Compositions and methods for detecting SARS-cov-2 nucleic acids

Abstract

To provide compositions and methods for detecting SARS-CoV-2 nucleic acids.SOLUTION: Nucleic acid oligomers, including amplification oligomers, detection probes, and capture probes, for the detection of SARS-CoV-2 nucleic acids are disclosed. Also disclosed are specific nucleic acid amplification and detection methods using the disclosed oligomers, as well as corresponding formulations, reaction mixtures and kits, and related methods for preparing aqueous reaction mixtures from dried formulations. In some aspects, the present invention provides compositions and / or kits for determining the presence or absence of SARS-CoV-2 in a sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 985,139, filed March 4, 2020, U.S. Provisional Patent Application No. 63 / 010,186, filed April 15, 2020, U.S. Provisional Patent Application No. 63 / 020,007, filed May 4, 2020, and U.S. Provisional Patent Application No. 63 / 078,790, filed September 15, 2020, each of which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing set forth in file DIA-0116-05_SeqList.txt is 65 kilobytes in size, was created on March 4, 2021, and is hereby incorporated by reference herein. [Background technology]

[0003] background Coronaviruses are a family of RNA viruses that infect birds and mammals, including humans. Coronaviruses belong to the family Coronaviridae, which has four major subgroups known as alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. Human coronaviruses include alphacoronaviruses 229E and NL63, and betacoronaviruses OC43, HKU1, SARS-CoV (the coronavirus that causes severe acute respiratory syndrome, or SARS), SARS-CoV-2 (formerly known as 2019-nCoV or Wuhan CoV), and MERS-CoV (the coronavirus that causes Middle East respiratory syndrome, or MERS).

[0004] Human coronaviruses cause approximately 10-15% of all upper and lower respiratory tract infections. They are a leading cause of significant hospitalization in children under 18 years of age, the elderly, and immunocompromised individuals. Four human coronaviruses (229E, HKU1, NL63, and OC43) have been associated with a variety of respiratory outcomes, including bronchiolitis and pneumonia (Gaunt et al., Journal of Clinical Microbiology 48:2940-2947, 2010). Up to 10% of acute respiratory illnesses are caused by human coronavirus NL63 (NL6 3) caused by type (Abdul-Rasool and Fielding, The Open Virology Journal 4:76-84, 2010). An important aspect of NL63 infection is its similarity to other human coronaviruses. Co-infections with influenza A, respiratory syncytial virus (RSV), parainfluenza virus, and human metapneumovirus are common. (See id.) In children, coronaviruses are associated with acute respiratory tract disease, pneumonia, and croup, which often lead to hospitalization. In one epidemiological study, of 1,471 hospitalized children (<2 years), 207 (14%) tested positive for human coronaviruses (Dijkman et al., Journal of Clinical Virology 53:135-139, 2012). In a large-scale study of 11,661 diagnostic respiratory specimens collected in Edinburgh, UK, between 2006 and 2009, 267 (2.30%) were positive for at least one coronavirus, accounting for 8.15% of all virus detections (see Gaunt et al., supra). 11–41% of detected coronaviruses were present in specimens that tested positive for other respiratory viruses (e.g., RSV) (see ibid.).

[0005] The emerging SARS-CoV-2 can cause severe lower respiratory tract infection (COVID-19) and has been declared a global emergency by the World Health Organization. SARS-CoV-2 is the cause of the recent pneumonia outbreak that began in early December 2019 in Wuhan, Hubei Province, China (Huang et al., Lancet (2020) v395, issue 10223, p.497). To date, thousands of human infections have been confirmed in China, in addition to many exported cases worldwide, including in the United States, Canada, Japan, Australia, and Europe (China CDC, 2020). Several commercially available assays based on nucleic acid detection are available for the detection of human coronaviruses OC43, HKU1, NL63, and 229E in clinical specimens. These include the NxTAG® Respiratory Pathogen Panel (Luminex), the BIOFIRE® FILMARRAY® Respiratory Panel (bioMerieux), and the ePlex® Respiratory Pathogen Panel (GenMark Diagnostics). The US Centers for Disease Control and Prevention has authorized the emergency use of its 2019-nCoV real-time RT-PCR diagnostic panel. Permission was granted. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gaunt et al., Journal of Clinical Microbiology 48:2940-2947, 2010 [Non-patent document 2] Abdul-Rasool and Fielding, The Open Virology Journal 4:76-84, 2010 [Non-patent document 3] Dijkman et al., Journal of Clinical Virology 53:135-139, 2012 [Non-patent document 4] Huang et al., Lancet (2020) v395, issue 10223, p.497 [Non-patent document 5] China CDC, 2020 Summary of the Invention [Means for solving the problem]

[0007] Abstract In some embodiments, the present invention provides compositions and / or kits for determining the presence or absence of SARS-CoV-2 in a sample. The compositions and / or kits generally include at least one amplification oligomer combination, including first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 nucleic acid. The described oligonucleotides, compositions, formulations, kits, and methods can be used for isothermal amplification of target nucleic acids in SARS-CoV-2. The isothermal reaction can be, but is not limited to, transcription-mediated amplification (TMA).

[0008] In another aspect, the present invention provides formulations for amplifying SARS-CoV-2 nucleic acids in a sample. The formulations generally include (i) at least one amplification oligomer combination, including first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, and (ii) a buffer. In some embodiments, the formulation is a dry composition. In some embodiments, kits are described that include such dry compositions for amplifying SARS-CoV-2 nucleic acids. In some embodiments, methods are described for preparing an aqueous reaction mixture for determining the presence or absence of SARS-CoV-2 in a sample, generally comprising combining the dry composition described with an aqueous reconstitution reagent.

[0009] In some aspects, methods are described for determining the presence or absence of SARS-CoV-2 in a sample. In some embodiments, the method includes performing an in vitro nucleic acid amplification reaction utilizing at least one amplification oligomer combination, including first and second SARS-CoV-2-specific amplification oligomers, to generate one or more amplicons corresponding to at least one SARS-CoV-2 target region, and detecting the presence or absence of the one or more amplicons. In some embodiments, the method is a multiplex method for determining the presence of SARS-CoV-2 and at least one other pathogen in a sample. Representative embodiments of these aspects are further presented below.

[0010] Embodiment 1. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) a first amplification oligomer combination comprising first and second SARS-CoV-2 Region 1-specific amplification oligomers capable of amplifying a first target region of a SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2 Region 1-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2 Region 1-specific target, wherein the sequence that hybridizes to this target is 18 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:26, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (ii) the second SARS-CoV-2 Region 1-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2 Region 1-specific target, wherein the sequence that hybridizes to this target is 18 to 23 contiguous nucleotides in length and contains a nucleotide sequence that is contained in SEQ ID NO: 85 and differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; a first amplification oligomer combination; (b) a second amplification oligomer combination comprising first and second SARS-CoV-2 region 2-specific amplification oligomers capable of amplifying a second target region of a SARS-CoV-2 target nucleic acid, (i) the first SARS-CoV-2 Region 2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2 Region 2-specific target, wherein the sequence that hybridizes to this target is 20 to 23 contiguous nucleotides in length, is contained in SEQ ID NO:29, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (ii) a combination of second amplification oligomers, wherein the second SARS-CoV-2 region 2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2 region 2-specific target, wherein the sequence that hybridizes to this target is 16 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:89, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:112, or SEQ ID NO:113; A composition or kit comprising:

[0011] Embodiment 2. The composition or kit of embodiment 1, further comprising a first detector probe oligomer configured to hybridize to a target sequence contained within an amplicon amplifiable by the combination of first amplification oligomers of (a), wherein the first detector probe oligomer comprises a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, the sequence being 19 to 27 contiguous nucleotides in length.

[0012] Embodiment 3. The composition or kit of embodiment 1 or 2, further comprising a second detector probe oligomer configured to hybridize to a target sequence contained in an amplicon amplifiable by the combination of second amplification oligomers of (b), wherein the second detector probe oligomer comprises a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, the sequence being 25 to 29 contiguous nucleotides in length.

