U.S. patent application number 17/325390 was filed with the patent office on 2021-11-04 for polynucleotides for amplification and detection of sars-cov-2.
The applicant listed for this patent is Talis Biomedical Corporation. Invention is credited to Nadya Andini, Kathy Chiu, Xuewen Jiang, Hedia Maamar.
Application Number | 20210340622 17/325390 |
Document ID | / |
Family ID | 1000005725246 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340622 |
Kind Code |
A1 |
Andini; Nadya ; et
al. |
November 4, 2021 |
POLYNUCLEOTIDES FOR AMPLIFICATION AND DETECTION OF SARS-COV-2
Abstract
Disclosed herein are primers and probes related to the detection
of SARS-CoV-2 via nucleic acid amplification testing (NAAT), for
example to amplify and determine the presence of SARS-CoV-2 in test
samples and/or to diagnose Covid-19. Specifically, the present
disclosure describes primers and probes that bind to the N gene,
ORF1ab, or E gene of SARS-CoV-2 coronavirus for detection via loop
mediated isothermal amplification (LAMP) and molecular beacon
hybridization.
Inventors: |
Andini; Nadya; (Livermore,
CA) ; Chiu; Kathy; (Irvine, CA) ; Jiang;
Xuewen; (Cupertino, CA) ; Maamar; Hedia; (El
Dorado Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Talis Biomedical Corporation |
Menlo Park |
CA |
US |
|
|
Family ID: |
1000005725246 |
Appl. No.: |
17/325390 |
Filed: |
May 20, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16912446 |
Jun 25, 2020 |
11047007 |
|
|
17325390 |
|
|
|
|
63009803 |
Apr 14, 2020 |
|
|
|
62993523 |
Mar 23, 2020 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2531/119 20130101;
C12Q 2521/107 20130101; C12Q 2521/101 20130101; C12Q 1/6813
20130101; C12Q 1/6876 20130101; C12Q 1/6844 20130101 |
International
Class: |
C12Q 1/6876 20060101
C12Q001/6876; C12Q 1/6813 20060101 C12Q001/6813; C12Q 1/6844
20060101 C12Q001/6844 |
Claims
1.-30. (canceled)
31. A composition comprising SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID
NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.
32. The composition of claim 31, further comprising a molecular
beacon comprising a fluorophore, a quencher and a
polynucleotide.
33. The composition of claim 32, wherein the polynucleotide
comprises a sequence selected from the group consisting of:
nucleotides 5-22 of SEQ ID NO: 75, nucleotides 5-22 of SEQ ID NO:
76, nucleotides 5-22 of SEQ ID NO: 80, nucleotides 5-22 of SEQ ID
NO: 81, nucleotides 4-22 of SEQ ID NO: 82, nucleotides 6-28 of SEQ
ID NO: 83, nucleotides 6-25 of SEQ ID NO: 84, and nucleotides 3-23
of SEQ ID NO: 85.
34. The composition of claim 33, wherein the polynucleotide
comprises a sequence selected from the group consisting of: SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,
SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.
35. The composition of claim 34, wherein the polynucleotide
consists a sequence selected from the group consisting of: SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,
SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.
36. The composition of claim 32, wherein the fluorophore is FAM and
the quencher is BHQ1.
37. The composition of claim 32, wherein the fluorophore is ATTO
565 or Alexa 594 and the quencher is BHQ1 or BHQ2.
38. The composition of claim 31, further comprising an
intercalating dye.
39. The composition of claim 31, further comprising a strand
displacement DNA polymerase and a reverse transcriptase.
40. A composition comprising a set of polynucleotides selected from
the group consisting of: Set-5 and Set-9.
41. A method of detecting SARS-CoV-2 in a test sample, the method
comprising: (a) extracting nucleic acid from the test sample; (b)
amplifying a target sequence by reacting the nucleic acid extracted
in step (a) with a reaction mixture comprising a strand
displacement DNA polymerase activity, a reverse transcriptase
activity, and a sequence-specific primer set, wherein said
sequence-specific primer set is selected from the group consisting
of: Set-5 and Set-9; and (c) detecting the presence or absence of
an amplification product from step (b); wherein the presence of
said amplification product is indicative of the presence of
SARS-CoV-2 in the test sample.
42. The method of claim 41, wherein the amplification in step (b)
of the target sequence is performed between about 60.degree. C. and
about 67.degree. C. for less than 30 minutes.
43. The method of claim 42, wherein the amplification step is
performed for less than fifteen minutes.
44. The method of claim 43, wherein the amplification step is
performed for less than twelve minutes.
45. The method of claim 44, wherein the amplification step is
performed for less than nine minutes.
46. The method of claim 41, wherein detecting the presence or
absence of the amplification product comprises hybridizing the
amplification product with a probe comprising a polynucleotide
attached to a label.
47. The method of claim 46, wherein the label is a fluorophore.
48. The method of claim 47, wherein the fluorophore is covalently
attached to a terminus of the polynucleotide.
49. The method of claim 46, wherein the labeled polynucleotide
comprises a sequence selected from the group consisting: of
nucleotides 5-22 of SEQ ID NO: 75, nucleotides 5-22 of SEQ ID NO:
76, nucleotides 5-22 of SEQ ID NO: 80, nucleotides 5-22 of SEQ ID
NO: 81, nucleotides 4-22 of SEQ ID NO: 82, nucleotides 6-28 of SEQ
ID NO: 83, nucleotides 6-25 of SEQ ID NO: 84, and nucleotides 3-23
of SEQ ID NO: 85.
50. The method of claim 49, wherein the labeled polynucleotide
comprises a sequence selected from the group consisting of: SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,
SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.
51. The method of claim 50, wherein the sequence-specific primer
set is Set-9 and the sequence of the labeled polynucleotide is SEQ
ID NO: 83.
52. A kit comprising the composition of claim 31.
53. The kit of claim 52, further comprising a strand displacement
polymerase and a reverse transcriptase.
54. The kit of claim 53, further comprising a molecular beacon
comprising a fluorophore, a quencher, and a polynucleotide.
55. The kit of claim 54, wherein the polynucleotide comprises a
sequence selected from the group consisting of: nucleotides 5-22 of
SEQ ID NO: 75, nucleotides 5-22 of SEQ ID NO: 76, nucleotides 5-22
of SEQ ID NO: 80, nucleotides 5-22 of SEQ ID NO: 81, nucleotides
4-22 of SEQ ID NO: 82, nucleotides 6-28 of SEQ ID NO: 83,
nucleotides 6-25 of SEQ ID NO: 84, and nucleotides 3-23 of SEQ ID
NO: 85.
56. The kit of claim 55, wherein the polynucleotide comprises a
sequence selected from the group consisting of: SEQ ID NO: 75, SEQ
ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO:
83, SEQ ID NO: 84, and SEQ ID NO: 85.
57. The kit of claim 56, wherein the polynucleotide consists a
sequence selected from the group consisting of: SEQ ID NO: 75, SEQ
ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO:
83, SEQ ID NO: 84, and SEQ ID NO: 85.
58. The kit of claim 57, wherein the polynucleotide consists of SEQ
ID NO: 83.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 16/912,446, filed Jun. 25, 2020, which claims the benefit of
U.S. Provisional Application No. 62/993,523, filed Mar. 23, 2020,
and U.S. Provisional Application No. 63/009,803, filed Apr. 14,
2020, the contents of which are each incorporated by reference in
their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 20, 2021, is named TSM-055US1 D1_SL.txt and is 23,625 bytes
in size.
FIELD OF THE INVENTION
[0003] The present invention relates to the fields of molecular
biology and nucleic acid chemistry. The invention provides methods
and reagents for detecting the SARS-CoV-2 virus, the causative
agent for the disease referred to as COVID-19. The invention also
relates to the fields of medical diagnostics and prognostics. In
particular, the invention relates to polynucleotides and methods
for amplifying and detecting certain genes in the SARS-CoV-2 viral
genome.
BACKGROUND
[0004] There is an urgent need for the development of a rapid,
affordable, sample-in answer-out point of care (POC) diagnostic
platform for the novel coronavirus spreading across the globe. On
11 Mar. 2020, the World Health Organization (WHO) declared
COVID-19, which is caused by the SARS-CoV-2 virus, to be a pandemic
based on 118,000 cases in 114 countries and 4,291 deaths. Ten days
later, the Johns Hopkins Coronavirus Resource Center reported
329,858 confirmed cases with 14,380 deaths worldwide. Three months
later, there are over 8.5 million confirmed cases worldwide and the
death toll is nearly half a million people with little indication
that spread of the disease is slowing. Throughout this public
health crisis, the inability to test members of the public for
infection with SARS-CoV-2 has significantly hampered efforts to
contain the pandemic.
[0005] The compositions and methods disclosed herein provide
primers and probes for the detection of the SARS-CoV-2 RNA virus
using loop-mediated isothermal amplification.
SUMMARY OF THE INVENTION
[0006] The present invention encompasses, in some embodiments, a
composition comprising a set of polynucleotides selected from the
group consisting of Set-1 through Set-17. In some embodiments, the
composition further comprises a probe. In some embodiments, the
probe comprises a label. In some embodiments, the probe is a
labeled polynucleotide. In a preferred implementation, the label is
a fluorophore, which preferably is covalently attached to a
terminus of the polynucleotide. In a particularly preferred
embodiment, the probe or polynucleotide is a molecular beacon
comprising a fluorophore, a quencher, and a polynucleotide. In one
embodiment, the fluorophore is FAM and the quencher is Black Hole
Quencher 1 (BHQ1; LGC Biosearch Technologies). In an alternate
implementation, the fluorophore is ATTO 565 or Alexa 594 or Cy5 and
the quencher is BHQ1 or BHQ2.
[0007] In some implementations, composition comprises a labeled
polynucleotide comprising a sequence selected from the group
consisting of nucleotides 5-27 of SEQ ID NO.: 55, nucleotides 7-25
of SEQ ID NO.: 56, nucleotides 5-22 of SEQ ID NO.: 75, nucleotides
5-22 of SEQ ID NO.: 76, nucleotides 5-29 of SEQ ID NO.: 77,
nucleotides 6-26 of SEQ ID NO.: 78, nucleotides 5-29 of SEQ ID NO.:
79, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID
NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 of
SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, nucleotides
3-23 of SEQ ID NO.: 85, and nucleotides 2-24 of SEQ ID NO.: 95. In
further implementations, the labeled polynucleotide can comprise a
sequence elected from the group consisting of SEQ ID NO.: 55, SEQ
ID NO.: 56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95. In
certain implementations, the sequence of the labeled polynucleotide
is selected from the group consisting of SEQ ID NO.: 55, SEQ ID
NO.: 56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95.
