen as disclosed herein. In another aspect,
the LAMP analysis can be reverse transcriptase LAMP (RT-LAMP).
[0154] In another aspect, the isothermal temperature sufficient to
facilitate a LAMP reaction can be in a temperature range from about
50.degree. C. to about 70.degree. C. In another aspect, the
isothermal temperature sufficient to facilitate a LAMP reaction can
be in a temperature range from about 60.degree. C. to about
70.degree. C. In another aspect, the isothermal temperature
sufficient to facilitate a LAMP reaction can be in a temperature
range from about 60.degree. C. to about 65.degree. C. The
isothermal temperature can be selected based on one or more of the
activity of the DNA polymerase, reverse transcriptase, or
combinations thereof.
[0155] In another example, a temperature sufficient to facilitate a
LAMP reaction can be in a temperature range of from about
60.degree. C. to about 70.degree. C. In another example, the
isothermal temperature can be a temperature within a range that
differs by less than 5 degrees Celsius.
[0156] In another embodiment, a system for performing a LAMP
analysis can comprise a composition as recited in this disclosure.
In another aspect, the system can comprise a solid phase medium on
to which the composition is deposited.
Maximizing pH-Sensitive Signal Output
[0157] When conducting a LAMP reaction, various indicators can be
used to read the results of the reaction. Three types of
colorimetric indicators include magnesium colorimetric indicators,
pH colorimetric indicators, and DNA intercalating colorimetric
indicators. Because magnesium can be a cofactor for DNA polymerase
and its concentration should be tightly controlled, magnesium-based
indicators can face various limitations when used in the context of
a LAMP reaction. DNA intercalating indicators can also face
limitations because of the number of variables in play. Although
all three indicators can be used in a LAMP Reaction, pH-based
indicators may be subject to fewer variables.
[0158] In one embodiment, a composition for loop-mediated
isothermal amplification (LAMP) analysis utilizing a pH-dependent
output signal can comprise a pH sensitive dye, and a plurality of
non-interfering LAMP reagents. In one aspect, the LAMP analysis can
be reverse transcription LAMP (RT-LAMP).
[0159] The selection of pH-sensitive dye can depend on various
factors, such as colorimetric range correlated to pH, degree of
contrast between color changes, level of pH for a color change,
uniformity of color change, reproducibility of color change, and
the like. For example, phenol red can have a colorimetric range
between a pH of about 6.8 and about 7.4. Below a pH of about 6.8,
phenol red can turn yellow and above a pH of about 7.4, phenol red
can turn red. The degree of difference between yellow and red can
be simple to read, and the pH change can occur at a pH level that
mimics physiological conditions.
[0160] In one aspect, the pH sensitive dye can be a pH indicator
with a color change around a pH of 6.5 to achieve a consistent and
contrasting color change (e.g., Phenol red). In one aspect, the pH
sensitive dye can be at least one of phenol red, litmus,
bromothymol blue, nitrazine yellow, cresol red, curcumin, brilliant
yellow, m-cresol purple, a-naphtholphthalein, phenolphthalein,
neutral red, acid fuchsin, azolitmin, the like, or combinations
thereof In one aspect, the concentration of the pH sensitive dye
can be from about 0.1 mM to about 1 mM when used on the solid-phase
medium. In another example, the concentration of the pH sensitive
dye can be from about 0.2 mM to about 0.8 mM when used on the
solid-phase medium. In yet another example, the concentration of
the pH sensitive dye can be from about 0.2 mM to about 0.3 mM when
used on the solid-phase medium.
[0161] To maximize the pH-sensitive signal output, the LAMP
reaction should be substantially free of reagents that would
introduce uncertainty into the signal by interfering with the LAMP
reaction (e.g., interfering with the DNA polymerase) or by
interfering with the signal from the LAMP reaction (e.g., the pH
signal). In one aspect, the plurality of non-interfering LAMP
reagents can comprise DNA polymerase, reverse transcriptase, target
primers, or combinations thereof. In another aspect, the plurality
of non-interfering LAMP reagents can be substantially free of
volatile reagents, pH-interfering reagents, magnesium-interfering
reagents, or combinations thereof.
[0162] In one example, the plurality of non-interfering LAMP
reagents can be substantially free of magnesium, ammonium sulfate,
or ammonium carbonate. Magnesium, as a cofactor of DNA polymerase,
should be tightly monitored to ensure that the LAMP reaction can
proceed as designed. Ammonium sulfate can ionize into the ammonium
ion, which can leave behind a sulfate ion that can react to form
sulfuric acid. Ammonium carbonate can also ionize into an ammonium
ion and leave behind a carbonate that can react to form carbonic
acid. Therefore, the plurality of non-interfering LAMP reagents
should be substantially free of these substances.
[0163] Because volatile agents can leave behind a composition that
can react to form an acid or base that can interfere with the
pH-dependent signal from the LAMP reaction, volatile agents should
be minimized. In one example, the plurality of non-interfering LAMP
reagents can be substantially free of volatile reagents including,
but not limited to: ammonium sulfate, and ammonium carbonate, the
like, or combinations thereof. In one aspect, the composition can
contain less than one or more of: 1.0 wt %, 0.5 wt %, 0.1 wt %, or
0.01 wt % of the volatile reagents.
[0164] Furthermore, any pH-interfering reagents can interfere with
the pH-dependent signal output when the pH-interfering reagents is
not compensated for. In one example, the plurality of
non-interfering LAMP reagents can be substantially free of
pH-interfering reagents including, but not limited to a number of
acids, bases, and combinations thereof. In one aspect, the
composition can contain less than one or more of: 1.0 wt %, 0.5 wt
%, 0.1 wt %, or 0.01 wt % of the pH-interfering reagents.
[0165] Even when the pH has been monitored, the pH-dependent signal
output can be negatively affected when the LAMP reaction is
interfered with. For example, magnesium, as a cofactor of DNA
polymerase, can interfere with the amplification from the LAMP
reaction when the concentration is outside a selected range. In one
example, the plurality of non-interfering LAMP reagents can be
substantially free of magnesium-interfering agents.
Magnesium-interfering agents can include magnesium-containing
agents including, but not limited to: Mg.sup.+, Mg.sup.30 ,
magnesium carbonate, magnesium chloride, magnesium citrate,
magnesium hydroxide, magnesium oxide, magnesium sulfate, magnesium
sulfate heptahydrate, the like, or combinations thereof. In one
aspect, the composition can contain less than one or more of: 1.0
wt %, 0.5 wt %, 0.1 wt %, or 0.01 wt % of magnesium. In another
example, magnesium-interfering agents can include chelating agents
that interfere with magnesium.
[0166] Even when the pH has been monitored and the LAMP reaction is
functioning properly, discoloration of the solid-phase medium can
result from other factors such as long-term storage. In one aspect,
the composition can comprise a non-discoloration additive. In one
example, the non-discoloration additive can comprise one or more of
a sugar, a buffer, a blocking agent, the like, or combinations
thereof. In one example, the sugar can stabilize the solid-based
medium and prevent discoloration under long-term storage
conditions. In one aspect, the sugar can comprise one or more of:
glucose, sucrose, trehalose, dextran, the like, or combinations
thereof.
[0167] In one aspect, the concentration of the sugar can be from
about 0.01 mM to about 1 M when used on the solid-phase medium. In
another example, the concentration of the sugar can be from about
10 mM to about 500 mM when used on the solid-phase medium. In yet
another example, the concentration of the sugar can be from about
200 mM to about 400 mM when used on the solid-phase medium.
[0168] A buffer can facilitate the stabilization of the LAMP
reaction by removing the variability from the saliva sample. In one
example, a buffer can include one or more of phosphate-buffered
saline (PBS), Dulbecco's PBS, Alsever's solution, Tris-buffered
saline (TBS), HEPES, BICINE, water, balanced salt solutions (BSS),
such as Hank's BSS, Earle's BSS, Grey's BSS, Puck's BSS, Simm's
BSS, Tyrode's BSS, BSS Plus, Ringer's lactate solution, normal
saline (i.e. 0.9% saline), 1/2 normal saline, the like, or
combinations thereof. In one aspect, the concentration of the
buffer can be from about 10 .mu.M to about 20 mM when used on the
solid-phase medium. In another example, the concentration of the
buffer can be from about 100 .mu.M to about 10 mM when used on the
solid-phase medium. In yet another example, the concentration of
the buffer can be from about 100 .mu.M to about 500 .mu.M when used
on the solid-phase medium.
[0169] A blocking agent can decrease the amount of RNase-based
degradation, DNase-based degradation, or other enzymatic
degradations. In one example, a blocking agent can include one or
more of bovine serum albumin, casein, or combinations thereof. In
one aspect, the concentration of the blocking agent can be from
about 0.01 wt % to about 5 wt % when used on the solid-phase
medium. In another example, the concentration of the blocking agent
can be from about 0.01 wt % to about 1 wt % when used on the
solid-phase medium. In yet another example, the concentration of
the blocking agent can be from about 0.02 wt % to about 0.06 wt %
when used on the solid-phase medium.
[0170] An antioxidant can increase the uniformity and contrast of
the pH-dependent signal on the solid-phase medium by eliminating
variables associated with oxidation reactions. In one example, the
composition can further comprise an antioxidant as disclosed
herein.