[0013] Embodiment 4. The composition or kit of embodiment 1, 2 or 3, wherein the sequence that hybridizes to the first SARS-CoV-2 Region 1-specific target in (a)(i) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2 Region 1-specific target in (a)(ii) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues.

[0014] Embodiment 5. The kit or composition of embodiment 1, 2, 3 or 4, wherein the sequence that hybridizes to the first SARS-CoV-2 region 2-specific target in (b)(i) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2 region 2-specific target in (b)(ii) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 or SEQ ID NO:84, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues.

[0015] Embodiment 5.1. (a)(i) the sequence that hybridizes to the first SARS-CoV-2 Region 1-specific target contains or consists of the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, or an RNA equivalent or DNA / RNA chimeric form thereof, including 0-7 nucleotide analogs; (a)(ii) the sequence that hybridizes to the second SARS-CoV-2 Region 1-specific target contains or consists of the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, including 0-7 nucleotide analogs; (b)(i) the sequence that hybridizes to the first SARS-CoV-2 Region 1-specific target contains or consists of the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, including 0-7 nucleotide analogs; 6. The composition or kit of embodiment 4 or 5, wherein the sequence that hybridizes to the target specific for region 2 contains or consists of the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues; and / or the sequence that hybridizes to the target specific for the second SARS-CoV-2 region 2 in (b)(ii) contains or consists of the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues.

[0016] Embodiment 6. The composition or kit of any one of embodiments 2, 4, or 5a, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:107, or SEQ ID NO:108, and includes 0-7 nucleotide analogs.

[0017] Embodiment 7. The composition or kit of any one of embodiments 3, 5 or 5a, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent or a DNA / RNA chimeric form thereof, or the like, which differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:17, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:109 or SEQ ID NO:110, and includes 0-7 nucleotide analogs.

[0018] Embodiment 8. The composition or kit of any one of embodiments 2, 3, 6, and 7, wherein the first and / or second detector probe oligomer further comprises a detectable label.

[0019] Embodiment 9. The composition or kit of embodiment 8, wherein the detectable label is a fluorescent or chemiluminescent label.

[0020] Embodiment 10. The composition or kit of embodiment 9, wherein the detectable label is a fluorescent label and the first and / or second detector probe oligomer further comprises a non-fluorescent quencher.

[0021] Embodiment 11. The composition or kit of any one of embodiments 2, 3, 6, 7, 8, 9 and 10, wherein the first and / or second detector probe oligomer further comprises a self-complementary sequence linked to a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0022] Embodiment 12. The composition or kit of embodiment 11, wherein the self-complementary sequence is linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe by a non-nucleotide linker.

[0023] Embodiment 13. The composition or kit of any one of embodiments 2-3 and 6-12, wherein the first and / or second detector probe oligomer is a molecular beacon or a molecular torch.

[0024] Embodiment 14. The composition or kit of any one of embodiments 1 to 13, wherein the sequence that hybridizes to the second SARS-CoV-2-specific target of (a)(ii) is linked at its 5' end to a promoter sequence.

[0025] Embodiment 15. The composition or kit of embodiment 14, wherein the promoter sequence is substantially identical to SEQ ID NO:11.

[0026] Embodiment 16. The composition or kit of embodiment 14, wherein the second SARS-CoV-2-specific amplification oligomer of (a)(ii) comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 or SEQ ID NO:62, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0027] Embodiment 17. The composition or kit of any one of embodiments 1 to 16, wherein the sequence that hybridizes to the second SARS-CoV-2-specific target of (b)(ii) is linked at its 5' end to a promoter sequence.

[0028] Embodiment 18. The composition or kit of embodiment 17, wherein the promoter sequence is substantially identical to SEQ ID NO:11.

[0029] Embodiment 19. The composition or kit of embodiment 17, wherein the second SARS-CoV-2-specific amplification oligomer of (b)(ii) comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:78, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0030] Embodiment 20. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising an amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, (a) a first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 18 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:26, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (b) the second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 18 to 23 contiguous nucleotides in length and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, or SEQ ID NO:88; Composition or kit.

[0031] Embodiment 21. The composition or kit of embodiment 20, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues.

[0032] Embodiment 22. The composition or kit of embodiment 20 or 21, wherein the sequence that hybridizes to a second SARS-CoV-2-specific target is attached at its 5' end to the promoter sequence.

[0033] Embodiment 23. The composition or kit of embodiment 22, wherein the promoter sequence is substantially identical to SEQ ID NO:11.

[0034] Embodiment 24. The composition or kit of embodiment 22, wherein the second SARS-CoV-2-specific amplification oligomer comprises a contiguous nucleotide sequence comprising or consisting of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 or SEQ ID NO:62, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0035] Embodiment 25. The composition or kit of any one of embodiments 20 to 24, further comprising a SARS-CoV-2-specific detector probe oligomer, wherein the detector probe oligomer comprises a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is 19 to 27 contiguous nucleotides in length and contains a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:114 by at most 0, 1, 2, 3, 4, or 5 nucleotides.

[0036] Embodiment 26. The composition or kit of embodiment 25, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:107, or SEQ ID NO:108, and includes 0 to 7 nucleotide analogs.

[0037] Embodiment 27. The composition or kit of embodiment 25, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe oligomer contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:107, or SEQ ID NO:108, and includes 0 to 7 nucleotide analogs.

[0038] Embodiment 28. The composition or kit of any one of embodiments 25 to 27, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0039] Embodiment 29. The composition or kit of embodiment 28, wherein the detectable label is a fluorescent or chemiluminescent label.

[0040] Embodiment 30. The composition or kit of embodiment 29, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0041] Embodiment 31. The composition or kit of any one of embodiments 25, 26, and 28-30, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, and optionally, the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0042] Embodiment 32. The composition or kit of embodiment 31, wherein the self-complementary sequence is linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe by a non-nucleotide linker.

[0043] Embodiment 33. The composition or kit of any one of embodiments 26 and 28-32, wherein the SARS-CoV-2-specific detector probe oligomer is a molecular beacon or a molecular torch.

[0044] Embodiment 34. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising an amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, (a) a first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 20 to 23 contiguous nucleotides in length and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 111; (b) the second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 16 to 27 contiguous nucleotides in length and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 112, or SEQ ID NO: 113; Composition or kit.

[0045] Embodiment 35. The composition or kit of embodiment 34, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues.

[0046] Embodiment 36. The composition or kit of embodiment 34 or 35, wherein the sequence that hybridizes to a second SARS-CoV-2-specific target is linked at its 5' end to the promoter sequence.

[0047] Embodiment 37. The composition or kit of embodiment 36, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0048] Embodiment 38. The composition or kit of embodiment 36, wherein the second SARS-CoV-2-specific amplification oligomer comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:78, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0049] Embodiment 39. The composition or kit of any one of embodiments 34 to 38, further comprising a SARS-CoV-2-specific detector probe oligomer, wherein the detector probe oligomer comprises a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is 25 to 29 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO:54 that differs from the nucleotide sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:115 by at most 0, 1, 2, 3, 4, or 5 nucleotides.

[0050] Embodiment 40. The composition or kit of embodiment 39, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 109, or SEQ ID NO: 110, and includes 0 to 7 nucleotide analogs.

[0051] Embodiment 41. The composition or kit of embodiment 39, wherein the sequence that hybridizes to the target of the second detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, or the like, that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:17, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:109, or SEQ ID NO:110, and includes 0 to 7 nucleotide analogs.

[0052] Embodiment 42. The composition or kit of any one of embodiments 39 to 41, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0053] Embodiment 43. The composition or kit of embodiment 42, wherein the detectable label is a fluorescent or chemiluminescent label.