[0008] In certain implementations, the composition targets the
first open reading frame of SARS-CoV2 (ORF1ab). In such
implementation, the set of polynucleotides can be Set-11 or Set-13,
and the labeled polynucleotide comprises a sequence selected from
the group consisting of nucleotides 5-29 of SEQ ID NO.: 77 and
nucleotides 5-29 of SEQ ID NO.: 79. In some embodiments, the
labeled polynucleotide comprises SEQ ID NO.: 79, or more preferably
SEQ ID NO.: 77. Preferably, the set of polynucleotides is Set-11.
In another implementation targeting ORF1ab, the set of
polynucleotides is Set-12 and the labeled polynucleotide comprises
nucleotides 6-26 of SEQ ID NO.: 78. In some implementations, the
labeled polynucleotide comprises SEQ ID NO.: 78.
[0009] In certain implementations, the composition targets gene N,
which encodes a nucleoprotein that packages the positive strand
viral genome RNA into a helical ribonucleocapsid (RNP) and plays a
fundamental role during virion assembly through its interactions
with the viral genome and membrane protein M. In such
implementations, the set of polynucleotides can be Set-5 or Set-9
and the labeled polynucleotide comprises a sequence selected from
the group consisting of nucleotides 5-22 of SEQ ID NO.: 75,
nucleotides 5-22 of SEQ ID NO.: 76, nucleotides 5-22 of SEQ ID NO.:
80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID
NO.: 82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides 6-25 of
SEQ ID NO.: 84, nucleotides 3-23 of SEQ ID NO.: 85. In some
embodiments, the labeled polynucleotide comprises a sequence
selected from the group consisting of SEQ ID NO.: 75, SEQ ID NO.:
76, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID NO.:83,
SEQ ID NO.: 84, and SEQ ID NO.: 85. Preferably, the set of
polynucleotides is Set-9. Similarly, the sequence of the labeled
polynucleotides preferably is SEQ ID NO.: 83. In a second
embodiment targeting the N gene, the set of polynucleotides is
Set-1 or Set-4 and the labeled polynucleotide comprises nucleotides
5-27 of SEQ ID NO.: 55. In such embodiments, the labeled
polynucleotide preferably comprises SEQ ID NO.: 55. In yet another
embodiment targeting the N gene, the set of polynucleotides is
Set-3 or Set-8 and the labeled polynucleotide comprises nucleotides
7-25 of SEQ ID NO.: 56. More preferably, the labeled polynucleotide
comprises SEQ ID NO.: 56.
[0010] In certain implementations, the composition targets gene E,
which encodes an envelope protein. In such implementations, the set
of polynucleotides is selected from the group consisting of Set-14,
Set-15, Set-16, and Set-17, and the labeled polynucleotide
comprises nucleotides 2-24 of SEQ ID NO.: 95. More preferably, the
labeled polynucleotide comprises SEQ ID NO.: 95. In a preferred
implementation, the polynucleotide sequence of the molecular beacon
consists of SEQ ID NO.:95.
[0011] In certain implementations, the composition for detecting
the presence of SARS-CoV-2 comprises SEQ ID NO.: 49, SEQ ID NO.:
50, SEQ ID NO.: 51, SEQ ID NO.: 52, SEQ ID NO.: 53, and SEQ ID NO.:
54. In such an implementation, the composition can further comprise
a molecular beacon comprising a fluorophore, a quencher, and SEQ ID
NO. 83. In another implementation, the composition further
comprises SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID
NO.: 66, SEQ ID NO.: 67, and SEQ ID NO.: 68.
[0012] In yet another embodiment, the composition for detecting the
presence of SARS-COV-2 comprises SEQ ID NO.: 63, SEQ ID NO.: 64,
SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67, and SEQ ID NO.: 68.
This composition can further comprise a molecular beacon comprising
a fluorophore, a quencher and SEQ ID NO. 77.
[0013] Yet another aspect of the invention provides a composition
comprising a first set of polynucleotides comprising SEQ ID NO.:
49, SEQ ID NO.: 50, SEQ ID NO.: 51, SEQ ID NO.: 52, SEQ ID NO.: 53
and SEQ ID NO.: 66; and a second set of polynucleotides comprising
SEQ ID NO.: 63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ
ID NO.: 67 and SEQ ID NO.: 68. In some implementations, the
composition further comprises a first labeled polynucleotide
comprising nucleotides 6-28 of SEQ ID NO.: 83 and a second labeled
polynucleotide comprising nucleotides 5-29 of SEQ ID NO.: 77. In
some implementations, the first polynucleotide comprises SEQ ID
NO.: 83 and the second labeled polynucleotide comprising SEQ ID
NO.: 77.
[0014] In many implementations described herein, the probe is a
molecular beacon comprising a fluorophore, a quencher, and a
polynucleotide. In implementations wherein the set of
polynucleotides is Set-5 or Set-9, the molecular beacon comprises a
sequence selected from the group consisting of nucleotides 5-22 of
SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides
5-22 of SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID NO.: 81,
nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 of SEQ ID NO.:
83, nucleotides 6-25 of SEQ ID NO.: 84, and nucleotides 3-23 of SEQ
ID NO.: 85. More preferably, the molecular beacon comprises a
sequence selected from the group consisting of SEQ ID NO.: 75, SEQ
ID NO: 76, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID
NO.: 83, SEQ ID NO.: 84 and SEQ ID NO.: 85. Even more preferably,
the polynucleotide sequence of the molecular beacon consists of a
sequence selected from the group consisting of SEQ ID NO.: 75, SEQ
ID NO: 76, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID NO.: 82, SEQ ID
NO.: 83, SEQ ID NO.: 84 and SEQ ID NO.: 85. In other
implementations, wherein the set of polynucleotides is Set-1 or
Set-4, the molecular beacon comprises nucleotides 5-27 of SEQ ID
NO.: 55, and more preferably comprises SEQ ID NO.: 55. In a
preferred implementation, the polynucleotide sequence of the
molecular beacon consists of SEQ ID NO.: 55. In implementations
wherein the set of polynucleotides is Set-3 or Set-8, the molecular
beacon comprises nucleotides 7-25 of SEQ ID NO.: 56, more
preferably the full sequence SEQ ID NO.: 56. In a preferred
implementation, the polynucleotide sequence of the molecular beacon
consists of SEQ ID NO.: 56. In implementations, wherein the set of
polynucleotides is Set-11 or Set-13, the molecular beacon comprises
a sequence selected from the group consisting of nucleotides 5-29
of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79.
Preferably, the molecular beacon comprises a sequence selected from
the group consisting of SEQ ID NO.: 77 and SEQ ID NO.: 79. In a
particularly preferred implementation, the polynucleotide sequence
of the molecular beacon consists of SEQ ID NO.: 77 or SEQ ID NO.:
79. In implementations wherein the set of polynucleotides is
Set-12, the molecular beacon comprises nucleotides 6-26 of SEQ ID
NO.: 78, more preferably the full sequence of SEQ ID NO.: 78. In a
preferred implementation, the polynucleotide sequence of the
molecular beacon consists of SEQ ID NO.: 78. In implementations,
wherein the set of polynucleotides is Set-14, Set-15, Set16 or
Set-17, the molecular beacon comprises nucleotides 2-24 of SEQ ID
NO.: 95, more preferably the full sequence of SEQ ID NO.: 95. In a
preferred implementation, The polynucleotide sequence of the
molecule beacon consists of SEQ ID NO.: 95.
[0015] One aspect of the invention provides molecular beacons
comprising a fluorophore, a quencher, and a polynucleotide, wherein
the polynucleotide comprises a sequence selected from the group
consisting of nucleotides 5-27 of SEQ ID NO.: 55, nucleotides 7-25
of SEQ ID NO.: 56, nucleotides 5-22 of SEQ ID NO.: 75, nucleotides
5-22 of SEQ ID NO.: 76, nucleotides 5-29 of SEQ ID NO.: 77,
nucleotides 6-26 of SEQ ID NO.: 78, nucleotides 5-29 of SEQ ID NO.:
79, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID
NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 of
SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, nucleotides
3-23 of SEQ ID NO.: 85, and nucleotides 2-24 of SEQ ID NO.: 95. In
a preferred implementation, the polynucleotide portion of the
molecular beacon comprises a sequence selected from the group
consisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NOS.: 75
through 85, and SEQ ID NO.: 95. More preferably, the sequence
selected from the group consisting of SEQ ID NO.: 55, SEQ ID NO.:
56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95.
[0016] Yet another aspect of the invention provides method of
detecting SARS-CoV-2 in a test sample, the method comprising (a)
extracting nucleic acid from the test sample, (b) amplifying a
target sequence by reacting the nucleic acid extracted in step (a)
with a reaction mixture comprising a strand displacement DNA
polymerase and a reverse transcriptase and a sequence specific
primer set, wherein said sequence-specific primer set is selected
from the group consisting of Set-1 through Set-17, and (c)
detecting the presence or absence of an amplified product of step
(b); wherein the presence of said amplification product is
indicative of the presence of SARS-CoV-2 in the test sample. In one
embodiment, the amplification in step (b) of the target sequence is
performed between about 60.degree. C. and about 67.degree. C. for
less than 30 minutes. Preferably, the amplification step is
performed for less than fifteen minutes. In some implementations,
the strand displacement DNA polymerase and the reverse
transcriptase activities are provided by a single enzyme.
[0017] In certain embodiments, detecting the presence or absence of
the amplification product comprises hybridizing the amplified
product with a probe comprising a polynucleotide attached to a
label. In a preferred implementation, the label is a fluorophore,
which is preferably attached to a terminus of the polynucleotide.
In a particularly preferred embodiment, the probe or polynucleotide
is a molecular beacon comprising a fluorophore, a quencher, and a
polynucleotide. In one embodiment, the fluorophore is FAM and the
quencher is BHQ1. In an alternate implementation, the fluorophore
is ATTO 565 or Alexa 594 or Cy5 and the quencher is BHQ1 or BHQ2.
In other implementations, detecting the presence or absence of the
amplification product comprises exposing the amplified product to
an intercalating dye.