[0171] In another example, the composition can further comprise a
solid phase medium. The solid-phase medium can include, but is not
limited to, one or more of: glass fiber, nylon, cellulose,
polysulfone, polyethersulfone, cellulose acetate, nitrocellulose,
polyester, hydrophilic polytetrafluoroethylene (PTFE), or
combinations thereof
[0172] Certain additives may increase the stability and uniformity
of the LAMP reaction. In one aspect, the composition can comprise
one or more of an enzyme, a nucleic acid, or combinations thereof
as disclosed herein. In one example, the enzyme can be an RNase
inhibitor or a DNase inhibitor. In another aspect, the composition
can comprise carrier DNA or carrier RNA. The carrier DNA or carrier
RNA can provide decoy substrate that sequesters the activity of
DNase or RNase, respectively.
[0173] In another example, a selected amount of guanidine
hydrochloride can stimulate the denaturing and exposing of RNA
molecules. In one aspect, the concentration of the guanidine
hydrochloride can be from about 1 mM to about 200 mM when used on
the solid-phase medium. In another example, the concentration of
the guanidine hydrochloride can be from about 10 mM to about 100 mM
when used on the solid-phase medium. In yet another example, the
concentration of the guanidine hydrochloride can be from about 20
mM to about 60 mM when used on the solid-phase medium.
[0174] The maximization of the pH-dependent output signal can also
be used in conjunction with other embodiments as disclosed herein.
In one embodiment, a method of performing a LAMP analysis with a
pH-dependent output signal can comprise providing an assembly of a
solid phase medium and a composition as recited herein. The method
can further comprise depositing a biological sample onto the solid
phase medium. The method can further comprise heating the assembly
to an isothermal temperature sufficient to facilitate a LAMP
reaction.
[0175] As disclosed herein, in one aspect, the biological sample
can be one or more of saliva, mucus, blood, urine, feces, sweat,
exhaled breath condensate, the like, or combinations thereof. In
another aspect, the biological sample can be saliva. In one aspect,
the method can comprise detecting a viral pathogen. In one aspect,
the viral pathogen can be a pathogen as otherwise disclosed
herein.
[0176] In one example, a temperature sufficient to facilitate a
LAMP reaction can be in a temperature range of from about
60.degree. C. to about 70.degree. C. In another example, the
isothermal temperature can be a temperature within a range that
differs by less than 5 degrees Celsius.
[0177] In another aspect, the isothermal temperature sufficient to
facilitate a LAMP reaction can be in a temperature range from about
50.degree. C. to about 70.degree. C. In another aspect, the
isothermal temperature sufficient to facilitate a LAMP reaction can
be in a temperature range from about 60 .degree. C. to about
70.degree. C. In another aspect, the isothermal temperature
sufficient to facilitate a LAMP reaction can be in a temperature
range from about 60.degree. C. to about 65.degree. C. The
isothermal temperature can be selected based on one or more of the
activity of the DNA polymerase, reversed transcriptase, or
combinations thereof.
[0178] In another embodiment, as depicted in FIG. 3, a method 300
of maximizing accuracy of an output signal in a pH-dependent LAMP
analysis can comprise providing a reagent mixture that minimizes
non-LAMP reaction produced discoloration from a signal output
medium as in block 310. The method can further comprise performing
the LAMP reaction, as shown in block 320. In one aspect, the method
can comprise controlling production of protons from a non-LAMP
reaction. In another aspect, the method can comprise controlling
oxidation from a non-LAMP reaction.
[0179] In another embodiment, a method of maximizing accuracy of an
output signal in a pH-dependent LAMP analysis can comprise
substantially eliminating non-LAMP reaction produced discoloration
from a signal output medium.
[0180] In another embodiment, a method of maximizing a level of
detection (LOD) in a pH-dependent LAMP analysis can comprise
substantially eliminating non-LAMP reaction produced discoloration
from a signal output medium. In one aspect, the color contrast can
be enhanced and the sample variability can be mitigated without
impacting the limit of detection by diluting the saliva to 5-10%
with water. In another example, the color contrast can be enhanced
and the sample variability can be mitigated without impacting the
limit of detection by filtering the saliva with a filter as
otherwise disclosed herein.
EXAMPLES
[0181] The following examples are provided to promote a more clear
understanding of certain embodiments of the present invention, and
are in no way meant as a limitation thereon.
[0182] Paper LAMP Analysis for Viral Targets in Diluted Saliva
Samples
Example 1
DNase/RNase-Free Distilled Water
[0183] DNase/RNase-free distilled water is prepared by filtration
with 0.1 .mu.m membrane and tested for DNase and RNase activity.
The DNase and RNase activity is tested in accordance with current
U.S. Pharmacopeia (USP) monograph test standards for Water for
Injection (WFI). Upon confirmation of no DNase, RNase, or protease
activity the water is considered contaminant free and ready for use
in preparing saliva samples.
Example 2
Amplification in Saliva
[0184] A nucleic acid sequence primer was designed to target RNaseP
in saliva as a positive control to confirm nucleotide amplification
from a saliva sample as illustrated in FIG. 4. FIG. 4A illustrates
flouorometric RT-qLAMP results for primer sets targeting RNaseP
POP7 in 18% saliva spiked with 105 genome equivalents/reaction of
heat-inactivated SARS-CoV-2. FIG. 4B illustrates flouorometric
RT-qLAMP results for primer sets targeting RNaseP POP7 in water
with 0.2 ng of synthetic RNaseP POP7 RNA.
[0185] As illustrated in FIG. 4A, 18% saliva that has been spiked
with 105 genome equivalents per reaction of heat-inactivated
SARS-CoV-2 was analyzed. In the left figure, no amplification
occurred because the primer (RNaseP.I) which was designed to target
RNaseP using the mRNA sequence for the POP7 gene which encodes for
the p20 subunit of RNaseP, did not adequately detect the low levels
of the RNaseP. In the middle figure, amplification occurred, as
shown in the blue lines without overlap with the black lines,
because the primer (RNaseP.II) was able to detect levels of RNaseP
without amplifying the no template control. In the right figure,
amplification occurred for both the black lines and the blue lines
because the primer (RNaseP.III) dimerized (e.g., showed
amplification in the no template control black lines).
[0186] As illustrated in FIG. 4B, water with 0.2 ng of synthetic
RNaseP POP7 RNA was analyzed. In the left figure, amplification
occurred in the blue lines and black lines because the primer
(RNaseP.I) dimerized (e.g., amplified the no template control). In
the middle figure, amplification occurred, as shown in the blue
lines without overlap with the black lines, because the primer
(RNaseP.II) was a was able to detect the levels of the RNaseP
without amplifying the no template control. In the right figure,
amplification occurred for the blue lines but not the black lines
because the primer (RNaseP.III) amplified the RNase P without
amplifying the no template control.
Example 3
Saliva Collection Devices
[0187] The type of saliva collection device can facilitate a saliva
sample in a LAMP reaction. In some cases, an operator may use
protective equipment to protect from a pathogen that can be spread
via airborne droplets (e.g., an aerosol virus). Therefore,
operators can wear personal protective equipment to protect against
the accidental contact with the aerosol virus. The specific saliva
collection device may be self-administered by the subject under the
guidance of a healthcare professional. The saliva collection device
has a proven efficacy and can fall into two categories:
sponge-based collection and passive drool collection as illustrated
in FIGS. 5A and 5B.
[0188] A sponge-collection device 500a uses a sponge-like
collection pad 504 to absorb saliva and includes a sample volume
adequacy indicator 512 to indicate when sufficient volume has been
collected. Once saturated, the sponge is inserted into a
compression tube 506 and compressed against a filter which strains
the saliva into a collection tube. A reason for this filtration
operation is that it strains mucins and high molecular weight
proteins out of the saliva and significantly reduces viscosity of
the specimens. As a result, the solid phase medium can take-up and
distribute the saliva in a more rapid, uniform, and reliable
fashion. The sponge-collection device 500a can also include: a
compression seal 508 on the compression tube 506 to form a seal
with the compression tube; a handle 510 to compress the compression
tube 506; and a sample volume adequacy indicator 512 to identify
when sufficient saliva has been collected.
[0189] A passive drool device 500b can provide unfiltered saliva,
with viscosity that slows absorption and distribution of the
sample. The passive drool device 500b can include: a collection
funnel 522 for collecting saliva; an indicator line 528 for
indicating when sufficient saliva has been collected; a collection
tube 524 to collect the saliva; a tube cap 526; a volume indicator
530; and a tube cap storage 532.
[0190] For both types of collection devices, residual risk of
exposure to the operator is minimized. With the sponge-based
devices, there is a hypothetical risk of aerosol release during the
compression operation, especially if a user is particularly abrupt
in the compression process. The healthcare operator may perform
this operation to control the risk of exposure. The collection
device may include a compression seal to prevent aerosol backflow.
With passive-drool collection, there may be some risk of
contaminating the outside of the device with stray saliva, which
could then transmit a cross-contamination to the operator if not
handled properly. In both cases, exposure risks are mitigated by
the patient self-collection of the saliva sample.
[0191] Three commercial saliva collection devices were selected to
evaluate their effect on the RT-LAMP reaction in saliva. The three
devices were "Saliva Sampler.TM." produced by StatSure Diagnostic
Systems, Inc., "Pure-SAL.TM." produced by Oasis Diagnostics, and
"Super-SAL.TM." also produced by Oasis Diagnostics. The StatSure
Saliva Sampler.TM. provides a tube containing a buffer (e.g.,
Buffer 2000) used to collect saliva from a patient. Super-SAL uses
a cylindrical absorbent pad and a collection tube to standardize
the collection of saliva by removing any solid contaminants and
mucinous material. Pure-SAL operates on a similar mechanism but
includes an additional filter in the collection tube to remove
contaminants.