[0054] Embodiment 44. The composition or kit of embodiment 43, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0055] Embodiment 45. The composition or kit of any one of embodiments 39, 40, and 42-44, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, and optionally, the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0056] Embodiment 46. The composition or kit of embodiment 45, wherein the self-complementary sequence is linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe by a non-nucleotide linker.

[0057] Embodiment 47. The composition or kit of any one of embodiments 40 and 42 to 46, wherein the SARS-CoV-2 specific detector probe oligomer is a molecular beacon or a molecular torch.

[0058] Embodiment 48. A composition or kit comprising one or more SARS-CoV-2-specific detector probe oligomers, wherein the SARS-CoV-2-specific detector probe oligomers are selected from the group consisting of: (a) a first detector probe oligomer comprising a sequence that hybridizes to a SARS-CoV-2-specific detector probe target, the sequence being 19 to 27 contiguous nucleotides in length and contained in SEQ ID NO:50, and which differs by no more than 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:114; and (b) a second detector probe oligomer comprising a sequence that hybridizes to a SARS-CoV-2-specific detector probe target, the sequence being 25 to 29 contiguous nucleotides in length and contained in SEQ ID NO:54, and which differs by no more than 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:115.

[0059] Embodiment 49. The composition or kit of embodiment 48, wherein the first and / or second detector probe oligomer comprises a sequence that hybridizes to a SARS-CoV-2-specific detector probe target, or a DNA equivalent, RNA equivalent, or DNA / RNA chimeric form thereof, that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, or SEQ ID NO:110, and includes 0 to 7 nucleotide analogs.

[0060] Embodiment 50. The composition or kit of embodiment 48, wherein the one or more SARS-CoV-2-specific detector probe oligomers independently comprise a sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, and DNA equivalents, RNA equivalents or DNA / RNA chimeric forms thereof, and those comprising 0-7 nucleotide analogs.

[0061] Embodiment 51. The composition or kit of embodiment 48 or 49, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0062] Embodiment 52. The composition or kit of embodiment 51, wherein the self-complementary sequence is linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe by a non-nucleotide linker.

[0063] Embodiment 53. The composition or kit of any one of embodiments 48 to 52, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0064] Embodiment 54. The composition or kit of embodiment 54, wherein the detectable label is a fluorescent or chemiluminescent label.

[0065] Embodiment 55. The composition or kit of embodiment 55, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0066] Embodiment 56. The composition or kit of any one of embodiments 48-49 and 51-55, wherein the SARS-CoV-2 specific detection probe oligomer is a molecular beacon or a molecular torch.

[0067] Embodiment 57. A formulation for amplifying SARS-CoV-2 nucleic acids in a sample, comprising: (a) a first amplification oligomer combination comprising first and second SARS-CoV-2 Region 1-specific amplification oligomers capable of amplifying a first target region of a SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2 Region 1-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2 Region 1-specific target, wherein the sequence that hybridizes to this target is 18 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:26, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (ii) the second SARS-CoV-2 Region 1-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2 Region 1-specific target, wherein the sequence that hybridizes to this target is 18 to 23 contiguous nucleotides in length and contains a nucleotide sequence that is contained in SEQ ID NO: 85 and differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; a first amplification oligomer combination; (b) a second amplification oligomer combination comprising first and second SARS-CoV-2 region 2-specific amplification oligomers capable of amplifying a second target region of a SARS-CoV-2 target nucleic acid, (i) the first SARS-CoV-2 Region 2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2 Region 2-specific target, wherein the sequence that hybridizes to this target is 20 to 23 contiguous nucleotides in length, is contained in SEQ ID NO:29, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (ii) a second SARS-CoV-2 Region 2-specific amplification oligomer comprising a sequence that hybridizes to a second SARS-CoV-2 Region 2-specific target, wherein the sequence that hybridizes to this target is 16 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:89, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:112, or SEQ ID NO:113; (c) buffering agent; 1. A formulation comprising:

[0068] Embodiment 58. The formulation of embodiment 57, wherein the sequence that hybridizes to the first SARS-CoV-2 region 1-specific target in (a)(i) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2 region 1-specific target in (a)(ii) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogues.

[0069] Embodiment 59. The formulation of embodiment 57 or 58, wherein the sequence that hybridizes to the first SARS-CoV-2 region 2-specific target in (b)(i) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2 region 2-specific target in (b)(ii) contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 or SEQ ID NO:84, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues.

[0070] Embodiment 60. The formulation of embodiment 57 or 58, wherein the sequence that hybridizes to the second SARS-CoV-2-specific target of (a)(ii) is linked at its 5' end to a promoter sequence.

[0071] Embodiment 61. The formulation of embodiment 60, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0072] Embodiment 62. (a)(ii) The formulation of embodiment 61, wherein the second SARS-CoV-2-specific amplification oligomer comprises a contiguous nucleotide sequence containing or consisting of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 or SEQ ID NO:62, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0073] Embodiment 63. The formulation of any one of embodiments 57 to 62, wherein the sequence that hybridizes to the second SARS-CoV-2-specific target of (b)(ii) is linked at its 5' end to a promoter sequence.

[0074] Embodiment 64. The formulation of embodiment 63, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0075] Embodiment 65. The formulation of embodiment 64, wherein the second SARS-CoV-2-specific amplification oligomer of (b)(ii) comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:78, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0076] Embodiment 66. The formulation of any one of embodiments 57 to 65, further comprising a first detector probe oligomer configured to hybridize to a target sequence contained in an amplicon amplifiable by the combination of first amplification oligomers of (a), wherein the first detector probe oligomer comprises a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, the sequence being 19 to 27 contiguous nucleotides in length.

[0077] Embodiment 67. The formulation of any one of embodiments 57 to 66, further comprising a second detector probe oligomer configured to hybridize to a target sequence contained in an amplicon amplifiable by the combination of second amplification oligomers of (b), wherein the detector probe oligomer comprises a sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, the sequence being 25 to 29 contiguous nucleotides in length.

[0078] Embodiment 68. The formulation of embodiment 66, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent or a DNA / RNA chimeric form thereof, or the like, which differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 107 or SEQ ID NO: 108, and includes 0 to 7 nucleotide analogs.

[0079] Embodiment 69. The formulation of embodiment 68, wherein the sequence that hybridizes to the target of the first detector probe oligomer contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:107 or SEQ ID NO:108, its DNA equivalent, RNA equivalent or DNA / RNA chimeric form, etc., and includes 0 to 7 nucleotide analogs.

[0080] Embodiment 70. The formulation of embodiment 67, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 109, or SEQ ID NO: 110, and includes 0 to 7 nucleotide analogs.

[0081] Embodiment 71. The formulation of embodiment 70, wherein the second detector probe oligomer contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:17, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:109 or SEQ ID NO:110, its DNA equivalent, RNA equivalent or DNA / RNA chimeric form, etc., and includes 0 to 7 nucleotide analogs.

[0082] Embodiment 72. The formulation of any one of embodiments 66 to 71, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0083] Embodiment 73. The formulation of embodiment 72, wherein the detectable label is a fluorescent or chemiluminescent label.

[0084] Embodiment 74. The formulation of embodiment 73, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0085] Embodiment 75. The formulation of any one of embodiments 57-74, further comprising one or more of a bulking agent, a surfactant, a nucleotide triphosphate, an enzyme, and an inorganic salt.

[0086] Embodiment 76. The formulation of any one of embodiments 57 to 75, which is a dry composition.

[0087] Embodiment 77. A formulation for amplifying SARS-CoV-2 nucleic acids in a sample, comprising: (a) an amplification oligomer combination comprising a first and a second SARS-CoV-2-specific amplification oligomer capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, wherein the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, the sequence that hybridizes to this target being 18 to 27 contiguous nucleotides in length and contained in SEQ ID NO:26 and having at most 0, 1, 2, or 3 nucleotides different from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; a second SARS-CoV-2-specific amplification oligomer comprising a sequence that hybridizes to a second SARS-CoV-2-specific target, the sequence hybridizing to this target being 18 to 23 contiguous nucleotides in length and contained in SEQ ID NO: 85, and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO: 88; (b) buffering agent; 1. A formulation comprising:

[0088] Embodiment 78. The formulation of embodiment 77, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues.