[0018] Another aspect of the invention provides methods of
detecting SARS-CoV-2 in a test sample, the method comprising (a)
extracting nucleic acid from the test sample, (b) amplifying a
target sequence by reacting nucleic acid extracted in step (a) for
less than ten minutes with a reaction mixture comprising a strand
displacement DNA polymerase and a sequence specific LAMP primer
set, and (c) detecting the presence or absence of an amplified
product of step (b); wherein the presence of said amplification
product is indicative of the presence of SARS-CoV-2 in the test
sample. In some implementations, the amplifying step comprises
reacting the nucleic acid extracted in step (a) with a reaction
mixture comprising a strand displacement DNA polymerase and a
sequence-specific primer set, wherein said sequence-specific primer
set is selected from the group consisting of Set-1 through Set-17.
In such implementations, detecting the presence or absence of the
amplification product can comprise hybridizing the amplified
product with a molecular beacon comprising a polynucleotide
sequence selected from the group consisting of nucleotides 5-27 of
SEQ ID NO.: 55, nucleotides 7-25 of SEQ ID NO.: 56, nucleotides
5-22 of SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76,
nucleotides 5-29 of SEQ ID NO.: 77, nucleotides 6-26 of SEQ ID NO.:
78, nucleotides 5-29 of SEQ ID NO.: 79, nucleotides 5-22 of SEQ ID
NO.: 80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of
SEQ ID NO.: 82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides
6-25 of SEQ ID NO.: 84, nucleotides 3-23 of SEQ ID NO.: 85, and
nucleotides 2-24 of SEQ ID NO.: 95. In such implementations,
detecting the presence or absence of the amplification product can
comprise hybridizing the amplified product with a molecular beacon
consisting of a polynucleotide sequence selected from the group
consisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NOS.: 75
through 85, and SEQ ID NO.: 95.
[0019] In certain implementations of the method to detect
SARS-CoV-2 in a test sample, the set of polynucleotides is Set-1 or
Set-4, and the labeled polynucleotide comprises nucleotides 5-27 of
SEQ ID NO.: 55. More preferably, the sequence of the labeled
polynucleotides is SEQ ID NO.: 55. In implementations wherein the
set of polynucleotides is Set-3 or Set-8, the labeled
polynucleotide comprises nucleotides 7-25 of SEQ ID NO.: 56. More
preferably, the sequence of the labeled polynucleotides is SEQ ID
NO.: 56. In implementations, wherein the set of polynucleotides is
Set-5 or Set-9, the labeled polynucleotide comprises a sequence
selected from the group consisting of nucleotides 5-22 of SEQ ID
NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides 5-22 of
SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides
4-22 of SEQ ID NO.: 82, nucleotides 6-28 of SEQ ID NO.: 83,
nucleotides 6-25 of SEQ ID NO.: 84, nucleotides 3-23 of SEQ ID NO.:
85. More preferably, the sequence of the labeled polynucleotide is
selected from the group consisting of SEQ ID NO.: 75, SEQ ID NO.:
76, SEQ ID NO.: 80, SEQ ID NO.: 82, SEQ ID NO.: 83, SEQ ID NO.: 84,
and SEQ ID NO.: 85. In implementations wherein the set of
polynucleotides is Set-9, the sequence of the labeled
polynucleotide preferably is SEQ ID NO.: 83. In implementations
wherein the set of polynucleotides is Set-11 or Set-13, the labeled
polynucleotide comprises a sequence selected from the group
consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides
5-29 of SEQ ID NO.: 79. Preferably, the labeled polynucleotide
comprises SEQ ID NO.: 77 or SEQ ID NO.: 79. In implementations,
wherein the set of polynucleotides is Set-12, the labeled
polynucleotide comprises nucleotides 6-26 of SEQ ID NO.: 78.
Preferably, the sequence of the labeled polynucleotides is SEQ ID
NO.: 78. In implementations wherein the set of polynucleotides is
selected from the group consisting of Set-14, Set-15, Set-16 and
Set-17, the labeled polynucleotide comprises nucleotides 2-24 of
SEQ ID NO.: 95. More preferably, the sequence of the labeled
polynucleotides is SEQ ID NO.: 95.
[0020] Yet another aspect of the invention provides kits comprising
the compositions comprising a set of polynucleotides selected from
the group consisting Set-1 through Set-17. In some embodiments, the
kit further comprises a strand displacement polymerase and a
reverse transcriptase. In certain embodiments, the kit comprises a
molecular beacon comprising a fluorophore, a quencher, and a
polynucleotide, wherein the polynucleotide comprises a sequence
selected from the group consisting of nucleotides 5-27 of SEQ ID
NO.: 55, nucleotides 7-25 of SEQ ID NO.: 56, nucleotides 5-22 of
SEQ ID NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides
5-29 of SEQ ID NO.: 77, nucleotides 6-26 of SEQ ID NO.: 78,
nucleotides 5-29 of SEQ ID NO.: 79, nucleotides 5-22 of SEQ ID NO.:
80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides 4-22 of SEQ ID
NO.: 82, nucleotides 6-28 of SEQ ID NO.: 83, nucleotides 6-25 of
SEQ ID NO.: 84, nucleotides 3-23 of SEQ ID NO.: 85, and nucleotides
2-24 of SEQ ID NO.: 95. The polynucleotide sequence of the
molecular beacon can comprise a sequence selected from the group
consisting of SEQ ID NO.: 55, SEQ ID NO.: 56, SEQ ID NOS.: 75
through 85, and SEQ ID NO.: 95. In some embodiments, the
polynucleotide sequence of the molecular beacon consists of a
sequence selected from the group consisting of SEQ ID NO.: 55, SEQ
ID NO.: 56, SEQ ID NOS.: 75 through 85, and SEQ ID NO.: 95.
[0021] In certain implementations of the kit, the set of
polynucleotides is Set-1 or Set-4. In such implementation, the kit
can further comprise a molecular beacon comprising a fluorophore, a
quencher, and a polynucleotide, wherein the polynucleotide
comprises nucleotides 5-27 of SEQ ID NO.: 55. Preferably, the
polynucleotide of the molecular beacon consists of SEQ ID NO.: 55.
In other implementations of the kit, the set of polynucleotides is
selected from the group consisting of Set-3 and Set-8. In such
implementations, the kit can further comprise a molecular beacon
comprising a fluorophore, a quencher, and a polynucleotide, wherein
the polynucleotide comprises nucleotides 7-25 of SEQ ID NO.: 56.
Preferably, the polynucleotide of the molecular beacon consists of
SEQ ID NO.: 56. In yet another implementation of the kit, the set
of polynucleotides is selected from the group consisting of Set-5
and Set-9. In such implementations, the kit can further comprise a
molecular beacon comprising a fluorophore, a quencher, and a
polynucleotide, wherein the polynucleotide comprises a sequence
selected from the group consisting of nucleotides 5-22 of SEQ ID
NO.: 75, nucleotides 5-22 of SEQ ID NO.: 76, nucleotides 5-22 of
SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID NO.: 81, nucleotides
4-22 of SEQ ID NO.: 82, nucleotides 6-28 of SEQ ID NO.: 83,
nucleotides 6-25 of SEQ ID NO.: 84, and nucleotides 3-23 of SEQ ID
NO.: 85. Preferably, the polynucleotide of the molecular beacon
consists of a sequence selected from the group consisting of SEQ ID
NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 80, SEQ ID NO.: 81, SEQ ID
NO.: 82, SEQ ID NO.: 83, SEQ ID NO.: 84, and SEQ ID NO.: 85. In
other implementations of the kit, the set of polynucleotides is
selected from the group consisting of Set-11 and Set-13. In such
implementations, the kit can further comprise a molecular beacon
comprising a fluorophore, a quencher, and a polynucleotide, wherein
the polynucleotide comprises a sequence selected from the group
consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides
5-29 of SEQ ID NO.: 79. Preferably, the polynucleotide of the
molecular beacon consists of a sequence selected from the group
consisting of SEQ ID NO.: 77 and SEQ ID NO.: 79. In yet another
implementation of the kit, the set of polynucleotides is Set-12. In
such implementations, the kit can further comprise a molecular
beacon comprising a fluorophore, a quencher, and a polynucleotide,
wherein the polynucleotide comprises nucleotides 6-26 of SEQ ID
NO.: 78. Preferably, the polynucleotide of the molecular beacon
consists of SEQ ID NO.: 78. In certain implementations of the kit,
the set of polynucleotides is selected from the group consisting of
Set-14, Set-15, Set-16, and Set-17. In such implementations, the
kit can further comprise a molecular beacon comprising a
fluorophore, a quencher, and a polynucleotide comprising
nucleotides 2-24 of SEQ ID NO.: 95. Preferably, the polynucleotide
of the molecular beacon consists of SEQ ID NO.: 95.
[0022] Another aspect of the invention provides a kit comprising a
set of polynucleotides comprising SEQ ID NO.: 49, SEQ ID NO.: 50,
SEQ ID NO.: 51, SEQ ID NO.: 52, SEQ ID NO.: 53, and SEQ ID NO.: 54.
The kit preferably, further comprises a strand displacement
polymerase and reverse transcriptase. In some implementations, the
kit further comprises a molecular beacon comprising a fluorophore,
a quencher, and SEQ ID NO. 83. In another implementation, the kit
further comprises a set of polynucleotides comprising SEQ ID NO.:
63, SEQ ID NO.: 64, SEQ ID NO.: 65, SEQ ID NO.: 66, SEQ ID NO.: 67,
and SEQ ID NO.: 68. This kit preferably further comprises a
molecular beacon comprising a fluorophore, a quencher and SEQ ID
NO. 77.
DETAILED DESCRIPTION
[0023] The present invention encompasses, in some embodiments, a
composition comprising a set of polynucleotides for priming a
nucleic acid amplification reaction and methods of using such. In
some embodiments, the composition further comprises a probe.
[0024] As used herein, "nucleic acid" includes both DNA and RNA,
including DNA and RNA containing non-standard nucleotides. A
"nucleic acid" contains at least one polynucleotide (a "nucleic
acid strand"). A "nucleic acid" may be single-stranded or
double-stranded. The term "nucleic acid" refers to nucleotides and
nucleosides which make up, for example, deoxyribonucleic acid (DNA)
macromolecules and ribonucleic acid (RNA) macromolecules. The most
common nucleic acids are deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). It should be further understood that the
present invention can be used for biological sequences containing
artificial nucleotides such as peptide nucleic acid (PNA),
morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA)
and threose nucleic acid (TNA), among others. Preferably, the
artificial nucleotides are locked nucleic acid molecules, including
[alpha]-L-LNAs. LNAs comprise ribonucleic acid analogues wherein
the ribose ring is "locked" by a methylene bridge between the
2'-oxygen and the 4'-carbon--i.e., oligonucleotides, containing at
least one LNA monomer, that is, one
2'-O,4'-C-methylene-.beta.-D-ribofuranosyl nucleotide. LNA bases
form standard Watson-Crick base pairs but the locked configuration
increases the rate and stability of the basepairing reaction
(Jepsen et al., Oligonucleotides, 14, 130-146 (2004)).