[0192] The saliva pH was measured from processed saliva samples,
and subsequent colorimetric and fluorescent RT-LAMP LOD assays were
run using processed saliva samples. This data is presented in FIG.
6A. This data illustrates the LoD in saliva processed using
different saliva collection devices (Pure-Sal, Super-Sal,
Stat-sure). The master-mix was treated with 0.6 microliter of HC1.
The Pure-SAL.TM. and Super-SAL saliva collection devices illustrate
a wider range of colorimetric response for a wider range of
concentrations (1 to 10k genome equivalents per reaction of heat
inactivated SARS-CoV-2).
Example 4
Saliva Collection Process
[0193] If a subject is self-testing, the subject collects a saliva
specimen under the guidance of the healthcare professional into a
specialized collection vessel which contains no additives and is
thus safe for the subject to use in the collection. The collected
volume of saliva is approximately 100 .mu.L. The sponge sampler for
example is inserted into the subject's mouth and saliva is
collected until the indicator on the sponge sampler changes color.
The sponge sampler is then inserted into a collection tube. The
sponge is then compressed to squeeze out the saliva (approximately
100 .mu.L) into a collection tube containing an amount of water in
it to dilute the saliva. The saliva is diluted in the water to a
saliva to water ratio of about 1:1 to about 1:20. The saliva is
transferred from the collection tube to a test site.
Example 5
RNase Inhibitor Effect On Saliva
[0194] An RNase inhibitor was added to untreated saliva at a
concentration of 1 .mu.L per mL of saliva to determine the effect
of the addition of an RNase Inhibitor on the RT-LAMP reaction.
[0195] The effect of RNAsecure.TM. (AM7006, Invitrogen.TM.) on
freshly collected saliva (5%) was tested to determine its
suitability as a single operation process for a point-of-care
RT-LAMP reaction. 1X RNAsecure.TM. was diluted from 25X stock using
1 ml of saliva. The treated saliva was used as a matrix to spike
heat-inactivated SARS-CoV-2 with a concentration range from 1000
copies to 62.5 copies/reaction into the Warmstart.TM. colorimetric
master mix along with 40mM of guanidine hydrochloride and
0.3ng/.mu.l carrier DNA with a pH of 7.6. The RNAsecure.TM.treated
RT-LAMP was incubated at 65.degree. C. to start the reaction. Fresh
saliva under these conditions was tested without RNAsecure.TM. as a
control.
[0196] 5 .mu.L of heat-inactivated virus was diluted in 5%
processed saliva (i.e., final reaction concentration) and added to
the RT-LAMP reaction to provide the indicated concentration for a
final reaction volume of 25 .mu.L. For negative reactions, 5 .mu.L
of processed saliva (5% final reaction concentration) was added in
lieu of diluted heat-inactivated virus to provide the same reaction
volume of 25 .mu.L. Heating was conducted in the incubator at
65.degree. C. for 60 mins. The colorimetric scans were taken using
the flatbed scanner before and after the RT-LAMP reaction. 1250
.mu.L of NEB colorimetric master mix was supplemented with 0.5
.mu.L of Antarctic Thermolabile, 3.5 .mu.L of dUTP. 25X. RNase
Inhibitor (RNASecure.TM.) was diluted in whole saliva to result in
a 1X concentration prior to adding to the reaction diluting to 5%
saliva.
[0197] RNAsecure.TM. did not show any significant increase in the
LoD of the reaction, as illustrated in FIG. 6B. That is, the
addition of RNase inhibitor did not result in an appreciable
increase in the measured parameters of the RT-LAMP reaction (e.g.,
the reaction speed, the false-positive rate, or the limit of
detection).
Example 6
Frozen Saliva Samples
[0198] In some instances, it may be desirable to freeze a saliva
sample for a period of time prior to its analysis due to logistics,
need for transport, etc. Such situations may merit specific care
when performing a LAMP analysis as recited herein. As illustrated
in FIG. 7, the pH of a frozen saliva sample can vary depending on
the number of days at -20 degrees C. before the saliva sample is
thawed and tested. In one example, the pH of the saliva sample from
Donor 1 varied from a pH of 7.21 without any days between
collection and testing to a pH of 7.46 after 6 days between
collection/freezing and testing. In another example, the pH of the
saliva sample from Donor 2 varied from a pH of 7.00 without any
days between collection and testing to a pH of 6.98 after 6 days
between collection/freezing and testing. In one example, the pH of
the saliva sample from Donor 3 varied from a pH of 7.18 without any
days between collection and testing to a pH of 7.18 after 6 days
between collection/freezing and testing. In one example, the pH of
the saliva sample from Donor 4 varied from a pH of 7.35 without any
days between collection and testing to a pH of 7.47 after 6 days
between collection/freezing and testing. In one example, the pH of
the saliva sample from Donor 1 varied from a pH of 7.22 without any
days between collection and testing to a pH of 7.24 after 6 days
between collection/freezing and testing.
Example 7
Limit of Detection in Fresh Saliva
[0199] FIG. 8 illustrates the limit of detection of fresh saliva.
Fresh saliva was collected using a drooling method and was diluted
in water in a 1:3 ratio to obtain 25% saliva and 75% water.
Heat-inactivated SARS-CoV-2 was spiked into the 25% saliva with
serial dilutions, as a control. 5 .mu.L of 25% saliva was added to
20 .mu.L of RT-LAMP reagents so that the final concentration of
saliva was 5%. After incubating at 65.degree. C. for 1 hour the
color changed. The number of copies on the y-axis represents what
the original concentration of 100% saliva would have be without
dilution. The limit of detection (LOD) for the primer was 250
copies/reaction in a volume of 25 .mu.L, which is equivalent to
about 200k copies/mL of saliva.
[0200] Thus, it was determined that diluting the saliva to 25% with
nuclease-free water and further diluting to a final concentration
of 5% saliva upon addition to the RT-LAMP reaction could obtain
results within 60 minutes. Dilution reduced the buffering capacity
of saliva and decreased the concentration of inhibitory components,
both of which would delay colorimetric reporting. Dilution is less
complex to the end-user compared to other pre-treatment operations
found in a variety of studies, such as pre-treatment with
proteases, Chelex.RTM. 100, or RNA extraction operations to
inactivate inhibitory components of saliva.
[0201] The LoD of the colorimetric assay in 5% saliva that has been
processed using Pure-SAL.TM. is 1000 copies/reaction (reaction
volume 25 .mu.L), which corresponds to 800 copies/.mu.L of patient
saliva after accounting for dilution (FIG. 6C).
[0202] As illustrated in FIG. 6C, different saliva collection
devices (Pure-SAL.TM., Super-Sal.TM., Stat-sure.TM.) can result in
varying LoD. 5% diluted saliva with water was tested for all the
processing techniques. Primer set orflab.2 was used. 5 .mu.L of
heat-inactivated virus diluted in 5% processed saliva (final
reaction concentration) was added to the RT-LAMP reaction to result
in the indicated concentration and a final reaction volume of 25
.mu.L. For negative reactions, 5 of processed saliva (5% final
reaction concentration) was added in lieu of diluted
heat-inactivated virus to provide the same reaction volume of 25 0.
Heating was conducted in an incubator at 65.degree. C. for 60 mins.
The colorimetric scans were taken using the flatbed scanner before
and after the RT-LAMP reaction. Reactions consisted of 12.5 .mu.L
of NEB 2X colorimetric master mix, 2.5 .mu.L of primer mix, 5 .mu.L
of water, and 5 .mu.L of sample.
[0203] This LoD is several orders of magnitude higher than RT-PCR
assays or other assays utilizing RNA extraction (on the order of 1
copy/reaction). However, these other assays were accompanied by
pretreatment protocols and/or RNA extraction operations to achieve
the reported LoD.
[0204] To enhance this LoD, we investigated the use of RNase
inhibitors, Guanidine HC1, and carrier DNA. The addition of RNase
inhibitors lowered the LoD in 5% saliva (FIG. 6B), which
contradicts literature reports in which RT-LAMP assays in saliva
utilize RNase inhibitors increased the LoD; this discrepancy may be
due to the type of RNase inhibitor used. Both
[0205] Guanidine HCl and carrier DNA increased the LoD (FIG. 6D and
FIG. 6E) and was added to our RT-LAMP reaction formulation for
colorimetric solution reactions. These components were not included
because of a color change when drying on paper.
[0206] As illustrated in FIG. 6D, 5 .mu.L of heat-inactivated virus
diluted in 5% processed saliva (final reaction concentration) was
added to the RT-LAMP reaction to result in the indicated
concentration and a final reaction volume of 25 .mu.L. For negative
reactions, 5 .mu.L of processed saliva (5% final reaction
concentration) was added in lieu of diluted heat-inactivated virus
to result in the same reaction volume of 25 .mu.l. Primer set
orflab.2 was used. Heating was conducted in an incubator
(Fisherbrand.TM. Isotemp.TM.) at 65.degree. C. for 60 mins. The
colorimetric scans were taken using the flatbed scanner before and
after the RT-LAMP reaction. 1250 .mu.L of NEB colorimetric master
mix was supplemented with 0.5 .mu.L of Antarctic Thermolabile UDG,
3.5 .mu.L of dUTP, and carrier DNA to result in a final reaction
concentration as indicated.