[0089] Embodiment 79. The formulation of embodiment 77 or 78, wherein the sequence that hybridizes to a second SARS-CoV-2-specific target is linked to the promoter sequence at its 5' end.

[0090] Embodiment 80. The formulation of embodiment 79, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0091] Embodiment 81. The formulation of embodiment 79, wherein the second SARS-CoV-2-specific amplification oligomer comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 or SEQ ID NO:62, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0092] Embodiment 82. The formulation of any one of embodiments 77 to 81, further comprising a SARS-CoV-2-specific detector probe oligomer, wherein the detector probe oligomer comprises a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is 19 to 27 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO:50 that differs from the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:114 by at most 0, 1, 2, 3, 4, or 5 nucleotides.

[0093] Embodiment 83. The formulation of embodiment 82, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent or a DNA / RNA chimeric form thereof, or the like, which differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 107 or SEQ ID NO: 108, and includes 0 to 7 nucleotide analogs.

[0094] Embodiment 83a. The formulation of embodiment 83, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0095] Embodiment 84. The formulation of embodiment 82, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe oligomer contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:107, or SEQ ID NO:108, and includes 0 to 7 nucleotide analogs.

[0096] Embodiment 85. The formulation of any one of embodiments 82 to 84, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0097] Embodiment 86. The formulation of embodiment 85, wherein the detectable label is a fluorescent or chemiluminescent label.

[0098] Embodiment 87. The formulation of embodiment 86, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0099] Embodiment 88. The formulation of any one of embodiments 77-87, further comprising one or more of a bulking agent, a surfactant, a nucleotide triphosphate, an enzyme, and an inorganic salt.

[0100] Embodiment 89. The formulation of any one of embodiments 77 to 88, which is a dry composition.

[0101] Embodiment 90. A formulation for amplifying SARS-CoV-2 nucleic acids in a sample, comprising: (a) an amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, wherein the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, and the sequence that hybridizes to this target is 20 to 23 contiguous nucleotides in length and is contained in SEQ ID NO:29 and has at most 0 to 10 nucleotides with the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; a combination of amplification oligomers containing a nucleotide sequence that differs by 1, 2, 3, 4 or 5 nucleotides, and a second SARS-CoV-2-specific amplification oligomer comprising a sequence that hybridizes to a second SARS-CoV-2-specific target, the sequence that hybridizes to this target being 16 to 27 contiguous nucleotides in length and contained in SEQ ID NO: 89, and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112 or SEQ ID NO: 113; (b) buffering agent; 1. A formulation comprising:

[0102] Embodiment 91. The formulation of embodiment 90, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues.

[0103] Embodiment 92. The formulation of embodiment 90 or 91, wherein the sequence that hybridizes to a second SARS-CoV-2-specific target is linked to the promoter sequence at its 5' end.

[0104] Embodiment 93. The formulation of embodiment 92, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0105] Embodiment 94. The formulation of embodiment 92, wherein the second SARS-CoV-2-specific amplification oligomer comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:78, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0106] Embodiment 95. The formulation of any one of embodiments 90 to 94, further comprising a SARS-CoV-2-specific detector probe oligomer, wherein the detector probe oligomer comprises a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is 25 to 29 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO:54 that differs from the nucleotide sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:115 by at most 0, 1, 2, 3, 4, or 5 nucleotides.

[0107] Embodiment 96. The formulation of embodiment 95, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a contiguous nucleotide sequence, its DNA equivalent, RNA equivalent or DNA / RNA chimeric form, etc., that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 109 or SEQ ID NO: 110, and includes 0 to 7 nucleotide analogs.

[0108] Embodiment 96. The formulation of embodiment 96, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0109] Embodiment 97. The formulation of embodiment 95, wherein the SARS-CoV-2-specific detector probe oligomer contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 109 or SEQ ID NO: 110, its DNA equivalent, RNA equivalent or DNA / RNA chimeric form, etc., and includes 0 to 7 nucleotide analogs.

[0110] Embodiment 98. The formulation of any one of embodiments 95 to 97, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0111] Embodiment 99. The formulation of embodiment 98, wherein the detectable label is a fluorescent or chemiluminescent label.

[0112] Embodiment 100. The formulation of embodiment 99, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0113] Embodiment 101. The formulation of any one of embodiments 90-100, further comprising one or more of a bulking agent, a surfactant, a nucleotide triphosphate, an enzyme, and an inorganic salt.

[0114] Embodiment 102. The formulation of any one of embodiments 90 to 101, which is a dry composition.

[0115] Embodiment 103. A formulation for detecting SARS-CoV-2 in a sample, comprising: (a) a buffer; (b) one or more SARS-CoV-2 specific detection probe oligomers; and (ii) a first detector probe oligomer comprising a sequence that hybridizes to a SARS-CoV-2-specific detector probe target that is 19 to 27 contiguous nucleotides in length and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:114; and (ii) a first detector probe oligomer comprising a sequence that hybridizes to a SARS-CoV-2-specific detector probe target that is 25 to 29 contiguous nucleotides in length and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:115.

[0116] Embodiment 104. The formulation of embodiment 103, wherein the first and / or second detector probe oligomer comprises a sequence that hybridizes to a SARS-CoV-2-specific detector probe target, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, or SEQ ID NO:110, its DNA equivalent, RNA equivalent, or DNA / RNA chimeric form, etc., and comprises 0 to 7 nucleotide analogs.

[0117] Embodiment 105. The formulation of embodiment 103, wherein the target-hybridizing sequence of at least one SARS-CoV-2-specific detector probe oligomer contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, or SEQ ID NO:110, and includes 0 to 7 nucleotide analogs.

[0118] Embodiment 106. The formulation of embodiment 103 or 104, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0119] Embodiment 107. The formulation of embodiment 106, wherein the self-complementary sequence is linked by a non-nucleotide linker to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0120] Embodiment 108. The formulation of any one of embodiments 103 to 107, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0121] Embodiment 109. The formulation of embodiment 108, wherein the detectable label is a fluorescent or chemiluminescent label.

[0122] Embodiment 110. The formulation of embodiment 109, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0123] Embodiment 111. The formulation of any one of embodiments 103-110, further comprising one or more of a bulking agent, a surfactant, and an inorganic salt.

[0124] Embodiment 112. The formulation of any one of embodiments 103 to 111, which is a dry composition.

[0125] Embodiment 113. A kit comprising a dry composition according to any one of embodiments 76, 89, 102 and 112.

[0126] Embodiment 114. The kit of embodiment 114, further comprising a reconstitution reagent, wherein the dry composition is in a first vial within the kit and the reconstitution reagent is in a second vial within the kit.

[0127] Embodiment 115. The kit of embodiment 113 or 114, wherein the dry composition comprises a bulking agent selected from the group consisting of trehalose, raffinose, mannitol, and combinations thereof.

[0128] Embodiment 116. The kit of any one of embodiments 113 to 115, wherein the dry composition comprises a non-linear surfactant.

[0129] Embodiment 117. The kit of embodiment 116, wherein the non-linear surfactant is a polyoxyethylene sorbitan fatty acid ester or digitonin.

[0130] Embodiment 118. The kit of any one of embodiments 113 to 117, wherein the reconstitution reagent comprises one or more of magnesium chloride, potassium chloride, and ethyl alcohol.

[0131] Embodiment 119. A method for preparing an aqueous reaction mixture for determining the presence or absence of SARS-CoV-2 in a sample, comprising combining a dry composition according to any one of embodiments 76, 89, 102 or 112 with a reconstitution reagent to produce an aqueous reaction mixture.