[0025] As used herein, a "polynucleotide" refers to a polymeric
chain containing two or more nucleotides, which contain
deoxyribonucleotides, ribonucleotides, and/or their analog, such as
those containing modified backbones (e.g. peptide nucleic acids
(PNAs) or phosphorothioates) or modified bases. "Polynucleotides"
includes primers, oligonucleotides, nucleic acid strands, etc. A
polynucleotide may contain standard or non-standard nucleotides.
Thus, the term includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA,
recombinant nucleic acids, branched nucleic acids, plasmids,
vectors, probes, primers, etc. Typically, a polynucleotide contains
a 5' phosphate at one terminus ("5' terminus") and a 3' hydroxyl
group at the other terminus ("3' terminus") of the chain. The most
5' nucleotide of a polynucleotide may be referred to herein as the
"5' terminal nucleotide" of the polynucleotide. The most 3'
nucleotide of a polynucleotide may be referred to herein as the "3'
terminal nucleotide" of the polynucleotide. Where nucleic acid of
the invention takes the form of RNA, it may or may not have a 5'
cap.
[0026] LAMP is a nucleic acid amplification method that relies on
auto-cycle strand-displacement DNA synthesis performed by Bst DNA
polymerase, or other strand displacement polymerases. The amplified
products are stem-loop structures with several repeated sequences
of the target and have multiple loops. The principal merit of this
method is that denaturation of the DNA template is not required,
and thus the LAMP reaction can be conducted under isothermal
conditions (ranging from 60 to 67.degree. C.). LAMP requires only
one enzyme and four types of primers that recognize six distinct
hybridization sites in the target sequence. The reaction can be
accelerated by the addition of two additional primers. The method
produces a large amount of amplified product, resulting in easier
detection, such as detection by visual judgment of the turbidity or
fluorescence of the reaction mixture.
[0027] In brief, the reaction is initiated by annealing and
extension of a pair of `loop-forming` primers (forward and backward
inner primers, FIP and BIP, respectively), followed by annealing
and extension of a pair of flanking primers (F3 and B3). Extension
of these primers results in strand-displacement of the loop-forming
elements, which fold up to form terminal hairpin-loop structures.
Once these key structures have appeared, the amplification process
becomes self-sustaining, and proceeds at constant temperature in a
continuous and exponential manner (rather than a cyclic manner,
like PCR) until all of the nucleotides (dATP, dTTP, dCTP &
dGTP) in the reaction mixture have been incorporated into the
amplified DNA. Optionally, an additional pair of primers can be
included to accelerate the reaction. These primers, termed Loop
primers, hybridize to non-inner primer bound terminal loops of the
inner primer dumbbell shaped products.
[0028] The term "primer" as used herein refers to an
oligonucleotide, which is capable of acting as a point of
initiation of synthesis when placed under conditions in which
synthesis of primer extension product which is complementary to a
nucleic acid strand (template) is induced, i.e., in the presence of
nucleotides and an agent for polymerization, such as DNA
polymerase, and at a suitable temperature and pH.
[0029] Applications for LAMP have been further extended to include
detection of RNA molecules by addition of Reverse Transcriptase
enzyme (RT). By including RNA detection, the types of targets for
which LAMP can be applied are also expanded and add the ability to
additionally target RNA based viruses, important regulatory
non-coding RNA (sRNA, miRNA), and RNA molecules that have been
associated with particular disease or physiological states. The
ability to detect RNA also has the potential to increase assay
sensitivity, for instance in choosing highly expressed, stable,
and/or abundant messenger RNA (mRNA) or ribosomal RNA (rRNA)
targets. This preliminary phase of amplification involves the
reverse transcription of RNA molecules to complementary DNA (cDNA).
The cDNA then serves as template for the strand displacing DNA
polymerase. Use of a thermostable RT enzyme (i.e., NEB RTx) enables
the reaction to be completed at a single temperature and in a one
step, single mix reaction.
[0030] A "target sequence," as used herein, means a nucleic acid
sequence of Neisseria gonorrhoeae, or complement thereof, that is
amplified, detected, or both amplified and detected using one or
more of the polynucleotides herein provided. Additionally, while
the term target sequence sometimes refers to a double stranded
nucleic acid sequence, those skilled in the art will recognize that
the target sequence can also be single stranded, e.g., RNA. A
target sequence may be selected that is more or less specific for a
particular organism. For example, the target sequence may be
specific to an entire genus, to more than one genus, to a species
or subspecies, serogroup, auxotype, serotype, strain, isolate or
other subset of organisms.
[0031] The speed, specificity and sensitivity of the primers/probe
compositions and method described herein result from several
aspects. Exemplary primers for use in the compositions and methods
according to the present invention include those provided in Table
1.
TABLE-US-00001 TABLE 1 Primer Sequences Sequence ID Target Sequence
(5' to 3') SEQ ID No.: 1 N gene GGACCAGGAACTAATCAGACA SEQ ID No.: 2
N gene TCTGCGGTAAGGCTTGAG SEQ ID No.: 3 N gene
ACCACGTTCCCGAAGGTGTCAGCGTTCTTCGGAATGTC SEQ ID No.: 4 N gene
TGACCTACACAGGTGCCATCAAGGCTCTGTTGGTGGGAAT SEQ ID No.: 5 N gene
GACTTCCATGCCAATGCG SEQ ID No.: 6 N gene GCTGAATAAGCATATTGACGCATAC
SEQ ID No.: 7 N gene ACCCCAAAATCAGCGAAA SEQ ID No.: 8 N gene
ATTGGAACGCCTTGTCC SEQ ID No.: 9 N gene
ATCGCGCCCCACTGCCACCCCGCATTACGTTT SEQ ID No.: 10 N gene
CGTCGGCCCCAAGGTTTATCCTTGCCATGTTGAGTG SEQ ID No.: 11 N gene
CAGTTGAATCTGAGGGTCCA SEQ ID No.: 12 N gene GTCTTGGTTCACCGCTCT SEQ
ID No.: 13 N gene AAAAGGCTTCTACGCAGAA SEQ ID No.: 14 N gene
CCTTTACCAGACATTTTGCTC SEQ ID No.: 15 N gene
ACTGCTGCCTGGAGTTGAATCAGTCAAGCCTCTTCTCG SEQ ID No.: 16 N gene
CTGCTAGAATGGCTGGCAATGTGGTTCAATCTGTCAAGCA SEQ ID No.: 17 N gene
ACTGTTGCGACTACGTGAT SEQ ID No.: 18 N gene GCGGTGATGCTGCTCT SEQ ID
No.: 19 N gene AGGAACTGATTACAAACATTGG SEQ ID No.: 20 N gene
TTTTGTATGCGTCAATATGCTT SEQ ID No.: 21 N gene
AAGGTGTGACTTCCATGCCAACAATTTGCCCCCAGC SEQ ID No.: 22 N gene
GGGAACGTGGTTGACCTACAGACTTGATCTTTGAAATTTGGATC SEQ ID No.: 23 N gene
TGCGCGACATTCCGAA SEQ ID No.: 24 N gene GGTGCCATCAAATTGGATGA SEQ ID
No.: 25 N gene TCAAAGATCAAGTCATTTTGCT SEQ ID No.: 26 N gene
GCCTGAGTTGAGTCAGC SEQ ID No.: 27 N gene
GTCTCTGCGGTAAGGCTTGAATACAAAACATTCCCACCAAC SEQ ID No.: 28 N gene
GCAAACTGTGACTCTTCTTCCTGTGCTCATGGATTGTTGCA SEQ ID No.: 29 N gene
ATCAGCCTTCTTCTTTTTGTCC SEQ ID No.: 30 N gene TGCAGATTTGGATGATTTCTCC
SEQ ID No.: 31 ORF1ab CAGAAATCAATACTGAGTCCTCT SEQ ID No.: 32 ORF1ab
GTAGCCAAATCAGATGTGAAC SEQ ID No.: 33 ORF1ab
CACAGAATTTTGAGCAGTTTCAAGAGTTCAGAGGCTGCTCGTG SEQ ID No.: 34 ORF1ab
CGTGTTTTACAGAAGGCCGCCATAGCATCAATGAGTCTCAGT SEQ ID No.: 35 ORF1ab
GCGGGAGAAAATTGATCGTAC SEQ ID No.: 36 ORF1ab
ATAACAATACTAGATGGAATTTCACAGT SEQ ID No.: 37 ORF1ab
CAAATTGTTGAATCCTGTGGT SEQ ID No.: 38 ORF1ab
AAATTCCATCTAGTATTGTTATAGCG SEQ ID No.: 39 ORF1ab
AATGCATAAAGAGGACTCAGTATTGATTTCAATTTTAAAGTTACAAAAGGAAAAGCT SEQ ID
No.: 40 ORF1ab CAGAGGCTGCTCGTGTTGTCACGCACAGAATTTTGAGC SEQ ID No.:
41 ORF1ab TGTTCACCAATATTCCAGGCA SEQ ID No.: 42 ORF1ab
CGATCAATTTTCTCCCGCAC SEQ ID No.: 43 N gene GTCAAGCCTCTTCTCGTTC SEQ
ID No.: 44 N gene CTTAGTGACAGTTTGGCCTT SEQ ID No.: 45 N gene