[0207] As illustrated in FIG. 6E, 5 .mu.L of heat-inactivated virus
diluted in 5% processed saliva (final reaction concentration) was
added to the RT-LAMP reaction to result in the indicated
concentration and a final reaction volume of 25 .mu.L. For negative
reactions, 5 .mu.L of processed saliva (5% final reaction
concentration) was added in lieu of diluted heat-inactivated virus
to result in the same reaction volume of 25 .mu.l. Primer set
orflab.2 was used. Heating was conducted in an incubator
(Fisherbrand.TM. Isotemp.TM.) at 65.degree. C. for 60 mins. The
colorimetric scans were taken using the flatbed scanner before and
after the RT-LAMP reaction. 1250 .mu.L of NEB colorimetric master
mix was supplemented with 0.5 .mu.L of Antarctic Thermolabile UDG,
3.5 .mu.L of dUTP, and Guanidine HCl (40 mM).
[0208] Finally, uracil-DNA glycosylase (UDG) and deoxyuridine
triphosphate (dUTP) (FIG. 6F) were included to reduce carryover
contamination. As illustrated in FIG. 6F, Colorimetric scans after
60 minutes of incubation at 65.degree. C. for 25uL reactions on the
thermomixer and incubator with and without the addition of UDGs and
dUTP. The Primer set used was orflab.II. The template was
heat-inactivated virus at the indicated concentration. For
reactions with UDG, 1250 .mu.L of NEB 2x colorimetric master mix
was supplemented with 0.5 .mu.L of Antarctic Thermolabile UDG, 3.5
.mu.L of dUTP. For all other reactions NEB 2x colorimetric master
mix was used.
[0209] When including Guanidine HC1, carrier DNA, and UDG, the LoD
of the RT-LAMP colorimetric assay in 5% processed saliva in
solution increased to 250 copies/reaction (FIG. 6G).
[0210] As illustrated in FIG. 6G, RT-LAMP colorimetric LoD using
saliva processed with Pure-SAL.TM. and saliva that was unprocessed.
Plates were heated in an incubator set at 65.degree. C. for 60
minutes. The Primer set used was orflab.II and the template was
heat-inactivated virus at the indicated concentration (positive
reactions) or nuclease-free water (negative reactions). Heating was
conducted in an incubator (Fisherbrand.TM. Isotemp.TM.) at
65.degree. C. for 60 mins. The colorimetric scans were taken using
the flatbed scanner before and after the RT-LAMP reaction. 1250
.mu.L of NEB colorimetric master mix was supplemented with 0.5
.mu.L of Antarctic Thermolabile UDG, 3.5 .mu.L of dUTP, carrier DNA
(0.3 ng/.mu.L), and Guanidine HCl (40 mM).
Example 8
Limit of Detection in Animal Nasal Swab
[0211] FIG. 9 illustrates the limit of detection in a bovine nasal
swab that was re-suspended in about 1 mL water. Heat-inactivated
SARS-CoV-2 was spiked into water with the re-suspended background
mucus and microbiome to obtain the same number of copies/reaction
as in the previous example with saliva. 5 .mu.L of sample was added
to 20 .mu.L of RT-LAMP. After incubating at 65.degree. C. for about
1 hour, the color changed. The LOD for the primer was about 250
copies/reaction in a volume of 25 .mu.L, which is equivalent to
about 5k copies/mL of nasal swab resuspension.
Example 9
Limit of Detection on Paper
[0212] FIG. 10 illustrates the limit of detection on paper. 20
.mu.L of RT-LAMP reagents were added to Grade 1 chromatography
paper. Heat-inactivated SARS-CoV-2 was spiked into the 100% pooled
saliva with serial dilutions of the virus. 15 .mu.L of about 100%
saliva was added to each piece of paper. After incubating at
65.degree. C. for 90 minutes the color changed. The LOD for the
primer was about 3k copies/reaction in a volume of 15 .mu.L saliva,
which is equivalent to about 20k copies/mL of saliva.
Reagent Compositions that Facilitate LAMP Analysis on Paper
Example 10
Sample Reagents
[0213] In one example, the reagents were included as shown in Table
A1. In another example, the reagents were included as shown in
Table A2.
TABLE-US-00001 TABLE A-1 Reagents Deoxynucleotide (dNTP) Solution
Set (100 mM of each individually dATP, dCTP, dGTP, and dTTP)
Deoxynucleotide (dNTP) Solution Mix TWEEN .RTM. 20 Bst 2.0
WarmStart .RTM. DNA Polymerase Bst 3.0 DNA Polymerase WarmStart
.RTM. RTx Reverse Transcriptase Tris-HCl Primer sets Target DNA for
QA testing
TABLE-US-00002 TABLE A-2 Reagents Potassium chloride ( Magnesium
sulfate Deoxynucleotide triphosphate (dNTP) Bst 2.0 DNA Polymerase
WarmStart .RTM. RTx Reverse Transcriptase Phenol Red Antarctic
Thermolabile uracil DNA glycosylase (UDG) Polysorbate 20 Betaine
Bovine Serum Albumin Trehalose Nuclease-Free Water RNase AWAY
Example 11
Buffer Selection and Concentration
[0214] Since the pH of saliva can vary from sample to sample, a
buffer was used on the paper-based device to maintain a consistent
starting pH. For phenol red, a pH of 7.6 was a suitable starting
point to enhance the colorimetric transition as illustrated in FIG.
11. A few buffers having a pKa of about 8 were screened because the
starting pH of 7.6 was close to the limits of the buffering range
to allow a color change when amplification happens. 10 mM of BICINE
buffer was used for the paper-based assay as illustrated in FIG.
12.
Example 12
Effect of Primers on Reaction Speed
[0215] In order to increase the speed of the RT-LAMP reaction, the
inclusion of multiple primer sets in the fluorescent RT-LAMP
reaction mix was investigated. The investigation was carried out in
water using NEB LAMP fluorescent dye as a fluorometric indicator.
The inclusion of multiple primer sets did not seem to increase the
reaction speed significantly. Rather, the reaction proceeded
primarily at the speed of the primer set that had the fastest
reaction time when used in isolation.
Example 13
Sample LAMP Protocol, Reagents, Validation, and Troubleshooting
Sample Lamp Protocol 13-A:
Primer Mix
[0216] 1. Obtain all 6 diluted primers from the freezer; 2. Mix 80
.mu.1 of FIP, 80 .mu.1 of BIP, 20 .mu.1 of FB, 20 .mu.1 LB, 10
.mu.1 of F3 and 10 .mu.1 of B3 in a tube; 3. Add enough PCR-grade
water to reach 500 .mu.1.
LAMP
[0217] 1. Obtain the NEB Bst 2.0 Warmstart kit and the primer mix;
2. While the reagents thaw and after at least 5 minutes of spraying
the DNAway, wipe the surfaces with a Kimwipe; 3. Label all the PCR
tubes needed with the DNA sample and primers that will be used.
Make sure to add a negative control which will not have DNA added;
4. Add 5 .mu.l of PCR-grade water (or dye), 12.5 .mu.1 of NEB Bst
2.0 Warmstart kit and 2.5 .mu.l of primer mix per reaction. A
master mix can be made for however many reactions will be run; 5.
If 5 .mu.1 of EBT dye are added, it should be in 1500 .mu.M
concentration so that the final concentration ends up being 300
.mu.M; 6.The reactions with no DNA should have an extra 5 .mu.l of
PCR-grade water added and not opened again until they have to be
loaded on a gel; 7. Once ready, the PCR tubes should be put in the
PCR tray previously left in the pass-through chamber and carried
out to BRK 2037; 8. Once in BRK 2037, obtain the sample DNA from
the -20.degree. C. freezer; 9. Spray your hands with DNAway spray
and rub your hands around the DNA sample tube so that it is covered
in the spray as well; 10. Add 5 .mu.l of the DNA sample where
appropriate and close the tubes. Never open 2 DNA tubes at the same
time and close the PCR tubes right after adding the DNA; 11. Put
the samples in a thermocycler set at 65.degree. C. for 1 hour and
80.degree. C. for 5 minutes (samples may be kept in at -20.degree.
C. overnight after this operation).
Sample Reagent Concentrations 13-B:
[0218] Colorimetric RT-LAMP master mix can be: KC1 (50 mM), MgSO4
(8 mM), dNTP mixture (1.4 mM each dNTP), Bst 2.0 WarmStart.RTM. DNA
Polymerase (0.32 U/.mu.L), WarmStart.RTM. RTx Reverse Transcriptase
(0.3 U/.mu.L), Phenol red (0.25 mM), dUTP (0.14 mM), Antarctic
Thermolabile UDG (0.0004 U/.mu.L), Tween.RTM. 20 (1% v/v), betaine
(20 mM), BSA (500 .mu.g/mL), and trehalose (10% w/v).
[0219] These components were titrated each from 0.25X liquid
concentration to 5X or higher for the paper LAMP assay. The
concentration was determined by the speed of the LAMP reaction, the
contrast between positive and negative LAMP results at 60 minutes
reaction time, and the reduced amount of non-specific
amplification.
[0220] To determine the concentration of protein stabilizing
additives, D-(+)-trehalose dihydrate was titrated from 0% to 15%
w/v using increments of 5% and lyophilized BSA was titrated from 0
to 1.25 mg/mL using increments of 0.2 mg/mL. The concentrations for
trehalose and BSA were 10% w/v and 0.626 mg/mL, respectively.