[0132] Embodiment 120. The method of embodiment 119, wherein the reconstitution reagent comprises one or more of magnesium chloride, potassium chloride, and ethyl alcohol.

[0133] Embodiment 121. The method of embodiment 119, wherein the combining step is performed using an automated system including a first holder for the dry composition, a second holder for the reconstitution reagent, a programmable controller, and a dispensing device in communication with the programmable controller, wherein the programmable controller is configured by software instructions to cause the dispensing device to transfer an aliquot of the reconstitution reagent from the second holder position to the first holder position, thereby creating the aqueous reaction mixture.

[0134] Embodiment 122. A method for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) contacting a sample containing or suspected of containing a SARS-CoV-2 target nucleic acid with at least a first amplification oligomer combination and a second amplification oligomer combination; (i) the first amplification oligomer combination comprises first and second SARS-CoV-2 region 1-specific amplification oligomers capable of amplifying a first target region of a SARS-CoV-2 target nucleic acid; (1) the first SARS-CoV-2 Region 1-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2 Region 1-specific target, wherein the sequence that hybridizes to this target is 18 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:26, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (2) the second SARS-CoV-2 Region 1-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2 Region 1-specific target, wherein the sequence that hybridizes to this target is 18 to 23 contiguous nucleotides in length, is contained in SEQ ID NO: 85, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; (ii) the second amplification oligomer combination comprises first and second SARS-CoV-2 region 2-specific amplification oligomers capable of amplifying a second target region of the SARS-CoV-2 target nucleic acid; (1) the first SARS-CoV-2 region 2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2 region 2-specific target, wherein the sequence that hybridizes to this target is 20 to 23 contiguous nucleotides in length, is contained in SEQ ID NO:29, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (2) a second SARS-CoV-2 region 2-specific amplification oligomer comprising a sequence that hybridizes to a second SARS-CoV-2 region 2-specific target, wherein the sequence that hybridizes to this target is 16 to 27 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO:89 that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:112, or SEQ ID NO:113; (b) incubating the sample under conditions suitable for nucleic acid amplification, wherein any SARS-CoV-2 target nucleic acid, if present in the sample, is used as a template to generate one or more amplicons corresponding to the first and / or second target regions of the SARS-CoV-2 target nucleic acid; (c) detecting the presence or absence of one or more amplicons, thereby determining the presence or absence of SARS-CoV-2 in the sample; A method comprising:

[0135] Embodiment 123. The method of embodiment 122, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target of (a)(i)(1) contains or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes from 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target of (a)(i)(2) contains or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes from 0 to 7 nucleotide analogs.

[0136] Embodiment 124. The method of embodiment 122 or 123, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target of (a)(ii)(1) contains or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target of (a)(ii)(2) contains or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs.

[0137] Embodiment 125. The method of any one of embodiments 122 to 124, wherein the sequence that hybridizes to the second SARS-CoV-2-specific target of (a)(i)(2) is linked at its 5' end to a promoter sequence.

[0138] Embodiment 126. The method of embodiment 125, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0139] Embodiment 127. The method of embodiment 125, wherein the second SARS-CoV-2-specific amplification oligomer of (a)(i)(2) comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs.

[0140] Embodiment 128. The method of any one of embodiments 122 to 127, wherein the sequence that hybridizes to the second SARS-CoV-2-specific target of (a)(ii)(2) is linked at its 5' end to a promoter sequence.

[0141] Embodiment 129. The method of embodiment 128, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0142] Embodiment 130. The method of embodiment 128, wherein the second SARS-CoV-2-specific amplification oligomer of (a)(ii)(2) comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:78, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs.

[0143] Embodiment 131. The detecting step (c) comprises contacting one or more amplicons, if present, with one or more SARS-CoV-2-specific detector probe oligomers, wherein the SARS-CoV-2-specific detector probe oligomers are (a) SARS-CoV-2-specific detector probe oligomers that are 19 to 27 contiguous nucleotides in length and contain a nucleotide sequence contained in SEQ ID NO:50 that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:114. 131. The method of any one of embodiments 122-130, wherein the SARS-CoV-2-specific detector probe oligomer is selected from the group consisting of: (i) a first detector probe oligomer comprising a sequence that hybridizes to a target of a SARS-CoV-2-specific detector probe; or (ii) a second detector probe oligomer comprising a sequence that hybridizes to a target of a SARS-CoV-2-specific detector probe, the second detector probe oligomer being 25 to 29 contiguous nucleotides in length and containing a nucleotide sequence contained in SEQ ID NO: 54 that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 115.

[0144] Embodiment 132. The method of embodiment 131, wherein the first and / or second detector probe oligomer comprises a sequence that hybridizes to a SARS-CoV-2-specific detector probe target, or a DNA equivalent, RNA equivalent, or DNA / RNA chimeric form thereof, that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, and SEQ ID NO:110, and includes 0 to 7 nucleotide analogs.

[0145] Embodiment 133. The method of embodiment 131, wherein the one or more SARS-CoV-2-specific detector probe oligomers independently comprise a sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, and DNA equivalents, RNA equivalents or DNA / RNA chimeric forms thereof, and those comprising 0-7 nucleotide analogs.

[0146] Embodiment 134. The method of embodiment 131 or 132, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0147] Embodiment 135. The method of embodiment 134, wherein the self-complementary sequence is linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe by a non-nucleotide linker.

[0148] Embodiment 136. The method of any one of embodiments 131 to 135, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0149] Embodiment 137. The method of embodiment 136, wherein the detectable label is a fluorescent or chemiluminescent label.

[0150] Embodiment 138. The method of embodiment 137, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0151] Embodiment 139. The method of any one of embodiments 134 to 138, wherein the SARS-CoV-2 specific detection probe oligomer is a molecular beacon or a molecular torch.

[0152] Embodiment 140. The method of any one of embodiments 122 to 139, wherein the SARS-CoV-2 target nucleic acid is separated from other components of the sample prior to the amplification step (b).

[0153] Embodiment 141. The method of embodiment 140, wherein the SARS-CoV-2 target nucleic acid is separated from other components of the sample prior to the contacting step (a).

[0154] Embodiment 142. The method of embodiment 140 or 141, wherein the separating step is carried out by directly or indirectly binding the SARS-CoV-2 target nucleic acid to a solid support, followed by washing the SARS-CoV-2 target nucleic acid-solid support complex to remove other components of the sample.

[0155] Embodiment 143. The method of embodiment 142, wherein the SARS-CoV-2 target nucleic acid is hybridized to a target capture oligonucleotide, and the target capture oligonucleotide is directly bound to a solid support.

[0156] Embodiment 144. The method of embodiment 142, wherein the SARS-CoV-2 target nucleic acid is hybridized to a target capture oligonucleotide, and the target capture oligonucleotide directly binds an immobilized probe attached to a solid support.

[0157] Embodiment 145. A target capture oligonucleotide comprising: (i) a target hybridizing sequence containing or consisting of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, or SEQ ID NO:102, its DNA equivalent, RNA equivalent, or DNA / RNA chimeric form, etc., and including 0-24 nucleotide analogs; and (ii) d(T) 0~3d(A) 14~30 and a solid support binding region which is

[0158] Embodiment 146. The method of any one of embodiments 143 to 145, wherein the target capture oligonucleotide comprises or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:96.

[0159] Embodiment 147. The method of any one of embodiments 122 to 146, wherein the in vitro nucleic acid amplification reaction is an isothermal amplification reaction.

[0160] Embodiment 148. The method of embodiment 147, wherein the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction or a nucleic acid sequence-based amplification (NASBA) reaction.

[0161] Embodiment 149. The method of any one of embodiments 122 to 146, wherein the in vitro nucleic acid amplification reaction is a PCR reaction.

[0162] Embodiment 150. The method of any one of embodiments 122 to 149, wherein the in vitro nucleic acid amplification reaction is a real-time reaction.