CCGCCATTGCCAGCCAAACAGTTCAAGAAATTCAACTCC SEQ ID No.: 46 N gene
GATGCTGCTCTTGCTTTGCTGGCCTTTACCAGACATTTTGC SEQ ID No.: 47 N gene
GAGAAGTTCCCCTACTGCTG SEQ ID No.: 48 N gene TGACAGATTGAACCAGCTTGA
SEQ ID No.: 49 N gene AATTTCAAAGATCAAGTCATTTTGC SEQ ID No.: 50 N
gene GTTGAGTCAGCACTGCTC SEQ ID No.: 51 N gene
AAGGCTTGAGTTTCATCAGCCTCGCATACAAAACATTCCCAC SEQ ID No.: 52 N gene
GCAGAGACAGAAGAAACAGCAAACATTGTTGCAATTGTTTGGAGAA SEQ ID No.: 53 N
gene TCTTTTTGTCCTTTTTAGGCTCTG SEQ ID No.: 54 N gene
CTCTTCTTCCTGCTGCAGATTTGG SEQ ID No.: 57 ORF1ab
CAAATTGTTGAATCCTGTGGT SEQ ID No.: 58 ORF1ab
AAATTCCATCTAGTATTGTTATAGCG SEQ ID No.: 59 ORF1ab
AATGCATAAAGAGGACTCAGTATTGATTTCAATTTTAAAGTTACAAAAGGAAAAGCT SEQ ID
No.: 60 ORF1ab CAGAGGCTGCTCGTGTTGTCACGCACAGAATTTTGAGC SEQ ID No.:
61 ORF1ab TGTTCACCAATATTCCAGGCA SEQ ID No.: 62 ORF1ab
CGATCAATTTTCTCCCGCAC SEQ ID No.: 63 ORF1ab TCTTATCAGAGGCACGTCA SEQ
ID No.: 64 ORF1ab TGTCTCACCACTACGACC SEQ ID No.: 65 ORF1ab
ACGTTTGATGAACACATAGGGCTTAAAGATGGCACTTGTGGC SEQ ID No.: 66 ORF1ab
TCGGATGCTCGAACTGCACTGCCTTCGAGTTCTGCTA SEQ ID No.: 67 ORF1ab
TTCAAGTTGAGGCAAAACGC SEQ ID No.: 68 ORF1ab CATGGTCATGTTATGGTTGAGC
SEQ ID No.: 69 ORF1ab ATGTCCAAATTTTGTATTTCCCTT SEQ ID No.: 70
ORF1ab GCTTTAACAAAATCGCCCG SEQ ID No.: 71 ORF1ab
GCAACTGGATAGACAGATCGAATTCTATCCATAATCAAGACTATTCAACCA SEQ ID No.: 72
ORF1ab CACCAAATGAATGCAACCAAATGTGTCTGCCATGAAGTTTCACC SEQ ID No.: 73
ORF1ab CATCAAGCTTTTTCTTTTCAACCC SEQ ID No.: 74 ORF1ab
CCTTTCAACTCTCATGAAGTGTG SEQ ID No.: 86 ORF1ab GAGGGACAAGGACACCAAG
SEQ ID No.: 87 ORF1ab TCGGATGCTCGAACTGCACTGTCTCACCACTACGACC SEQ ID
No.: 88 ORF1ab GGTAGCAGAACTCGAAGGCAT SEQ ID No.: 89 E gene
AAGAGACAGGTACGTTAATAGT SEQ ID No.: 90 E gene TTAGACCAGAAGATCAGGAAC
SEQ ID No.: 91 E gene AAGCGCAGTAAGGATGGCTATAGCGTACTTCTTTTTCTTGC SEQ
ID No.: 92 E gene TGCGTACTGCTGCAATATTGTTTTAACACGAGAGTAAACGTAAA SEQ
ID No.: 93 E gene TGTAACTAGCAAGAATACCACG SEQ ID No.: 94 E gene
CGTGAGTCTTGTAAAACCTTCT SEQ ID No.: 96 E gene GAAGATCAGGAACTCTAGAAGA
SEQ ID No.: 97 E gene ACACAATCGAAGCGCAGTAAGTTTTCTTGCTTTCGTGGTAT SEQ
ID No.: 98 E gene GCGTACTGCTGCAATATTGTTTTAACACGAGAGTAAACGTAAA SEQ
ID No.: 99 E gene TGGCTAGTGTAACTAGCAAGA SEQ ID No.: 100 E gene
ACGTTAATAGTTAATAGCGTACTT SEQ ID No.: 101 E gene
TCAGGAACTCTAGAAGAATTCA SEQ ID No.: 102 E gene
ACAATCGAAGCGCAGTAAGGTTTTCTTGCTTTCGTGGTAT SEQ ID No.: 103 E gene
TGCGTACTGCTGCAATATTGTTTTTAACACGAGAGTAAACGTAAA
[0032] Detection of the LAMP amplified products can be achieved via
a variety of methods. In some implementations, LAMP amplified
products are detected using intercalating dyes. Intercalating dyes
are generally aromatic cations with planar structures that insert
between stacked base pairs in the DNA duplex, an arrangement that
provides an environmentally dependent fluorescence enhancement for
dye molecules and creates a large increase in the fluorescence
signal relative to the free dye in solution. The signal enhancement
provides a proportional response, allowing direct quantitative DNA
measurements. Preferred intercalating dyes in the present
disclosure include fluorescent dyes. The dye can be a cyanine or a
non-cyanine intercalating die. In some cases, the intercalating dye
is a cyanine dye. In some cases, the cyanine dye can be Thiazole
Orange, SYBR.RTM. (e.g. Sybr Green I, Sybr Green II, Sybr Gold,
SYBR DX), Oil Green, CyQuant GR, SYTOX Green, SYTO9, SYT010,
SYTO17, SYBR14, Oxazile Yellow, Thiazone Orange, SYTO, TOTO, YOYO,
BOBO, and POPO. In some cases, the dye is a non-cyanine dye. In
some cases, the non cyanine dye is pentacene, anthracene,
naphthalene, ferrocene, methyl viologen, tri-morpholino ammonium,
propidium (e.g., propidium iodide) or another aromatic or
heteroaromatic derivative.
[0033] In a preferred embodiment, detection of product is conducted
by adding a fluorescently-labeled probe to the primer mix. The term
used herein "probe" refers to a single-stranded nucleic acid
molecule comprising a portion or portions that are complementary,
or substantially complementary, to a target sequence. In certain
implementations, the fluorescently-labeled probe is a molecular
beacon.
[0034] As used herein, "molecular beacon" refers to a single
stranded hairpin-shaped oligonucleotide probe designed to report
the presence of specific nucleic acids in a solution. A molecular
beacon consists of four components; a stem, hairpin loop, end
labelled fluorophore and opposite end-labelled quencher (Tyagi et
al., (1998) Nature Biotechnology 16:49-53). When the hairpin-like
beacon is not bound to a target, the fluorophore and quencher lie
close together and fluorescence is suppressed. In the presence of a
complementary target nucleotide sequence, the stem of the beacon
opens to hybridize to the target. This separates the fluorophore
and quencher, allowing the fluorophore to fluoresce. Alternatively,
molecular beacons also include fluorophores that emit in the
proximity of an end-labelled donor. "Wavelength-shifting Molecular
Beacons" incorporate an additional harvester fluorophore enabling
the fluorophore to emit more strongly. Current reviews of molecular
beacons include Wang et al., 2009, Angew Chem Int Ed Engl,
48(5):856-870; Cissell et al., 2009, Anal Bioanal Chem
393(1):125-35; Li et al., 2008, Biochem Biophys Res Comm
373(4):457-61; and Cady, 2009, Methods Mol Biol 554:367-79.
Exemplary probes for use in the compositions and methods according
to the present invention include those provided in Table 2. In
certain implementations, the probes may include one or more linked
nucleic acids (LNA) as indicated by "[+X]", where X indicates the
identity of the nucleobase. Bold indicates the portion of the
molecular beacon that hybridizes to sequences found in the target
coronavirus genome.
TABLE-US-00002 TABLE 2 Probe Sequences ID Fluor Quench Sequence (5'
to 3') Sequence ID MB1 FAM BHQ1
CACGCCAAA[+T]TT[+C]AAA[+G]AT[+C]AAGTCATGGCGTG SEQ ID NO.: 55 MB2
FAM BHQ1 CAGCTGCTTGA[+C]AGA[+T]TGA[+A]CCAGCAGCTG SEQ ID NO.: 56 MB3
FAM BHQ1 CACGGTGACT[+C]TT[+C]TT[+C]CTGCTCACCGTG SEQ ID NO.: 75 MB4
FAM BHQ1 CGAGTCCTGC[+T]GC[+A]GA[+T]TTGGACTCG SEQ ID NO.: 76 MB5 FAM
BHQ1 CAGCTCATGG[+T]CAT[+G]TTAT[+G]GTTGAGCTG SEQ ID NO.: 77 MB6 FAM
BHQ1 CACACTTT[+C]AA[+C]TC[+T]CA[+T]GAAGTGTG SEQ ID NO.: 78 MB7 FAM
BHQ1 CAGCTCATGGTCATGTTATGGTTGAGCTG SEQ ID NO.: 79 MB8 FAM BHQ1
CGAGTCCTGC[+T]GC[+A]GA[+T]TTGGACTCG SEQ ID NO.: 80 MB9 FAM BHQ1
CATGTCCTGC[+T]GC[+A]GA[+T]TTGGACATG SEQ ID NO.: 81 MB10 FAM BHQ1
CACTTCCTGC[+T]GC[+A]GA[+T]TTGGAAGTG SEQ ID NO.: 82 MB11 FAM BHQ1
CACGCGTGACT[+C]TTCTT[+C]CTGC[+T]GCAGACGCGTG SEQ ID NO.: 83 MB12 FAM
BHQ1 CACGCTTC[+T]TC[+C]TGC[+T]GC[+A]GA[+T]TTGAAGCGTG SEQ ID NO.: 84
MB13 FAM BHQ1 CAGACTCTTCT[+T]CC[+T]GC[+T]GCAGAGTCTG SEQ ID NO.: 85
MB14 FAM BHQ1 CACGTGAGT[+C]TT[+G]TAA[+A]ACC[+T]TCTCACGTG SEQ ID
NO.: 95
[0035] In one implementation, the molecular beacon comprises a
fluorophore, a quencher, and a polynucleotide, wherein the
polynucleotide comprises a sequence selected from the group
consisting of nucleotides 5-27 of SEQ ID NO.: 55, nucleotides 7-25
of SEQ ID NO.: 56, nucleotides 5-22 of SEQ ID NO.: 75, nucleotides
5-22 of SEQ ID NO.: 76, nucleotides 5-29 of SEQ ID NO.: 77,
nucleotides 6-26 of SEQ ID NO.: 78, nucleotides 5-29 of SEQ ID NO.:
79, nucleotides 5-22 of SEQ ID NO.: 80, nucleotides 5-22 of SEQ ID
NO.: 81, nucleotides 4-22 of SEQ ID NO.: 82, nucleotides 6-28 of
SEQ ID NO.: 83, nucleotides 6-25 of SEQ ID NO.: 84, nucleotides
3-23 of SEQ ID NO.: 85. In one embodiment, the polynucleotide
comprises a sequence selected from the group consisting of SEQ ID
NO.: 55, SEQ ID NO.: 56 and SEQ ID NOS.: 75-85. In another
embodiment, the polynucleotide consists of a sequence selected from
the group consisting of SEQ ID NO.: 55, SEQ ID NO.: 56 and SEQ ID
NOS.: 75-85.