Sample Lamp Protocol 13-C:
Reagents
[0221] Reagents as shown in Table A-2 of Example 10.
Equipment
[0222] Forceps, 0.5-10 .mu.L Pipette, 2-20 .mu.L Pipette, 20-200
.mu.L Pipette, 100-1000 .mu.L Pipette, Ahlstrom-Munksjo Grade 222,
pH probe, a heat source that could reach 65.degree. C. (e.g.,
incubator, water bath), PCR hood.
Sanitization:
[0223] Spray pipettes and all workbench (PCR hood) surfaces with
RNase AWAY. Wipe thoroughly after applying RNase AWAY. The RNase
AWAY can interfere with the reaction if there is any remaining. Use
separate rooms for manufacturing the paper-based device and loading
the samples to aid in the prevention of cross-contamination.
Pre-cut 5 mm x 6 mm chromatography paper.
LAMP Preparation:
[0224] 1. Prepare 2X LAMP mix as indicated in Table 13B-1 inside a
PCR hood. 2. Adjust pH with 1M KOH (approximately 1-2 .mu.L) to
pH.about.7.5-8.0 (a red but not pink color). Does not need to be
precise. After adjusting pH, 2X LAMP Mix can be stored at
-20.degree. C.
TABLE-US-00003 TABLE 13B-1 2X LAMP Mix Stock Final Components
Volume Unit concentration Unit concentration Unit KCL 100 .mu.L
1000 mM 100 mM MgSO.sub.4 160 .mu.L 100 mM 16 mM dNTPs 280 .mu.L 10
mM 2.8 mM Bst 2.0 DNA Polymerase 10.8 .mu.L 120 U/.mu.L 1.296
U/.mu.L RTx Reverse Transcriptase 40 .mu.L 15 U/.mu.L 0.6 U/.mu.L
Phenol red 20 .mu.L 25 mM 0.2 mM dUTPs 2.8 .mu.L 100 mM 0.28 mM
Antarctic Thermolabile 0.4 .mu.L 1 U/.mu.L 0.0004 U/.mu.L UDG
Polysorbate 20 100 .mu.L 20 % 2 % Nuclease-Free Water 286 .mu.L --
-- -- -- Total 1000 .mu.L
[0225] 3. Prepare a master mix according to Table 13B-2.
TABLE-US-00004 TABLE 13B-2 Complete mix for paper LAMP Stock Final
Components Volume Unit concentration Unit concentration Unit 2X
LAMP Mix 125 .mu.L -- -- -- -- 10X Primer Mix .sup.a 25 .mu.L -- --
-- -- Betaine 1 .mu.L 5 M 20 mM BSA 3.13 .mu.L 40 mg/mL 0.626 mg/mL
Trehalose 36 .mu.L 689 mg/mL -- -- Water 9.2 .mu.L Total 200 .mu.L
.sup.a Stocks of the LAMP primers can be made at a workable
concentration in water for ease of setup. A 10X Primer Mix
containing all 6 LAMP primers. 10X Primer mix: 16 .mu.M FIP/BIP, 2
.mu.M F3/B3, 4 .mu.M Loop F/B can be made.
[0226] 4. Adjust pH to 8.0 with 0.1M KOH. Use a micro-pH electrode.
5. Mix thoroughly. Lay paper pads out on a clean surface inside the
PCR hood. Add 30 .mu.L of complete mix on pre-cut grade 222 paper
pads. 6. Dry under PCR hood at room temperature for 60 minutes. 7.
After drying, collect paper pads into a clean centrifuge tube or a
clean re-sealable plastic bag.
Sample Loading
[0227] 1. Spray down the working bench with RNase AWAY and clean it
with wipers. 2. Take out templates (DNA, RNA, heat-inactivated
virus) from the freezer. 3. Lay out the reaction pads on a clean
surface. You can lay your pads on a new transparency film and
discard it after use. 4. Prepare negative control pads first.
Reconstitute pads with 25 .mu.L non-template solvent (water,
saliva). The reconstitution process should be gentle, avoid washing
out the regents from the pad. 5. Place the negative control pads
inside a clean container (e.g., 1'' x 1'' resealable plastic bag,
centrifuge tube) using forceps. 6. Dilute template with solvent
into desire concentration. 7. Lay out more reaction pads and
reconstitute pads with 25 .mu.L diluted template. 8. Place the
positive pads inside a clean container (e.g., 1'' x 1'' resealable
plastic bag, centrifuge tube) using forceps. 9. Clean up the
workspace and bring the pads for imaging and incubating.
Imaging and incubation:
[0228] Note: There are multiple imaging methods (e.g., time-lapse
video, scanning) and heat sources (e.g., incubator, water bath). In
this protocol, a tabletop scanner and a microbiological incubator
can be used. 1. Arrange the pads on top of the scanner. Scan the
pads before the reaction (0 min). 2. Preheat the incubator to
65.degree. C. 3. Place the pads into the incubator. Separate the
pads. The heating uniformity can affect the result consistency. 4.
Take out pads and repeat scanning at different time points (usually
every 30 minutes). 5. After final scanning, discard the reaction
pads inside biohazard trash.
Validation:
[0229] To verify the occurrence of LAMP amplification, each
reaction pad was transferred to a clean 1.5 mL microcentrifuge
tube. 100 .mu.L Buffer EB was added to each tube. Reaction pads
were submerged in Buffer EB overnight for eluting nucleic acids.
Gel electrophoresis (2% agarose gel) was done with the eluent to
verify the occurrence of LAMP amplification. A ladder-like pattern
(typical LAMP product pattern) was shown in each positive pad lane
while there was no obvious band in each negative lane (FIG. 13A and
13B).
[0230] As illustrated in FIGS. 13A and 13B, paper LAMP validation
was performed. As shown in FIG. 13A, LAMP on paper with two
conditions (with and without BSA in the reaction mix) was
performed. As shown in FIG. 13B associated gel electrophoresis (2%
agarose) was performed. The orf7ab.1 primer set targeting
SARS-CoV-2 was used. Negative reaction pads were reconstituted with
25 .mu.L nuclease free water. Positive reaction pads were
reconstituted with 25 .mu.L 400 copies/.mu.L heat-inactivated
SARS-CoV-2 virus. Heating was carried out in an incubator set at
65.degree. C. and scanned in a flatbed scanner.
[0231] BSA is a reagent that can be used in the LAMP mix. Adding
BSA can speed up reactions and increase sensitivity, as illustrated
in FIG. 14A and FIG. 14B. In these reactions, a low template
concentration LAMP on paper was performed subject to two conditions
(with and without BSA in the reaction mix). FIG. 14A shows a
0-minute time point. FIG. B shows a 60-minute time point. The
orf7ab.1 primer was used in this experiment. Negative reaction pads
were reconstituted with 25 .mu.L nuclease free water. Positive
reaction pads were reconstituted with 25 .mu.L 8 copies/.mu.L and
16 copies/.mu.L heat-inactivated SARS-CoV-2 virus (to reach a final
concentration of 200 copies/reaction and 400 copies/reaction),
respectively.
[0232] However, BSA can also introduce pH variations to the device.
FIG. 13A and FIG. 14B show that after incubation (60 min) negative
paper pads containing BSA have a yellowish edge. After elution and
running the eluent with gel electrophoresis, there was no DNA
product visible on the gel, as shown in FIG. 13B, indicating that
the yellow color at the edge is not caused by off-target
amplification or contamination. A heterogeneous distribution of BSA
can lead to the yellow color at the edges upon application of
heat.
Troubleshooting:
[0233] Unusual pink color on paper pads: During the process of
preparing LAMP paper pads, there can be unusual pink spots
different from the surrounding color, which can be caused by
residual RNase AWAY either directly sprayed onto the pads and/or
transferred via the forceps. RNase AWAY can degrade any RNA/DNA
template added. If this occurs, thoroughly dry all equipment and
surfaces, cut new 5x6 mm paper pads, and restart the `LAMP
preparation` section from Operation 5.
[0234] Overflowing of reagents after pad reconstitution: During the
sample loading operation, the pad can be unable to absorb the
entire sample volume added to it for reconstitution. The template
concentration may not be accurately represented by overflowed pads.
Overflowing can be caused by insufficient drying of the pads. If
this occurs: 1) dry for a longer time, 2) use an enhanced drying
method such as heat drying (place on a clean microbiological
incubator at 37.degree. C.; do NOT set the temperature higher than
45.degree. C. to prevent activation of the Bst 2.0 WarmStart.RTM.
polymerase) or convective drying (use small fans to enhance airflow
during drying), or 3) reduce reconstitution volume to 20 .mu.L.
[0235] Negative controls exhibit color change: During the imaging
and incubation operation, the negative pads can change at the same
time or shortly after the sample-containing pads. This can be
caused by either primer dimerization/non-specific amplification or
carryover contaminants of previous LAMP reactions. To resolve,
validate the primer in liquid-based LAMP prior to using them on
paper. To control carryover contaminations, 1) implement dUTPs and
UDG in all LAMP reactions, 2) maintain separate working stations
for LAMP mixture preparation and sample addition, and 3) aliquot
reagent stocks and use new aliquots if contamination is suspected
to have occurred. Over-incubating the reaction can also induce
non-specific amplification. Do not exceed an incubation time of 75
minutes.