[0163] Embodiment 151. The method of any one of embodiments 122 to 150, wherein the in vitro nucleic acid amplification reaction is a quantitative reaction.

[0164] Embodiment 152. The method of any one of embodiments 122 to 151, wherein the in vitro nucleic acid amplification reaction is a multiplex reaction.

[0165] Embodiment 153. A method for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) contacting a sample containing or suspected of containing a SARS-CoV-2 target nucleic acid with a combination of amplification oligomers comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of the SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 18 to 27 contiguous nucleotides in length, is contained in SEQ ID NO:26, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (ii) the second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 18 to 23 contiguous nucleotides in length and contains a nucleotide sequence that is contained in SEQ ID NO: 85 and differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; Steps and (b) incubating the sample under conditions suitable for nucleic acid amplification, wherein any SARS-CoV-2 target nucleic acid, if present in the sample, is used as a template to generate amplicons corresponding to target regions of the SARS-CoV-2 target nucleic acid; (c) detecting the presence or absence of the amplicon, thereby determining the presence or absence of SARS-CoV-2 in the sample; A method comprising:

[0166] Embodiment 154. The method of embodiment 153, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues.

[0167] Embodiment 155. The method of embodiment 153 or 154, wherein the sequence that hybridizes to a second SARS-CoV-2-specific target is linked at its 5' end to the promoter sequence.

[0168] Embodiment 156. The method of embodiment 155, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0169] Embodiment 157. The method of embodiment 155, wherein the second SARS-CoV-2-specific amplification oligomer comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 or SEQ ID NO:62, or an RNA equivalent or a DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs.

[0170] Embodiment 158. The method of any one of embodiments 153 to 157, wherein the detecting step (c) comprises contacting the amplicon, if present, with a SARS-CoV-2-specific detector probe oligomer comprising a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is 19 to 27 contiguous nucleotides in length, is contained in SEQ ID NO: 50, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 114.

[0171] Embodiment 159. The method of embodiment 158, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 107, or SEQ ID NO: 108, and includes 0 to 7 nucleotide analogs.

[0172] Embodiment 160. The method of embodiment 158, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe oligomer contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:107 or SEQ ID NO:108, its DNA equivalent, RNA equivalent or DNA / RNA chimeric form, etc., and includes 0 to 7 nucleotide analogs.

[0173] Embodiment 161. The method of embodiment 158 ​​or 159, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, optionally wherein the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0174] Embodiment 162. The method of embodiment 161, wherein the self-complementary sequence is linked by a non-nucleotide linker to the sequence that hybridizes to the target of the SARS-CoV-2-specific detection probe.

[0175] Embodiment 163. The method of any one of embodiments 158 to 162, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0176] Embodiment 164. The method of embodiment 163, wherein the detectable label is a fluorescent or chemiluminescent label.

[0177] Embodiment 165. The method of embodiment 164, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0178] Embodiment 166. The method of embodiment 164, wherein the SARS-CoV-2 specific detection probe oligomer is a molecular beacon or a molecular torch.

[0179] Embodiment 167. The method of any one of embodiments 153 to 166, wherein the SARS-CoV-2 target nucleic acid is separated from other components of the sample prior to the amplification step (b).

[0180] Embodiment 168. The method of embodiment 167, wherein the SARS-CoV-2 target nucleic acid is separated from other components of the sample prior to the contacting step (a).

[0181] Embodiment 169. The method of embodiment 167 or 168, wherein the separating step is carried out by directly or indirectly binding the SARS-CoV-2 target nucleic acid to a solid support, followed by washing the SARS-CoV-2 target nucleic acid-solid support complex to remove other components of the sample.

[0182] Embodiment 170. The method of embodiment 169, wherein the SARS-CoV-2 target nucleic acid is hybridized to a target capture oligonucleotide, and the target capture oligonucleotide is directly bound to a solid support.

[0183] Embodiment 171. The method of embodiment 169, wherein the SARS-CoV-2 target nucleic acid is hybridized to a target capture oligonucleotide, and the target capture oligonucleotide directly binds an immobilized probe attached to a solid support.

[0184] Embodiment 172. A target capture oligonucleotide comprising: (i) a target hybridizing sequence containing or consisting of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, or SEQ ID NO:102, its DNA equivalent, RNA equivalent, or DNA / RNA chimeric form, etc., and including 0-24 nucleotide analogs; and (ii) d(T) 0~3 d(A) 14~30 172. The method of embodiment 170 or 171, comprising a solid support binding region which is

[0185] Embodiment 173. The method of any one of embodiments 170 to 172, wherein the target capture oligonucleotide comprises a sequence containing or consisting of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:96.

[0186] Embodiment 174. The method of any one of embodiments 153 to 173, wherein the in vitro nucleic acid amplification reaction is an isothermal amplification reaction.

[0187] Embodiment 175. The method of embodiment 174, wherein the isothermal amplification reaction is a TMA reaction or a NASBA reaction.

[0188] Embodiment 176. The method of any one of embodiments 153 to 173, wherein the in vitro nucleic acid amplification reaction is a PCR reaction.

[0189] Embodiment 177. The method of any one of embodiments 153 to 176, wherein the in vitro nucleic acid amplification reaction is a quantitative reaction.

[0190] Embodiment 178. The method of any one of embodiments 153 to 177, wherein the in vitro nucleic acid amplification reaction is a real-time reaction.

[0191] Embodiment 179. A method for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) contacting a sample containing or suspected of containing a SARS-CoV-2 target nucleic acid with a combination of amplification oligomers comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of the SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 20 to 23 contiguous nucleotides in length and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (ii) the second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target, wherein the sequence that hybridizes to this target is 16 to 27 contiguous nucleotides in length and contains a nucleotide sequence that is contained in SEQ ID NO:89 and differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:112, or SEQ ID NO:113; Steps and (b) incubating the sample under conditions suitable for nucleic acid amplification, wherein any SARS-CoV-2 target nucleic acid, if present in the sample, is used as a template to generate amplicons corresponding to target regions of the SARS-CoV-2 target nucleic acid; (c) detecting the presence or absence of the amplicon, thereby determining the presence or absence of SARS-CoV-2 in the sample; A method comprising:

[0192] Embodiment 180. The method of embodiment 179, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues.

[0193] Embodiment 181. The method of embodiment 179 or 180, wherein the sequence that hybridizes to a second SARS-CoV-2-specific target is linked to the promoter sequence at its 5' end.

[0194] Embodiment 182. The method of embodiment 181, wherein the promoter sequence is substantially identical to SEQ ID NO: 11.

[0195] Embodiment 183. The method of embodiment 181, wherein the second SARS-CoV-2-specific amplification oligomer comprises or consists of a contiguous nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:78, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs.

[0196] Embodiment 184. The method of any one of embodiments 179 to 183, wherein the detecting step (c) comprises contacting the amplicon with a SARS-CoV-2-specific detector probe oligomer comprising a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is 25 to 29 contiguous nucleotides in length and contains a nucleotide sequence that is contained in SEQ ID NO: 54 and differs from the nucleotide sequence of SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 115 by at most 0, 1, 2, 3, 4, or 5 nucleotides.

[0197] Embodiment 185. The method of embodiment 184, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 109, or SEQ ID NO: 110, including 0 to 7 nucleotide analogs.

[0198] Embodiment 186. The method of embodiment 184, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detector probe oligomer contains or consists of a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 109, or SEQ ID NO: 110, including 0 to 7 nucleotide analogs.

[0199] Embodiment 187. The method of embodiment 184 or 185, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a self-complementary sequence linked to the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe, and optionally, the self-complementary sequence is linked to the 5' end of the sequence that hybridizes to the target of the SARS-CoV-2-specific detector probe.

[0200] Embodiment 188. The method of embodiment 187, wherein the self-complementary sequence is linked by a non-nucleotide linker to the sequence that hybridizes to the target of the SARS-CoV-2-specific detection probe.