[0036] The molecular beacon is preferably used in a composition
also comprising a set of sequence-specific LAMP primers. In one
implementation, the molecular beacon comprises a sequence selected
from the group consisting of nucleotides 5-27 of SEQ ID NO.: 55 and
nucleotides 7-25 of SEQ ID NO.: 56. In such an implementation, the
molecular beacon can comprise a sequence selected from the group
consisting of SEQ ID NO.: 55, SEQ ID NO.: 56 and SEQ ID NOS.:
75-85. More preferably, polynucleotide sequence of the molecular
beacon consists of a sequence selected from the group consisting of
SEQ ID NO.: 55, SEQ ID NO.: 56 and SEQ ID NOS.: 75-85.
[0037] The term "label" as used herein means a molecule or moiety
having a property or characteristic which is capable of detection
and, optionally, of quantitation. A label can be directly
detectable, as with, for example (and without limitation),
radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal
particles, fluorescent microparticles and the like; or a label may
be indirectly detectable, as with, for example, specific binding
members. It will be understood that directly detectable labels may
require additional components such as, for example, substrates,
triggering reagents, quenching moieties, light, and the like to
enable detection and/or quantitation of the label. When indirectly
detectable labels are used, they are typically used in combination
with a "conjugate". A conjugate is typically a specific binding
member that has been attached or coupled to a directly detectable
label. Coupling chemistries for synthesizing a conjugate are well
known in the art and can include, for example, any chemical means
and/or physical means that does not destroy the specific binding
property of the specific binding member or the detectable property
of the label. As used herein, "specific binding member" means a
member of a binding pair, i.e., two different molecules where one
of the molecules through, for example, chemical or physical means
specifically binds to the other molecule. In addition to antigen
and antibody specific binding pairs, other specific binding pairs
include, but are not intended to be limited to, avidin and biotin;
haptens and antibodies specific for haptens; complementary
nucleotide sequences; enzyme cofactors or substrates and enzymes;
and the like.
[0038] The molecular beacon can be composed of nucleic acid only
such as DNA or RNA, or it can be composed of a peptide nucleic acid
(PNA) conjugate. The fluorophore can be any fluorescent organic dye
or a single quantum dot. The quenching moiety desirably quenches
the luminescence of the fluorophore. Any suitable quenching moiety
that quenches the luminescence of the fluorophore can be used. A
fluorophore can be any fluorescent marker/dye known in the art.
Examples of suitable fluorescent markers include, but are not
limited to, Fam, Hex, Tet, Joe, Rox, Tamra, Max, Edans, Cy dyes
such as Cy5, Fluorescein, Coumarin, Eosine, Rhodamine, Bodipy,
Alexa, Cascade Blue, Yakima Yellow, Lucifer Yellow, Texas Red, and
the family of ATTO dyes. A quencher can be any quencher known in
the art. Examples of quenchers include, but are not limited to,
DABCYL (4-(dimethylaminoazo)benzene-4-carboxylic acid), dark
quenchers such as ECLIPSE Dark Quencher, ElleQuencher, Tamra
(tetramethylrhodamine), Black Hole quenchers and QSY dyes. The
skilled person would know which combinations of dye/quencher are
suitable when designing a probe. In an exemplary embodiment,
fluorescein (FAM) is used in conjunction with Blackhole
Quencher.TM. (BHQ.TM.). Binding of the molecular beacon to
amplified product can then be directly, visually assessed.
Alternatively, the fluorescence level can be measured by
spectroscopy in order to improve sensitivity.
[0039] A variety of commercial suppliers produce standard and
custom molecular beacons, including Abingdon Health (UK), Attostar
(US, MN), Biolegio (NLD), Biomers.net (DEU), Biosearch Technologies
(US, CA), Eurogentec (BEL), Gene Link (US, NY) Integrated DNA
Technologies (US, IA), Isogen Life Science (NLD), Midland Certified
Reagent (US, TX), Eurofins (DEU), Sigma-Aldrich (US, TX), Thermo
Scientific (US, MA), TIB MOLBIOL (DEU), TriLink Bio Technologies
(US, CA). A variety of kits, which utilize molecular beacons are
also commercially available, such as the Sentinel.TM. Molecular
Beacon Allelic Discrimination Kits from Stratagene (La Jolla,
Calif.) and various kits from Eurogentec SA (Belgium) and Isogen
Bioscience BV (The Netherlands).
[0040] The oligonucleotide probes and primers of the invention are
optionally prepared using essentially any technique known in the
art. In certain embodiments, for example, the oligonucleotide
probes and primers described herein are synthesized chemically
using essentially any nucleic acid synthesis method, including,
e.g., according to the solid phase phosphoramidite triester method
described by Beaucage and Caruthers (1981), Tetrahedron Letts.
22(20):1859-1862, which is incorporated by reference, or another
synthesis technique known in the art, e.g., using an automated
synthesizer, as described in Needham-VanDevanter et al. (1984)
Nucleic Acids Res. 12:6159-6168, which is incorporated by
reference. A wide variety of equipment is commercially available
for automated oligonucleotide synthesis. Multi-nucleotide synthesis
approaches (e.g., tri-nucleotide synthesis, etc.) are also
optionally utilized. Moreover, the primer nucleic acids described
herein optionally include various modifications. To further
illustrate, primers are also optionally modified to improve the
specificity of amplification reactions as described in, e.g., U.S.
Pat. No. 6,001,611, issued Dec. 14, 1999, which is incorporated by
reference. Primers and probes can also be synthesized with various
other modifications as described herein or as otherwise known in
the art.
[0041] In addition, essentially any nucleic acid (and virtually any
labeled nucleic acid, whether standard or non-standard) can be
custom or standard ordered from any of a variety of commercial
sources, such as Integrated DNA Technologies, the Midland Certified
Reagent Company, Eurofins, Biosearch Technologies, Sigma Aldrich
and many others.
[0042] The term "test sample" as used herein, means a sample taken
from an organism or biological fluid that is suspected of
containing or potentially contains a target sequence. The test
sample can be taken from any biological source, such as for
example, tissue, blood, saliva, sputa, mucus, sweat, urine,
urethral swabs, cervical swabs, vaginal swabs, urogenital or anal
swabs, conjunctival swabs, ocular lens fluid, cerebral spinal
fluid, milk, ascites fluid, synovial fluid, peritoneal fluid,
amniotic fluid, fermentation broths, cell cultures, chemical
reaction mixtures and the like. The test sample can be used (i)
directly as obtained from the source or (ii) following a
pre-treatment to modify the character of the sample. Thus, the test
sample can be pre-treated prior to use by, for example, preparing
plasma or serum from blood, disrupting cells or viral particles,
preparing liquids from solid materials, diluting viscous fluids,
filtering liquids, distilling liquids, concentrating liquids,
inactivating interfering components, adding reagents, purifying
nucleic acids, and the like.
[0043] Advantageously, the invention enables reliable rapid
detection of SARS-CoV-2 in a clinical sample, such as sputum or a
nasal or pharyngeal swab.
[0044] To further illustrate, prior to analyzing the target nucleic
acids described herein, those nucleic acids may be purified or
isolated from samples that typically include complex mixtures of
different components. Cells in collected samples are typically
lysed to release the cell contents, including target nucleic acids.
For example, a test sample suspected of containing virus, can be
lysed by contacting viral particles with various enzymes,
chemicals, and/or lysed by other approaches known in the art, which
degrade, e.g., viral particle walls. In some embodiments, nucleic
acids are analyzed directly in the cell lysate. In other
embodiments, nucleic acids are further purified or extracted from
lysates prior to detection. Essentially any nucleic acid extraction
methods can be used to purify nucleic acids in the samples utilized
in the methods of the present invention. Exemplary techniques that
can be used to purifying nucleic acids include, e.g., affinity
chromatography, hybridization to probes immobilized on solid
supports, liquid-liquid extraction (e.g., phenol-chloroform
extraction, etc.), precipitation (e.g., using ethanol, etc.),
extraction with filter paper, extraction with micelle-forming
reagents (e.g., cetyl-trimethyl-ammonium-bromide, etc.), binding to
immobilized intercalating dyes (e.g., ethidium bromide, acridine,
etc.), adsorption to silica gel or diatomic earths, adsorption to
magnetic glass particles or organo silane particles under
chaotropic conditions, and/or the like. Sample processing is also
described in, e.g., U.S. Pat. Nos. 5,155,018, 6,383,393, and
5,234,809, which are each incorporated by reference.
[0045] A test sample may optionally have been treated and/or
purified according to any technique known by the skilled person, to
improve the amplification efficiency and/or qualitative accuracy
and/or quantitative accuracy. The sample may thus exclusively, or
essentially, consist of nucleic acid(s), whether obtained by
purification, isolation, or by chemical synthesis. Means are
available to the skilled person, who would like to isolate or
purify nucleic acids, such as DNA, from a test sample, for example
to isolate or purify DNA from pharyngeal scrapes (e.g., QIAamp-DNA
Mini-Kit; Qiagen, Hilden, Germany).
Equivalents and Scope
[0046] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0047] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0048] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0049] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0050] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
[0051] Section and table headings are not intended to be
limiting.
EXAMPLES
[0052] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1: Target Selection and Primer Probe Design
[0053] Gene sequences for multiple isolates of SARS-CoV-2, closely
related species such as common human coronavirus, severe acute
respiratory syndrome (SARS)-related coronavirus, and Middle East
respiratory syndrome-related coronavirus (MERS-CoV), and for other
species commonly found in the nasal or pharyngeal samples acquired
from patients with upper respiratory symptoms were retrieved from
the NCBI database. Sequences were aligned using Clustal omega
(Sievers, et al. 2011. Molecular Systems Biology 7:539) and regions
with unique specific bases to SARS-CoV-2 species were identified.
Loop mediated amplification primers were designed using LAMP
designer (Premier Biosoft) or an in-house design algorithm. For
added specificity, molecular beacons or probes targeting the
amplified products were designed manually or using Beacon designer
(Premier Biosoft). Designed primer sets and beacons were further
analyzed for specificity using BLAST against the NCBI nucleotide
database, including human transcriptome, influenza virus, and
non-human coronavirus. Various primer sets and probes were designed
and screened for reaction speed.
[0054] The inventive primer sets are summarized in Table 3, which
include, at a minimum, a forward inner primer (FIP) and backward
inner primer (BIP). Additionally, the primer sets typically also
include at least two additional primers selected from the forward
outer primer (F3), backward outer primer (B3), forward loop primer
(LF) and backward loop primer (LB).
