POLYNUCLEOTIDES FOR AMPLIFICATION AND DETECTION OF SARS-COV-2
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.
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° C. and about 67° 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.