[0236] Sample pH and buffering capacity will influence colorimetric
readouts: Since phenol red is a pH indicator, the pH of the sample
and its buffering capacity can have a significant impact on the
assay. It has been confirmed that saliva concentrations of 5-10%
v/v (diluted with water) work with the reagent composition
presented here. 5% saliva was selected because that concentration
has a faster response time and produces consistent results. Human
saliva has a sophisticated buffering system that includes
bicarbonate, phosphate, and proteins, which prevents pH change (and
thus color change) at high saliva concentration. The paper LAMP
device with nasal swab resuspended in water was tested and not show
any inhibition from the sample matrix. Colorimetric readouts can
also be hampered by buffered salt solution (e.g., transport
media).
Example 14
Limit of Detection on Paper of Untreated Saliva With Inactivated
Virus
[0237] As illustrated in FIG. 15, whole untreated saliva with heat
inactivated SARS-CoV-2 virus validated the limit of detection of
about 20 copies per .mu.L saliva. Various sample concentrations
including 20 copies/.mu.L (1.times. LoD--10 samples), 40
copies/.mu.L (2.times. LoD--10 samples), 100 copies/.mu.L (2
samples), 1000 copies/.mu.L (2 samples), 10,000 copies/.mu.L (2
samples), 100,000 copies/.mu.L (2 samples), 1,000,000 copies/.mu.L
(2 samples) were created using aliquots of pooled saliva (30
aliquots) as the negative samples. The results were confirmed using
image processing.
Reagent Compositions that Maximize PH-Sensitive Signal Output
Example 15
Sample LAMP Dyes
TABLE-US-00005 [0238] TABLE B Dye Phenol red solution Litmus
Bromothymol Blue Nitrazine Yellow Cresol Red, Sodium salt Curcumin
Brilliant Yellow m-Cresol Purple .alpha.-Naphtholphthalein Neutral
Red Acid fuchsin Acid fuchsin, Calcium salt
Example 16
[0239] Fluorescent reporters would use an additional ultraviolet
(UV) light source to be read without specialized instrumentation.
However, a colorimetric assay using phenol red as an indicator
would not use UV light, and can be interpreted by the naked eye.
Polymerization of DNA produces protons and phenol red is responsive
to pH. Diluted saliva (5% final concentration) was used to overcome
the buffering capacity of saliva to measure changes in pH. Diluting
saliva to a 5% final concentration also reduced the concentration
of interferents (e.g., RNase).
[0240] Incorporating carrier DNA and guanidine hydrochloride also
enhanced the LoD and provided a colorimetric response that was
comparable in water and in saliva. The mechanism by which carrier
DNA increase the LAMP results was unclear. This mechanism was
explored by using NEB 1kb DNA ladder (NEB-N3232L). Different
concentrations of carrier DNA were used (0.3ng/.mu.l and
0.75ng/.mu.l) to study the effect on the LoD. Guanidine chloride
(40mM) was also used. The pH of the complete master mix was
maintained at 7.6., and the same condition was also tested without
carrier DNA. Fresh saliva (5%) with a pH of 6.5 was used to test
the effect of carrier DNA along with guanidine chloride with the
heat-inactivated SARS-CoV-2 at a concentration range of from 1000
copies to 62.5 copies/reaction (FIG. 6D).
[0241] Guanidine hydrochloride was reported to increase the
sensitivity of LAMP. Its performance with our primer set was tested
by adding 40mM of guanidine hydrochloride to the NEB Warmstart.TM.
colorimetric master mix with a pH of 7.6. Pooled saliva (5%) with a
pH of 6.5 was used to test the effect of guanidine chloride with
the heat-inactivated SARS-CoV-2 with a concentration range of from
1000 copies to 62.5 copies/reaction. This same composition was also
tested without adding guanidine chloride as a control. Guanidine
chloride increased the replicate sensitivity and has a consistent
amplification across the replicate (FIG. 6E).
[0242] Due to the different mechanisms in which phenol red and
fluorescent dye report LAMP-based nucleic acid amplification, these
differences in signal measurement over time were investigated.
Reactions were prepped on FrameStar 96-well skirted optical bottom
plates with a combination of Warmstart.TM. Colorimetric LAMP 2x
Master Mix and LAMP Fluorescent Dye, sealed with Thermo
Scientific.TM. Adhesive Plate Seals, and placed in a Clariostar.TM.
Plus Microplate Reader (BMG) to for incubation and measurement of
absorbance and fluorescent intensity. Color change over time was
expressed as a ratio of A.sub.432 nm/A.sub.560 nm. Absorbance
values were baseline-corrected by subtracting A.sub.432 nm and
A.sub.560 nm by A.sub.620 nm.
[0243] Based on FIG. 16, color changes in reactions occurred later
compared to fluorescence changes. Colorimetric and fluorometric
data was collected on BMG CLARIOstar.RTM. Plus plate reader.
Reaction base mix consisted of NEB 2x Colorimetric LAMP master mix,
2.5 .mu.L of primer mix, and 5 .mu.L of a 1:100 dilution of NEB
LAMP Fluorescent Dye (NEB B1700A). The chamber temperature of the
plate reader was allowed to equilibrate to 65.degree. C. prior to
inserting the plate. 5 .mu.L of heat-inactivated virus diluted in
water was added to the reaction base mix to result in the final
reaction concentrations as indicated (positive reactions). For NTC
reactions, 5 .mu.L of nuclease-free water was added to the reaction
base mix. This difference in change suggested that pH-based
reporters give a slower response to LAMP-based DNA amplification
than fluorescent reporters.
Example 17
[0244] Paper can be scaled up to millions of devices, but when
RT-LAMP reagents were placed on paper, the paper changed color even
when a negative control was used even though no amplification was
occurring. One possibility for this color change is oxidation of
cellulose caused by the heat and the oxidizing nature of ammonium
sulfate present in the RT-LAMP mixture. Another possibility for
this color change is acidification of the reagents due to degassing
of ammonia from the RT-LAMP mixture. Eliminating ammonium sulfate
maintained the color for a negative control. Increasing the
concentration of phenol red, which acts as an antioxidant, also
maintained the color for the negative control as illustrated in
FIG. 11.
Example 18
Screening of Colorimetric Dyes
[0245] Three classes of colorimetric indicators were evaluated for
a paper-based assay: (i) magnesium colorimetric indicators, (ii) pH
colorimetric indicators, and (iii) DNA intercalating colorimetric
indicators.
[0246] For magnesium indicators, Calmagite (CAS# 3147-14-6),
Xylidly blue I (CAS# 14936-97-1), Chlorophosphonazo III (CAS#
1914-99-4), o-Cresolpthalein Complexone (CAS# 2411-89-4),
Eriochrome.RTM. Black T (EBT, CAS# 1787-61-7), and Hydroxynapthol
blue (HNB, CAS# 63451-35-4) were screened.
[0247] For pH indicators, Bromothymol Blue (CAS# 76-59-5), Acid
Fuchsin (CAS# 3244-88-0), Nitrazine yellow (CAS# 5423-07-4), Cresol
red (CAS# 1733-2-6), Cresol red sodium salt (CAS# 62625-29-0),
Curcumin (CAS# 458-37-7), Phenol red (CAS# 143-74-8), Phenol red
sodium salt (CAS# 34487-61-1), Brilliant yellow (CAS# 3051-11-4),
o-Cresolphthalein (CAS# 596-27-0), m-Cresol purple (CAS#
2303-01-7), m-Cresol purple sodium salt (CAS# 62625-31-4),
.alpha.-Naptholphthalein (CAS# 596-01-0), and Neutral red (CAS#
553-24-2) were screened.
[0248] For DNA intercalating dyes, Crystal violet (CAS# 548-62-9)
was screened.
[0249] Many magnesium indicators did not produce a consistent color
change on paper. The metal ion indicators (calmagite and EBT)
interacted with magnesium(II) ions in solution whose concentration
decreased throughout the RT-LAMP experiment due to the formation of
magnesium pyrophosphate, a byproduct of the polymerase
reaction.
[0250] FIG. 17A shows the colorimetric response of calmagite at
varying concentrations throughout the LAMP reaction using genomic
DNA as template. LAMP detection was performed with increasing
concentrations of calmagite (magnesium indicator). The lo1B.3
primer set targeting Histophilus somni genomic DNA was used.
Positive reactions were spiked with 5 .mu.L of HS gDNA at a
concentration of 0.2 ng/.mu.L. Negative reactions used 5 .mu.L of
nuclease-free water. The total reaction volume was 25 .mu.L.
Reactions were prepared using 12.5 of NEB Warmstart 2x master mix,
2.5 .mu.L or primer mix, and 5 .mu.L of either template (positive
reactions) or water (negative) as above, and 5 .mu.L of Calmagite
prepared in water to produce a final concentration as
indicated.
[0251] At the concentrations tested, a visual change was not
detected during the LAMP reaction. EBT showed a detectible color
change from violet to a dark blue between the 0 minute and 60
minute time points of the LAMP reaction; however, the color change
was not clear, which can interfere with interpretations in clinical
settings.
[0252] As illustrated in FIG. 17B), LAMP detection was performed
with increasing concentrations of Eriochrome.RTM. Black T
(magnesium indicator). The lo1B.3 primer set targeting Histophilus
somni genomic DNA (gDNA) was used. Positive reactions were spiked
with 5 .mu.L of HS gDNA at a concentration of 0.2 ng/.mu.L.
Negative reactions used 5 .mu.L of nuclease-free water. Total
reaction volume is 25 .mu.L. Reactions were prepared using 12.5
.mu.L of NEB Warmstart 2x master mix, 2.5 .mu.L or primer mix, and
5 .mu.L of either template (positive reactions) or water (negative)
as above, and 5 .mu.L of EBT prepared in water to produce a final
concentration as indicated.