[0201] Embodiment 189. The method of any one of embodiments 184 to 188, wherein the SARS-CoV-2-specific detector probe oligomer further comprises a detectable label.

[0202] Embodiment 190. The method of embodiment 189, wherein the detectable label is a fluorescent or chemiluminescent label.

[0203] Embodiment 191. The method of embodiment 190, wherein the detectable label is a fluorescent label and the SARS-CoV-2-specific detector probe oligomer further comprises a non-fluorescent quencher.

[0204] Embodiment 192. The method of embodiment 190, wherein the SARS-CoV-2 specific detection probe oligomer is a molecular beacon or a molecular torch.

[0205] Embodiment 193. The method of any one of embodiments 179 to 192, wherein the SARS-CoV-2 target nucleic acid is separated from other components of the sample prior to the amplification step (b).

[0206] Embodiment 194. The method of embodiment 193, wherein the SARS-CoV-2 target nucleic acid is separated from other components of the sample prior to the contacting step (a).

[0207] Embodiment 195. The method of embodiment 193 or 194, wherein the separating step is carried out by directly or indirectly binding the SARS-CoV-2 target nucleic acid to a solid support, followed by washing the SARS-CoV-2 target nucleic acid-solid support complex to remove other components of the sample.

[0208] Embodiment 196. The method of embodiment 195, wherein the SARS-CoV-2 target nucleic acid is hybridized to a target capture oligonucleotide, and the target capture oligonucleotide is directly bound to a solid support.

[0209] Embodiment 197. The method of embodiment 195, wherein the SARS-CoV-2 target nucleic acid is hybridized to a target capture oligonucleotide, and the target capture oligonucleotide directly binds an immobilized probe attached to a solid support.

[0210] Embodiment 198. A target capture oligonucleotide comprising: (i) a target hybridizing sequence containing or consisting of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, or SEQ ID NO:102, its DNA equivalent, RNA equivalent, or DNA / RNA chimeric form, etc., and including 0-24 nucleotide analogs; and (ii) d(T). 0~3 d(A) 14~30 198. The method of embodiment 196 or 197, comprising a solid support binding region which is

[0211] Embodiment 199. The method of any one of embodiments 196 to 198, wherein the target capture oligonucleotide comprises or consists of a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:96.

[0212] Embodiment 200. The method of any one of embodiments 179 to 199, wherein the in vitro nucleic acid amplification reaction is an isothermal amplification reaction.

[0213] Embodiment 201. The method of embodiment 200, wherein the isothermal amplification reaction is a TMA reaction or a NASBA reaction.

[0214] Embodiment 202. The method of any one of embodiments 179 to 199, wherein the in vitro nucleic acid amplification reaction is a PCR reaction.

[0215] Embodiment 203. The method of any one of embodiments 179 to 202, wherein the in vitro nucleic acid amplification reaction is a quantitative reaction.

[0216] Embodiment 204. The method of any one of embodiments 179 to 203, wherein the in vitro nucleic acid amplification reaction is a real-time reaction.

[0217] Embodiment 205. A multiplex method for determining the presence or absence of SARS-CoV-2 and at least one other pathogen in a sample, wherein the presence or absence of SARS-CoV-2 is determined using the method of any one of embodiments 153 to 178.

[0218] Embodiment 206. The multiplex method of embodiment 205, for determining the presence or absence of SARS-CoV-2 and at least one respiratory pathogen in a sample.

[0219] Embodiment 207. The multiplex method of embodiment 205, wherein the presence or absence of SARS-CoV-2 and one or more pathogens selected from the group consisting of influenza A, influenza B, respiratory syncytial virus A, respiratory syncytial virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, adenovirus, metapneumovirus, rhinovirus, coronavirus 229E, coronavirus NL63, coronavirus HKU1, coronavirus OC43, SARS-CoV (SARS), and MERS-CoV is determined.

[0220] Embodiment 208. A multiplex method for determining the presence or absence of SARS-CoV-2 and at least one other pathogen in a sample, wherein the presence or absence of SARS-CoV-2 is determined using the method of any one of embodiments 179 to 204.

[0221] Embodiment 209. The multiplex method of embodiment 208, for determining the presence or absence of SARS-CoV-2 and at least one respiratory pathogen in a sample.

[0222] Embodiment 210. The multiplex method of embodiment 208, wherein the presence or absence of SARS-CoV-2 and one or more pathogens selected from the group consisting of influenza virus A, influenza virus B, respiratory syncytial virus A, respiratory syncytial virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, adenovirus, metapneumovirus, rhinovirus, coronavirus 229E, coronavirus NL63, coronavirus HKU1, coronavirus OC43, SARS-CoV (SARS), and MERS-CoV is determined.

[0223] Embodiment 211. A multiplex method for determining the presence or absence of SARS-CoV-2 and at least one other pathogen in a sample, wherein the presence or absence of SARS-CoV-2 is determined using the method of any one of embodiments 122 to 152.

[0224] Embodiment 212. The multiplex method of embodiment 211, for determining the presence or absence of SARS-CoV-2 and at least one respiratory pathogen in a sample.

[0225] Embodiment 213. The multiplex method of embodiment 211, wherein the presence or absence of SARS-CoV-2 and one or more pathogens selected from the group consisting of influenza A, influenza B, respiratory syncytial virus A, respiratory syncytial virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, adenovirus, metapneumovirus, rhinovirus, coronavirus 229E, coronavirus NL63, coronavirus HKU1, coronavirus OC43, SARS-CoV (SARS), and MERS-CoV is determined.

[0226] Embodiment 214. The method of any one of embodiments 122 to 223, wherein the sample is a pooled sample.

[0227] These and other aspects and embodiments will become evident upon reference to the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0228] Figures 1 and 2 are linearity plots showing the performance of primer and probe (PPR) mixtures for the amplification and detection of SARS-CoV-2 organisms.

[0229] [Figure 1]Figure 1 shows linearity data for the response to dilutions of in vitro transcripts (IVT) using combined PPR mixes containing each of SARS-CoV-2 PPR mix 1 and SARS-CoV-2 PPR mix 5 as shown in Table 1. See Example 2 below.

[0230] [Figure 2] Figure 2 shows linearity data for response to dilutions of specific virus strains using the same combined PPR mix. See Example 3 below. DETAILED DESCRIPTION OF THE INVENTION

[0231] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions being described relate. As used herein, the following terms and phrases have the meanings ascribed to them unless otherwise specified.

[0232] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a nucleic acid," as used herein, is understood to refer to one or more nucleic acids. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0233] When a value is expressed as "about" X or "approximately" X, the stated value of X is to be understood to be accurate to ±10%.

[0234] All ranges shall be construed as including the endpoints unless there is an explicit exclusion, such as "excluding the endpoints," so, for example, "within 10 and 15" includes the values ​​10 and 15. Those of ordinary skill in the art will understand that the stated ranges include the endpoints, as well as integers between the endpoints and rational numbers within the range, where permissible (e.g., the range 5 to 10 includes 5, 6, 7, 8, 9, and 10, and values ​​such as 6.8, 9.35, etc., where permissible).

[0235] A "sample" includes any specimen containing or suspected of containing a coronavirus that causes COVID-19, such as SARS-CoV-2, including components thereof, e.g., nucleic acid or nucleic acid fragments. A sample also includes a "biological sample," which includes any tissue or material derived from a living or dead mammal (e.g., human) or organism. Biological samples include, but are not limited to, nasopharyngeal swabs, nasal swabs, middle turbinate swabs, oropharyngeal swabs, throat swabs, nasal washes, bronchial washes, nasal aspirates, sputum, blood, plasma, serum, blood cells, saliva, mucus, respiratory tissue, exudates (e.g., bronchoalveolar lavage fluid), sputum, tracheal aspirates, lymph nodes, gastrointestinal tissue, feces, urine, urogenital fluids, and biopsied cells or tissues. A sample can be processed or altered by sample preparation. A sample can be an individual sample (ie, a sample derived from a single subject) or a pooled sample (ie, a sample prepared by pooling multiple individual samples).