TABLE-US-00003 TABLE 3 LAMP Primer Sets Set F3 B3 FIP BIP LF LB
Set-1 SEQ ID No.: 1 SEQ ID No.: 2 SEQ ID No.: 3 SEQ ID No.: 4 SEQ
ID No.: 5 SEQ ID No.: 6 Set-2 SEQ ID No.: 7 SEQ ID No.: 8 SEQ ID
No.: 9 SEQ ID No.: 10 SEQ ID No.: 11 SEQ ID No.: 12 Set-3 SEQ ID
No.: 13 SEQ ID No.: 14 SEQ ID No.: 15 SEQ ID No.: 16 SEQ ID No.: 17
SEQ ID No.: 18 Set-4 SEQ ID No.: 19 SEQ ID No.: 20 SEQ ID No.: 21
SEQ ID No.: 22 SEQ ID No.: 23 SEQ ID No.: 24 Set-5 SEQ ID No.: 25
SEQ ID No.: 26 SEQ ID No.: 27 SEQ ID No.: 28 SEQ ID No.: 29 SEQ ID
No.: 30 Set-6 SEQ ID No.: 31 SEQ ID No.: 32 SEQ ID No.: 33 SEQ ID
No.: 34 SEQ ID No.: 35 SEQ ID No.: 36 Set-7 SEQ ID No.: 37 SEQ ID
No.: 38 SEQ ID No.: 39 SEQ ID No.: 40 SEQ ID No.: 41 SEQ ID No.: 42
Set-8 SEQ ID No.: 43 SEQ ID No.: 44 SEQ ID No.: 45 SEQ ID No.: 46
SEQ ID No.: 47 SEQ ID No.: 48 Set-9 SEQ ID No.: 49 SEQ ID No.: 50
SEQ ID No.: 51 SEQ ID No.: 52 SEQ ID No.: 53 SEQ ID No.: 54 Set-10
SEQ ID No.: 57 SEQ ID No.: 58 SEQ ID No.: 59 SEQ ID No.: 60 SEQ ID
No.: 61 SEQ ID No.: 62 Set-11 SEQ ID No.: 63 SEQ ID No.: 64 SEQ ID
No.: 65 SEQ ID No.: 66 SEQ ID No.: 67 SEQ ID No.: 68 Set-12 SEQ ID
No.: 69 SEQ ID No.: 70 SEQ ID No.: 71 SEQ ID No.: 72 SEQ ID No.: 73
SEQ ID No.: 74 Set-13 SEQ ID No.: 63 SEQ ID No.: 86 SEQ ID No.: 65
SEQ ID No.: 87 SEQ ID No.: 67 SEQ ID No.: 88 Set-14 SEQ ID No.: 89
SEQ ID No.: 90 SEQ ID No.: 91 SEQ ID No.: 92 SEQ ID No.: 93 SEQ ID
No.: 94 Set-15 SEQ ID No.: 89 SEQ ID No.: 96 SEQ ID No.: 97 SEQ ID
No.: 98 SEQ ID No.: 99 SEQ ID No.: 94 Set-16 SEQ ID No.: 100 SEQ ID
No.: 101 SEQ ID No.: 102 SEQ ID No.: 103 SEQ ID No.: 99 SEQ ID No.:
94 Set-17 SEQ ID No.: 89 SEQ ID No.: 96 SEQ ID No.: 91 SEQ ID No.:
92 SEQ ID No.: 93 SEQ ID No.: 94
[0055] Typically, 3 to 5 .mu.L of RNA standards or genomic RNA or
genomic RNA extracted from negative nasal swab matrix or negative
controls (NTC=nuclease free water or Tris buffer, no template
control) served as template for RTLAMP reactions. 10-25 .mu.l total
volume reactions were prepared on ice as mixes containing
formulations including 1.times. amplification buffer comprising
10-40 mM Tris-HCl, 0-0.5% Tween 20, 0-300 mM Trehalose, 5-70 mM
KCl, 4-41 mM MgSO.sub.4, 10-20 mM (NH.sub.4).sub.2SO.sub.4, 0-2 mM
TCEP and 1.6-2 mM each dCTP, dGTP, dATP and dTTP. NEB Bst2
polymerase (NEB CN #M0537L) and RTx Warmstart reverse transcriptase
(NEB CN #M0380S) enzymes. Primers (2 .mu.M inner primers, 0.2 .mu.M
outer primers, and 0.8 .mu.M Loop primers) were added to individual
reactions or directly to master mixes as required per experimental
design. Molecular beacons (0.2 .mu.M) or 200 nM To-Pro dye was also
added to the master mix, as indicated in the examples below.
Amplification reactions were prepared with the standard 6-primer
composition. Master mixes were distributed to individual sample
templates, vortexed and centrifuged briefly and each reaction
loaded into individual wells of a 96 or 384 well plate (Roche CN
#4729692001 or BioRad CNhsI9605). Reactions were carried out at
temperatures ranging from 60-67.degree. C. and fluorescence
monitored on either a Roche LightCycler 96 Real-Time PCR instrument
or a BioRad CFX96 real time cycler. Target amplification was
monitored via intercalating dye or molecular beacon probe binding
to target resulting in release of molecular beacon fluorescence
intramolecular quenching.
Example 2: Amplification Reaction Kinetics
[0056] Input samples were RNA molecular standards generated from in
vitro transcription of 900-1500 bp gene fragments, including those
of SARS-CoV-2 and common human coronavirus.
[0057] An RNA molecular standard or genomic RNA was diluted to
various concentrations ranging from 50-5000 copies/reaction to
assess the sensitivity of the indicated primer set (Table 3).
Standards were serially diluted with 0.1 mg/mL Poly A carrier RNA
in PBS (Sigma) and used as template for amplification in RTLAMP
reactions. ToPro.TM. or Syto.TM. dye or a compatible wavelength
version within the same dye set family (Life Technologies; green or
red fluorescent carbocyanine nucleic acid stain) was used for the
detection of the amplified product. The master mix was prepared as
described in Example 1.
[0058] This example shows that using this set of primers and the
loop mediated amplification method, fast amplification kinetics are
achieved. Results are summarized in Table 4, in which the Time to
Positive (T.sub.p) was calculated by using an in-house developed
algorithm. NT indicates concentrations not tested. Results are
classified by the time to positive (NT means "not tested" and "no
call" indicates that no amplification was detected).
TABLE-US-00004 TABLE 4 Time to Positive Dye Detection Time to
Positive (minutes) Primer Set 5,000/reaction 500/reaction
50/reaction NTC Set-1 7.8 9.1 13.0 37.2 Set-2 5.8 6.6 8.4 no call
Set-3 NT 8.2 9.2 no call Set-4 8.6 9.7 13.3 no call Set-5 8.54 10.0
no call no call Set-6 6.8 8.0 no call no call Set-7 7.7 8.8 12.4 no
call Set-8 6.7 8.3 10.5 24.0 Set-9 6.5 7.8 9.6 no call Set-10 5.6
6.5 9.6 28.3 Set-11 4.8 5.3 6.5 no call Set-12 7.7 9.0 11.9 no call
Set-14 8.5 9.7 11 no call
[0059] A negative nasal swab matrix was spiked with genomic
material from common human coronaviruses strain 229E, NL63, and
OC43. The corresponding extracted nucleic acids or DNAs were used
as templates in RT-LAMP reactions containing the LAMP primers and
compared to 500-5000 copies/reaction of SARS-COV-2 RNA standard for
specificity.
TABLE-US-00005 TABLE 5 Cross-Reactivity Dye Detection Primers 5000
500 229E NL63 OC43 Set-1 7 8.5 no call no call no call Set-4 7 7.9
no call no call no call Set-8 6.3 7.2 no call no call no call
Set-11 4.8 5.3 no call no call no call
Example 3: Molecular Beacon Detection
[0060] To provide an additional level of direct sequence based
detection of amplified product (as opposed to indirect dye
detection), molecular beacons (MB1-MB14; see Table 2) targeting
unique nucleotides within the SARS-CoV-2 amplicon of primer sets
with promising times-to-positive combined with sensitivity, were
designed and utilized for detection of amplification from RNA
standards (Table 4). The molecular beacon probe was designed with
5' fluorophore/3' quencher modifications (6-Carboxyfluorescein
(FAM)) and Black Hole Quencher 1 (BHQ1) and Locked Nucleic Acid (+)
included to provide target-specific fluorescent detection.
[0061] 10-25 .mu.L total volume reactions were evaluated utilizing
25 to 500 copies/reaction of SARS-CoV-2 RNA standard or genomic RNA
as template input according to the methods described in Examples 1
and 2. While use of a Molecular Beacon for detection resulted in a
slight increase in reaction Tp, the ability to directly detect
amplification products based on sequence, and thereby distinguish
closely related species, provides a reasonable tradeoff.
TABLE-US-00006 TABLE 6 Time to Positive Probe Detection Time to
Positive (minutes) Primers Beacon 500 50 25 NTC Set-1 MB1 10.1 14.7
13.9 no call Set-8 MB2 9.1 12.0 12.4 no call Set-9 MB4 9.4 14.9
14.5 no call Set-9 MB8 8.1 12.7 NT no call Set-9 MB9 8.2 10.6 NT no
call Set-9 MB10 9.2 11.6 NT no call Set-11 MB5 7.1 8.3 8.9 no call
Set-11 MB7 NT 7.8 8.5 no call Set-12 MB6 11.3 13.7 14.7 no call
Set-9 MB11 7.1 8.5 12.6 no call Set-14 MB14 NT 13.7 13.7 no
call
[0062] Selected primer set and Molecular Beacon pair was
additionally tested for specificity by comparing reactions with 500
to 5000 copies/reaction of SARS-CoV-2 RNA standard to reactions
with approximately 5.times.10.sup.5 copies/reaction of RNA
standards or extracted nucleic acids from negative nasal swab
matrix spiked with closely related common human coronavirus strain
229E, NL63, OC43, or SARS-related coronavirus. When the
amplification reactions were performed as described in Example 1
and 2, the primer and Molecular Beacon pair tested had no
cross-reactivity against common human coronavirus (Table 7).