[0253] Additionally, using LAMP on paper using EBT did not result
in a detectable color change. As illustrated in FIG. 17C, LAMP
detection was performed with increasing concentrations of
Eriochrome.RTM. Black T on chromatography paper in PCR tubes. The
lo1B.3 primer set targeting Histophilus somni genomic DNA was used.
Positive reactions were spiked with 5 .mu.L of H. somni gDNA at a
concentration of 0.2 ng/.mu.L. Negative reactions used 5 .mu.L of
nuclease-free water. Total reaction volume was 25 .mu.L. Reactions
were prepared using 12.5 of NEB Warmstart 2x master mix, 2.5 .mu.L
or primer mix, and 5 .mu.L of either template (positive reactions)
or water (negative) as above, and 5 .mu.L of EBT (300 .mu.M)
prepared in nuclease-free water. For EBT reactions, the reaction
consisted of 25 .mu.L of EBT (300 .mu.M) prepared in nuclease-free
water.
[0254] By screening several different types of paper and confirming
via gel electrophoresis that amplification was occurring on PES and
polysulfone BTS 0.8, a colorimetric change on any paper could not
be detected (FIG. 17D and FIG. 17E).
[0255] As shown in FIG. 17D, LAMP detection was performed on
multiple papers: chromatography grade 1, anionic exchange nylon,
cationic exchange nylon, polyether sulfone membrane, asymmetric
sub-micron polysulfone (BTS 0.8), asymmetric sub-micron polysulfone
(BTS 100) and hydroxylated nylon 1.2. b) Endpoint scans of the
papers in panel a at 60 minutes and c) gel electrophoresis (2%
agarose) scan of the extracted LAMP products at 60 minutes. The
lo1B.3 primer set targeting Histophilus somni (HS) genomic DNA was
used. Positive reactions were spiked with 5 .mu.L of H. somni gDNA
at a concentration of 0.2 ng/.mu..mu.L. Negative reactions used 5
.mu.L of nuclease-free water. Total reaction volume is 25 .mu.L.
Reactions were prepared using 12.5 .mu.L of NEB Warmstart 2x master
mix, 2.5 .mu.L or primer mix, and 5 .mu.L of either template
(positive reactions) or water (negative) as above, and 5 .mu.L of
EBT (300 .mu.M) prepared in nuclease-free water. Papers (as
indicated) were placed in a PCR tube containing 25 .mu.L of
reaction and was wicked by the paper over the course of reaction.
After 60 minutes, the papers were removed and scanned. Gel was
extracted using 30.
[0256] As illustrated in FIG. 17E, results were generated for
overtime in a) PCR tubes, b) gel electrophoresis (2% agarose) of
the extracted DNA from papers at 60 minutes, and c) scanned papers
at 60 minutes. The lo1B.3 primer set targeting Histophilus somni
(HS) genomic DNA was used. Positive reactions were spiked with 5
.mu.LL of H. somni gDNA at a concentration of 0.2 ng/.mu.L.
Negative reactions used 5 .mu.L of nuclease-free water. Total
reaction volume is 25 .mu.L. Reactions were prepared using 12.5
.mu.L of NEB Warmstart 2x master mix, 2.5 .mu.L or primer mix, and
5 .mu.L of either template (positive reactions) or water (negative)
as above, and 5 .mu.L of EBT (300 .mu.M) prepared in nuclease-free
water. Biodyne A amphoteric paper were placed in a PCR tube
containing 25 .mu.L of reaction and was wicked by the paper over
the course of reaction. After 60 minutes, the papers were removed
and scanned.
[0257] Additionally, it was difficult to stabilize the Crystal
violet indicator in solution for the RT-LAMP reaction. For Leuco
crystal violet (LCV), an unstable derivative of crystal violet,
excess sodium sulfite was used to maintain stability colorlessness
in solution. To solubilize in water, LCV used sodium sulfite (SS)
and beta-cycLoDextrin (BCD). Upon binding to dsDNA, LCV converted
back to crystal violet (e.g., violet in solution). Consequently,
throughout the RT-LAMP reaction as more dsDNA was produced as a
result of amplification, the solution was expected to change color
from colorless to violet. When LAMP reactions were performed at
varying concentrations of CV, however, changes in color occurred in
both positive and negative reactions.
[0258] As shown in FIG. 17F, LAMP detection was performed with
intercalating dye, Crystal violet in solution and b) associated gel
electrophoresis (2% agarose) scan of the products at 60 minutes.
The lo1B.3 primer set targeting H. somni gDNA was used. Positive
reactions were spiked with 5 .mu.L of H. somni gDNA at a
concentration of 0.2 ng/.mu.L. Negative reactions used 5 .mu.L of
nuclease-free water. Total reaction volume was 25 .mu.L. Reactions
were prepared using 12.5 .mu.L of NEB Warmstart 2x master mix, 2.5
.mu.L or primer mix, and 5 .mu.L of either template (positive
reactions) or water (negative) as above, and 5 .mu.L of Crystal
violet prepared in nuclease-free water to result in a final
concentration as indicated. Papers were placed in a PCR tube
containing 25 .mu.L of reaction and was wicked by the paper over
the course of reaction. After 60 minutes, the papers were removed
and scanned. Sodium sulfite and cyclodextrin were used to
solubilize crystal violet.
[0259] To confirm that amplification was occurring in the positive
reactions but not the negative reactions, the RT-LAMP solution was
run in a 2% agarose gel, which showed that the positive reactions
showed amplification at all tested concentrations of CV, while the
negative reactions did not show amplification at any tested
concentrations of CV. Thus, the color change in the negative
reactions was caused by the degradation of LCV to CV, not because
of binding of amplified DNA.
[0260] Further testing for CV on paper at different concentrations
of CV, SS, and BCD, provided results that were indistinguishable
between negative and positive reactions. As shown in FIG. 17G,
endpoint colorimetric scans were performed for LAMP detection with
intercalating dye, Crystal violet (CV), on paper. Sodium sulfite
(SS) and Beta-cyclodextrin (BCD) were used to solubilize CV. Papers
were loaded with 25 .mu.l of LAMP reaction prepared using 12.5
.mu.L of NEB Warmstart.TM. 2x master mix, 2.5 .mu.L or primer mix,
and 5 .mu.L of either template (positive reactions) or water
(negative) as above, and 5 .mu.L of CV prepared in nuclease-free
water at the indicated concentrations.
[0261] Finally, several colorimetric pH indicators which cover a
range of about 2 pH units with a color transition pH of about 7.0
were tested to match the anticipated pH range change and transition
point of a RT-LAMP reaction. These ranges were chosen based on the
initial starting pH of our LAMP colorimetric master mix and also to
overcome the buffering capacity of saliva. One exception to this
selection process was Acid fuchsin, which covers a range of 3.0 pH
and has a color transition pH of 5.0. FIGS. 17H-17K show varying
concentrations of the selected pH indicators along with the
associated gel electrophoresis results of the LAMP reaction at 60
minutes.
[0262] As illustrated in FIG. 17H, RT-LAMP detection was performed
with increasing concentrations of cresol red sodium salt, Neutral
red, Phenol red sodium salt, m-cresol purple, and m-cresol purple
sodium salt in solution (pH indicator). The N.10 primer set
targeting the N gene of SARS-CoV-2 was used. Positive reactions
were spiked with 5 .mu.L of in-vitro transcribed N gene RNA at a
concentration of 0.2 ng/.mu.L. Negative reactions used 5 .mu.L of
nuclease-free water. Total reaction volume was 25 .mu.L. Reactions
were prepared using 12.5 .mu.L of NEB Warmstart.TM. 2x master mix,
2.5 .mu.L or primer mix, and 5 .mu.L of either template (positive
reactions) or water (negative) as above, and 5 .mu.L of the
indicated pH indicator at the designated concentration in
nuclease-free water. Reactions were carried out in an incubator and
scanned every 20 minutes using a flatbed scanner.
[0263] As illustrated in FIG. 17I, LAMP detection with increasing
concentrations of Cresol red (pH indicator) was performed. The
lo1B.3 primer set targeting H. somni gDNA was used. Positive
reactions were spiked with 5 .mu.L of H. somni gDNA at a
concentration of 0.2 ng/.mu.L. Negative reactions used 5 .mu.L of
nuclease-free water. Total reaction volume is 25 .mu.L. Reactions
were prepared using 12.5 .mu.L of NEB Warmstart.TM. 2x master mix,
2.5 .mu.L or primer mix, 5 .mu.L of cresol red resulting in a final
reaction concentration as indicated, and 5 .mu.L of either template
(positive reactions) or water (negative) as above.
[0264] As illustrated in FIG. 17J, RT-LAMP detection was performed
with increasing concentrations of Cresol red, sodium salt, m-cresol
purple, Bromothymol blue and Acid fuchsin in solution and b)
associated gel electrophoresis (2% agarose) scan of products at 60
mins. The N.10 primer set targeting the N gene of SARS-CoV-2 was
used. Positive reactions were spiked with 5 .mu.L of in-vitro
transcribed N gene RNA at a concentration of 0.2 ng/.mu.L. Negative
reactions used 5 .mu.L of nuclease-free water. Reactions were
prepared using 12.5 .mu.L of NEB Warmstart 2x master mix, 2.5 .mu.L
or primer mix, and 5 .mu.L of either template (positive reactions)
or water (negative) as above, and 5 .mu.L of the indicated pH
indicator to result in a final reaction concentration as indicated.
Heating was carried out in an incubator set at 65.degree. C. and
scanned in a flatbed scanner every 20 minutes.
[0265] As illustrated in FIG. 17K, endpoint gel electrophoresis
scans of the RT-LAMP products (60 mins) were performed on a 2%
Agarose gel.
[0266] As shown, one pH indicator that produced a distinct
colorimetric response between positive and negative reactions was
cresol red. Additionally, phenol red (the pH indicator used in
NEB's colorimetric RT-LAMP kit) was also evaluated with respect to
varying initial pH values resulting from the addition of HCl and
KOH to the solution to provide the indicated initial pH value.
[0267] As illustrated in FIG. 17L, RT-LAMP detection with Phenol
red at pH 8.1, 8.5, and 8.8 in solution (pH indicator) was
performed. Adjustments were made with HCl and KOH prior to the
addition of template RNA. The N.10 primer set targeting the N gene
of SARS-CoV-2 was used. Positive reactions were spiked with 5 .mu.L
of in-vitro transcribed N gene RNA at a concentration of 0.2
ng/.mu.L. Negative reactions used 5 .mu.L of nuclease-free water.
Reactions consisted of 20 master mix and 5 .mu.L of template as
described above (positive) or nuclease-free water (negative). 10 mL
of master mix was made with (NH4)2SO4 (20 mM), KC1 (100 mM), MgSO4
(16 mM), dNTP mix (28 mM each dNTP), tween 20 (0.2% v/v).
Therefore, phenol red resulted in a higher level of contrast
between positive and negative reactions when compared to cresol
red.
[0268] Consequently, pH indicators with a color change around pH
6.5 had the most consistent and the most contrasting color change
(e.g., Phenol red).
Example 19
Effect of Starting pH on paper
[0269] To evaluate the color stability of incorporating drying into
our process, the pH of the LAMP master mix was adjusted to 8.0,
8.5, or remained unadjusted (e.g., 7.6) and the water or synthetic
RNA (N gene, 0.2 ng/.mu.L) used for rehydration was also adjusted
to 8.0, 8.5, or remained unadjusted (e.g., 5.5).
[0270] As illustrated in FIG. 18, pH 7.6 is the unadjusted pH of
the RT-LAMP reaction mixture. Wet setup indicates 5 .mu.L of
synthetic RNA (N gene, 0.2 ng/.mu.L, `+`) or water (`-`) were added
immediately after adding 20 .mu.L of LAMP reaction master mix.
Dried setup indicates paper strips were left to dry for 30 minutes
at room temperature after applying 20 .mu.L LAMP master mix and
then rehydrated with 25 .mu.L synthetic RNA (`+`) or water
(`-`).
[0271] Adjusting the pH to 8.0 resulted in a better color stability
in the negative controls, whereas a pH of 8.5 was too high to allow
for discernible color change the standard and the dry set ups after
120 minutes of incubation. When the pH was left unadjusted, the
color changed even when a control was loaded.
Example 20
Effect of Trehalose and Tween 20 on RT-LAMP Colorimetric
Response
[0272] FIG. 19B shows colorimetric RT-LAMP results with the
inclusion of Trehalose or Tween 20 at the given concentration. The
orflab.II primer set was used. 20 .mu.L of RT-LAMP master mix
containing a base formulation of KCl (50 mM), MgSO.sub.4 (8 mM),
equimolar dNTP mixture (1.4 mM each dNTP), WarmStart BST 2.0 (0.32
U/.mu.L), Warm Start RTx (0.3 U/.mu.L), Phenol red (0.25 mM), dUTP
(0.14 mM), Antarctic UDG (0.0004 U/.mu.L), Tween 20 (1% v/v, if
indicated), Betaine (20 mM), BSA (40 mg/mL), and Trehalose (10%
w/v, if indicate) was added to Grade 1 chromatography paper (5
mm.times.20 mm) and allowed to dry inside a PCR preparation hood
for 60 minutes. 25 .mu.L of Heat-inactivated SARS-CoV-2 at a final
concentration of 1.times.10.sup.5 copies per reaction in 25%
processed saliva (positive reactions) or nuclease-free water
(negative reactions) was added to the dry reaction pads. The pads
were heated in an incubator set at 65.degree. C. for 60 minutes and
then scanned using a flatbed scanner.
[0273] Inclusion of ammonium sulfate caused a color from red to
yellow upon drying of RT-LAMP reagents when no template was
present. This color change was prevented by increasing the phenol
red concentration and replacing ammonium sulfate with betaine (FIG.
1 9A). Furthermore, the addition of trehalose and bovine serum
albumin (BSA) increased the reaction speed and increased LoD (FIG.
19B).
Example Embodiments
[0274] In one example there is provided, a method of preparing a
saliva sample for loop-mediated isothermal amplification (LAMP)
detection of a pathogen target, that can include providing an
amount of saliva from a test subject; and diluting the saliva in
water to a degree that reduces a buffering capacity of the saliva
while maintaining a sufficient concentration to allow for detection
of the pathogen target.
[0275] In one example of a method of preparing a saliva sample for
loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the method can further comprise reducing a
viscosity of the saliva as compared to an original viscosity.
[0276] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viscosity can be reduced by one or more of
dilution, filtering, or combinations thereof.
[0277] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viscosity can be reduced using filtering.
[0278] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viscosity can be reduced using a 10 micron
filter.
[0279] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viscosity can be reduced to a degree that
increases flowability through a solid phase medium as compared to
an original viscosity.
[0280] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viscosity can be reduced to a range of from
about 1.0 cP to about 50 cP.
[0281] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the method can further comprise filtering the
saliva sample to a degree that adjusts a saliva sample pH to a test
sample target range.
[0282] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the test sample target range can be from about 7.2
to about 8.6.
[0283] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the saliva can be diluted in the water to a saliva
to water ratio of about 1:1 to about 1:20.
[0284] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the saliva can be diluted in the water to a degree
that provides the sample with an optical density at 600 nm
(OD.sub.600) of less than 0.2.
[0285] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the water can have a pH greater than 6.0 and is
substantially free of contaminants.
[0286] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the saliva sample can consist essentially of
saliva and water.
[0287] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the saliva can have a volume of from about 50
.mu.l to about 100 .mu.l.
[0288] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the saliva sample can have a volume of from about
100 .mu.l to about 1 ml.
[0289] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the saliva can be collected using sponge-based
collection.
[0290] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the pathogen target can comprise a viral pathogen,
a bacterial pathogen, a fungal pathogen, or a protozoa
pathogen.
[0291] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the pathogen target can be a viral target.
[0292] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viral target can comprise a dsDNA virus, an
ssDNA virus, a dsRNA virus, a positive-strand ssRNA virus, a
negative-strand ssRNA virus, an ssRNA-RT virus, or a ds-DNA-RT
virus.
[0293] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the viral target can comprise H1N1, H2N2, H3N2,
H1N1pdm09, or SARS-CoV-2.
[0294] In another example of a method of preparing a saliva sample
for loop-mediated isothermal amplification (LAMP) detection of a
pathogen target, the LAMP detection can comprise reverse
transcription LAMP (RT-LAMP) detection.
[0295] In one example there is provided a test sample composition
for loop-mediated isothermal amplification (LAMP) analysis which
can comprise or include an amount of a test subject's saliva that
is sufficient to detect a pathogen target via a LAMP analysis in
combination with an amount of water that reduces a buffering
capacity of the saliva.
[0296] In one example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
composition can have a viscosity of from about 1.0 cP to about 50
cP.
[0297] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
composition can have a pH of from about 7.2 to about 8.6.
[0298] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
composition can have a saliva to water ratio of about 1:1 to about
1:20.
[0299] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
composition can have an optical density at 600 nm (0D600) of less
than 0.2.
[0300] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the water
can have a pH greater than 6.0 and is substantially free of
contaminants.
[0301] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
composition can consist essentially of saliva and water.
[0302] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the saliva
can have a volume ranging from about 50 .mu.l to about 100 pl.
[0303] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the saliva
sample can have a volume of from about 100 .mu.1 to about 1 ml.
[0304] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
pathogen target can comprise a viral pathogen, a bacterial
pathogen, a fungal pathogen, or a protozoa pathogen.
[0305] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
pathogen target can be a viral target.
[0306] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the viral
target can comprise a dsDNA virus, an ssDNA virus, a dsRNA virus, a
positive-strand ssRNA virus, a negative-strand ssRNA virus, an
ssRNA-RT virus, or a ds-DNA-RT virus.
[0307] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the viral
target can comprise H1N1, H2N2, H3N2, H1N1pdm09, or SARS-CoV-2.
[0308] In another example of a test sample composition for
loop-mediated isothermal amplification (LAMP) analysis, the
buffering capacity of the composition can be less than 5 mM.
[0309] It should be understood that the above-described methods are
only illustrative of some embodiments of the present invention.
Numerous modifications and alternative arrangements may be devised
by those skilled in the art without departing from the spirit and
scope of the present invention and the appended claims are intended
to cover such modifications and arrangements. Thus, while the
present invention has been described above with particularity and
detail in connection with what is presently deemed to be the most
practical and preferred embodiments of the invention, it will be
apparent to those of ordinary skill in the art that variations
including, may be made without departing from the principles and
concepts set forth herein.
* * * * *