TABLE-US-00007 TABLE 7 Cross Reactivity Probe Detection Primers
Beacon 5000 500 229E NL63 OC43 SARS Set-8 MB2 8.4 9.1 no call no
call no call NT Set-10 MB3 6.3 7.1 NT NT NT no call Set-12 MB6 10.1
11.3 NT NT NT no call
Example 4: Multi-Target Amplification
[0063] The genomic sequence of SARS-CoV-2, like all viruses, is
subject to mutation and natural selection. Given the potential
lethality and realtively high transimissability of SARS-CoV-2, it
would be beneficial for an assay to detect the presence of more
than one genomic target. Thus, if one portion of the virus has
mutated, the virus can still be detecte via a second location. This
can be achieved by performing two separate amplification assays.
However, time, personnel and resources are limited. Accordingly, it
is advantageous if two or more genomic targets can be assessed
simultaneously in the same reaction well. This is commonly
performed with standard PCR assays, which typically utilize two
primers per amplification locus. LAMP, however, typically includes
six primers comprising eight separate hybridization sites. This
creates significantly higher potential for primer:primer
interactions that could interfere with targeted amplification or
lead to off-target amplification that could be confused for
on-target amplification (leading to a mistaken positive diagnosis
for COVID-19). Set-9 and Set-11 were confirmed in silico to present
little potential for off-target amplication from primer-primer
interactions.
[0064] First, Set-9 and Set-11 were combined to amplify an RNA
molecular standard, diluted to 25, 50, or 500 copies per reaction
various concentrations ranging from 5-500 copies/reaction to assess
the sensitivity combined primer set. Standards were serially
diluted with 0.1 mg/mL Poly A carrier RNA in PBS (Sigma) and used
as template for amplification in RTLAMP reactions. ToPro.TM. or
Syto.TM. dye was used for the detection of the amplified product.
The master mix was prepared as described in Example 1.
Amplification was detected at 5.3 minutes (time to positive) for
500 copies/reaction; 6.2 minutes for 50 copies/reaction and 6.5
minutes for 25 copies/reaction. No amplication was detection with
the negative control, as expected.
[0065] A negative nasal swab matrix was spiked with genomic
material from common human coronaviruses strain 229E, NL63, and
OC43. The corresponding extracted nucleic acids or DNAs were used
as templates in RT-LAMP reactions containing combined LAMP primer
set and compared to 50-5000 copies/reaction of SARS-COV-2 RNA
standard or extracted genomic RNA from negative nasal swab matrix
for specificity. No amplification was detected for any potentially
cross-reacting species.
[0066] To provide an additional level of direct sequence-based
detection of amplified product (as opposed to indirect dye
detection), molecular beacons (MB5 and MB11; See Table 2) targeting
unique nucleotides within the SARS-CoV-2 ORF1b and N gene
amplicons, respectively, were included to provide target-specific
fluorescent detection. 10-25 .mu.L total volume reactions were
evaluated utilizing 25, 50 or 500 copies/reaction of SARS-CoV-2 RNA
standard as template input according to the methods described in
Examples 1 and 2. The combined primer set (Set-9 and Set-11) plus
two beacons (MB5 and MB11) detected application at 6.5 minutes
(time to positive) at 500 copies/reaction; at 7.7 minutes at 50
copies/reaction and at 7.8 minutes at 25 copies/reaction. No
amplification was detected with a no template (negative)
control.
[0067] The combined primer set and Molecular Beacon pair was
additionally tested for specificity by comparing reactions with 50
to 5000 copies/reaction of SARS-CoV-2 RNA standard or extracted
genomic RNA from negative nasal swab matrix to reactions with
approximately 5.times.10.sup.5 copies/reaction of RNA standards or
extracted nucleic acids from negative nasal swab matrix spiked with
closely related common human coronavirus strain 229E, NL63, OC43,
or SARS-related coronavirus. When the amplification reactions were
performed as described in Example 1 and 2, the primer (Set-9 and
Set-11, together) and Molecular Beacon pair (MB5 and MB 11,
together) detected no cross-reactivity against common human
coronavirus 229E, human coronavirus NL83, human coronavirus OC43,
and SARS-CoV (2003).
[0068] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0069] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
Sequence CWU 1
1
103121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ggaccaggaa ctaatcagac a 21218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2tctgcggtaa ggcttgag 18338DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3accacgttcc cgaaggtgtc
agcgttcttc ggaatgtc 38440DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4tgacctacac aggtgccatc
aaggctctgt tggtgggaat 40518DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 5gacttccatg ccaatgcg
18625DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6gctgaataag catattgacg catac 25718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7accccaaaat cagcgaaa 18817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 8attggaacgc cttgtcc
17932DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9atcgcgcccc actgccaccc cgcattacgt tt
321036DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10cgtcggcccc aaggtttatc cttgccatgt tgagtg
361120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11cagttgaatc tgagggtcca 201218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12gtcttggttc accgctct 181319DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13aaaaggcttc tacgcagaa
191421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14cctttaccag acattttgct c 211538DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15actgctgcct ggagttgaat cagtcaagcc tcttctcg 381640DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16ctgctagaat ggctggcaat gtggttcaat ctgtcaagca 401719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17actgttgcga ctacgtgat 191816DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18gcggtgatgc tgctct
161922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19aggaactgat tacaaacatt gg 222022DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20ttttgtatgc gtcaatatgc tt 222136DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 21aaggtgtgac ttccatgcca
acaatttgcc cccagc 362244DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 22gggaacgtgg ttgacctaca
gacttgatct ttgaaatttg gatc 442316DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 23tgcgcgacat tccgaa
162420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 24ggtgccatca aattggatga 202522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
25tcaaagatca agtcattttg ct 222617DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 26gcctgagttg agtcagc
172741DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27gtctctgcgg taaggcttga atacaaaaca ttcccaccaa c
412841DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28gcaaactgtg actcttcttc ctgtgctcat ggattgttgc a
412922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29atcagccttc ttctttttgt cc 223022DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30tgcagatttg gatgatttct cc 223123DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 31cagaaatcaa tactgagtcc tct
233221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32gtagccaaat cagatgtgaa c 213343DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33cacagaattt tgagcagttt caagagttca gaggctgctc gtg
433442DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34cgtgttttac agaaggccgc catagcatca atgagtctca gt
423521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35gcgggagaaa attgatcgta c 213628DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36ataacaatac tagatggaat ttcacagt 283721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37caaattgttg aatcctgtgg t 213826DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 38aaattccatc tagtattgtt
atagcg 263957DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 39aatgcataaa gaggactcag tattgatttc
aattttaaag ttacaaaagg aaaagct 574038DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
40cagaggctgc tcgtgttgtc acgcacagaa ttttgagc 384121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
41tgttcaccaa tattccaggc a 214220DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 42cgatcaattt tctcccgcac
204319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 43gtcaagcctc ttctcgttc 194420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
44cttagtgaca gtttggcctt 204539DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 45ccgccattgc cagccaaaca
gttcaagaaa ttcaactcc 394641DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 46gatgctgctc ttgctttgct
ggcctttacc agacattttg c 414720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 47gagaagttcc cctactgctg
204821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 48tgacagattg aaccagcttg a 214925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
49aatttcaaag atcaagtcat tttgc 255018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
50gttgagtcag cactgctc 185142DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 51aaggcttgag tttcatcagc
ctcgcataca aaacattccc ac 425246DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 52gcagagacag aagaaacagc
aaacattgtt gcaattgttt ggagaa 465324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53tctttttgtc ctttttaggc tctg 245424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
54ctcttcttcc tgctgcagat ttgg 245533DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
55cacgccaaat ttcaaagatc aagtcatggc gtg 335630DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
56cagctgcttg acagattgaa ccagcagctg 305721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
57caaattgttg aatcctgtgg t 215826DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 58aaattccatc tagtattgtt
atagcg 265957DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 59aatgcataaa gaggactcag tattgatttc
aattttaaag ttacaaaagg aaaagct 576038DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
60cagaggctgc tcgtgttgtc acgcacagaa ttttgagc 386121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
61tgttcaccaa tattccaggc a 216220DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 62cgatcaattt tctcccgcac
206319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 63tcttatcaga ggcacgtca 196418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
64tgtctcacca ctacgacc 186542DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 65acgtttgatg aacacatagg
gcttaaagat ggcacttgtg gc 426637DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 66tcggatgctc gaactgcact
gccttcgagt tctgcta 376720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 67ttcaagttga ggcaaaacgc
206822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 68catggtcatg ttatggttga gc 226924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
69atgtccaaat tttgtatttc cctt 247019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
70gctttaacaa aatcgcccg 197151DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 71gcaactggat agacagatcg
aattctatcc ataatcaaga ctattcaacc a 517244DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
72caccaaatga atgcaaccaa atgtgtctgc catgaagttt cacc
447324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 73catcaagctt tttcttttca accc 247423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
74cctttcaact ctcatgaagt gtg 237529DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 75cacggtgact cttcttcctg
ctcaccgtg 297626DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 76cgagtcctgc tgcagatttg gactcg
267729DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 77cagctcatgg tcatgttatg gttgagctg
297826DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 78cacactttca actctcatga agtgtg 267929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
79cagctcatgg tcatgttatg gttgagctg 298026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
80cgagtcctgc tgcagatttg gactcg 268126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
81catgtcctgc tgcagatttg gacatg 268226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
82cacttcctgc tgcagatttg gaagtg 268334DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
83cacgcgtgac tcttcttcct gctgcagacg cgtg 348432DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
84cacgcttctt cctgctgcag atttgaagcg tg 328528DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
85cagactcttc ttcctgctgc agagtctg 288619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
86gagggacaag gacaccaag 198737DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 87tcggatgctc gaactgcact
gtctcaccac tacgacc 378821DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 88ggtagcagaa ctcgaaggca t
218922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 89aagagacagg tacgttaata gt 229021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
90ttagaccaga agatcaggaa c 219141DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 91aagcgcagta aggatggcta
tagcgtactt ctttttcttg c 419244DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 92tgcgtactgc tgcaatattg
ttttaacacg agagtaaacg taaa 449322DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 93tgtaactagc aagaatacca cg
229422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 94cgtgagtctt gtaaaacctt ct 229530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
95cacgtgagtc ttgtaaaacc ttctcacgtg 309622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
96gaagatcagg aactctagaa ga 229741DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 97acacaatcga agcgcagtaa
gttttcttgc tttcgtggta t 419843DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 98gcgtactgct gcaatattgt
tttaacacga gagtaaacgt aaa 439921DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 99tggctagtgt aactagcaag a
2110024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 100acgttaatag ttaatagcgt actt 2410122DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
101tcaggaactc tagaagaatt ca 2210240DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
102acaatcgaag cgcagtaagg ttttcttgct ttcgtggtat 4010345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
103tgcgtactgc tgcaatattg tttttaacac gagagtaaac gtaaa 45
* * * * *