[0236] "Sample preparation" refers to any steps or methods required to prepare a sample for amplification and / or detection. Samples can be treated to chemically, physically, and / or mechanically disrupt tissues, cells, or cellular components, releasing intracellular components into aqueous or organic solutions that may further contain enzymes, buffers, salts, surfactants, etc., used to prepare the biological sample for analysis. Samples can also be chemically, physically, and / or mechanically treated to remove cellular components or debris. Samples can be processed by passing the sample over or through a filtration device, by centrifugation, or by attachment to a medium, matrix, or support. Sample preparation includes methods for concentrating components such as polynucleotides from a larger sample volume, for example, by filtration from a larger volume sample, by centrifugation, or by isolating microorganisms from the sample using standard microbiological methods. Sample preparation can also include the use of polynucleotides to specifically or nonspecifically capture target nucleic acids and separate them from other sample components.

[0237] The term "target capture" refers to the selective separation or isolation of a target nucleic acid from other components of a sample mixture, such as cell fragments, organelles, proteins, lipids, carbohydrates, or other nucleic acids. Target capture systems can be specific and can selectively separate a given target nucleic acid from other sample components (e.g., by using a sequence specific to the intended target nucleic acid, such as a TCO TS sequence). Target capture methods and compositions have been previously described in detail (U.S. Patent Nos. 6,110,678 and 6,534,273, and U.S. Patent Application Publication No. 2008 / 0286775A1). In some embodiments, target capture utilizes a solution-phase TCO and an immobilized capture probe attached to a support to form a complex with the target nucleic acid and separate the captured target from other components.

[0238] A "target capture oligonucleotide" (TCO) is a nucleic acid oligonucleotide that specifically hybridizes to a sequence in a target nucleic acid through standard base pairing and ligates to a binding partner on an immobilized probe to capture the target nucleic acid to a support. TCOs can be used to capture or isolate target nucleic acids from a sample. A TCO comprises a target-specific (TS) nucleotide sequence that hybridizes (i.e., is complementary to) a region of the target nucleic acid. In some embodiments, the TCO TS sequence comprises a 10-35 nucleotide sequence that is at least 90%, at least 95%, or 100% complementary to a nucleotide sequence present in the target nucleic acid and hybridizes to a region in the target nucleic acid sequence (the TCO binding site). A TCO comprises an immobilized capture probe binding region that binds to an immobilized capture probe (e.g., via a specific binding pair interaction). In some embodiments, the TCO TS sequence is linked to the capture probe binding region. In some embodiments, the TCO TS sequence and the capture probe binding region are present on two different oligonucleotides linked together by one or more linkers. In some embodiments, the capture probe binding region comprises a polyA sequence, a polyT sequence, or a polyT-polyA sequence. In some embodiments, the polyT-polyA sequence is (dT) 0~3 (dA) 14~30 or (dT)3(dA) 30 Includes:

[0239] An "immobilized capture probe" provides a means for linking a TCO to a solid support. In some embodiments, the immobilized capture probe contains a base sequence recognition molecule linked to the solid support, which facilitates separation of the bound target polynucleotide from unbound material. Any known solid support may be used, such as matrices and particles free in solution. For example, the solid support may be nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and magnetically attractable particles. In some embodiments, the support comprises monodisperse (i.e., uniform in size within about ±5%) magnetic spheres. The immobilized capture probe may be directly (e.g., by covalent linkage or ionic interaction) or indirectly linked to the solid support. Common examples of useful solid supports include magnetic particles or beads.

[0240] A "nucleotide" is a subunit of a nucleic acid (also referred to herein as a "nucleobase") consisting of a phosphate group, a pentose sugar, and a nitrogenous base. The pentose sugar found in RNA is ribose. In DNA, the pentose sugar is 2'-deoxyribose.

[0241] "Nucleic acid" and "polynucleotide" refer to polymeric compounds containing nucleotides and / or nucleotide analogs linked together to form a biopolymer. Biopolymers include conventional RNA, conventional DNA, mixed RNA-DNA, and nucleotide analog-containing versions thereof. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids," or PNA), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions or modifications, such as analogs with a methoxy, fluoro, or halide group at the 2' position of the ribose (also referred to herein as "2'-O-Me" or "2'-methoxy," or 2'-fluoro, or "2'-halide"). The nitrogenous bases include the common bases adenine (A), uracil (U), guanine (G), thymine (T), and cytosine (C), as well as their analogs (e.g., inosine, 5-methyl-2'-deoxycytosine ("5-Me-dC" or "5 MeC), propyne dU, and isoguanine). Nucleic acids may contain one or more "abasic" residues, in which case the backbone does not contain a nitrogenous base at position(s) of the polymer.

[0242] Sequence identity can be determined by aligning sequences using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.) using default gap parameters, or by inspection and best alignment (i.e., the alignment that results in the highest percentage of sequence similarity over the comparison window). The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions where identical residues appear in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions (i.e., the window size) within the comparison window, not counting gaps, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified, the comparison window between two sequences is defined by the total length of the shorter of the two sequences.

[0243] The term "complementarity" refers to the ability of a polynucleotide to form hydrogen bonds (hybridize) with another polynucleotide sequence through either traditional Watson-Crick base pairing or other non-traditional base pairing. Two complementary polynucleotide strands are antiparallel to each other. The percentage of complementarity indicates the percentage of bases in a contiguous strand in a first nucleic acid sequence that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 would be 50%, 60%, 70%, 80%, 90%, and 100% complementary). The percentage of complementarity is calculated in a similar manner to the percentage of identity.

[0244] "RNA equivalent" and "DNA equivalent" refer to RNA and DNA molecules that have essentially the same nucleic acid sequence or complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. Differences between RNA and DNA equivalents do not contribute to differences in homology, as equivalents have the same degree of complementarity to a particular sequence. "DNA / RNA chimeric form" refers to a nucleic acid that contains both DNA and RNA nucleotides. Unless the context clearly dictates otherwise, reference to coronavirus nucleic acids includes RNA and DNA equivalents, as well as their DNA / RNA chimeric forms.

[0245] "Oligomer," "oligonucleotide," or "oligo" generally refers to a nucleic acid of less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 5 nt and an upper limit of about 900 nt. In some embodiments, oligomers range in size from 5 to 15 nt and an upper limit of 50 to 500 nt. In some embodiments, oligomers range in size from 10 to 100 nucleobases, 10 to 90 nucleobases, 10 to 80 nucleobases, 10 to 70 nucleobases, or 10 to 60 nucleobases. Oligonucleotides can be DNA and / or RNA and / or analogs thereof. The term oligonucleotide does not denote any specific function for the reagent, but is used generically to encompass all such reagents described herein. Oligomers may be referred to by their functional name (e.g., capture probe, primer, or promoter primer), although those skilled in the art will understand that such terms refer to oligomers. Described oligomers include RNA polymerase promoter-containing oligomers (also referred to as promoter primers, for example, T7 primers), non-RNA polymerase promoter-containing oligomers (also referred to as non-T7 primers, NT7 primers, or non-promoter primers), probe oligomers (also referred to as detection oligomers or detection probes, probes, or torches), target capture oligonucleotides (TCO), forward primers (non-T7 primers or NT7 primers), and reverse primers (including T7 primers).Oligomers can be synthetically produced using any well-known in vitro chemical or enzymatic method, and can be purified after synthesis by standard methods, for example, by high performance liquid chromatography (HPLC).

[0246] Any of the described oligonucleotides may contain at least one modified nucleotide (i.e., at least one nucleotide analog). The modified nucleotide may be, but is not limited to, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5-methylcytosine. In some embodiments, the amplification oligonucleotide comprises two or more modified nucleotides. The two or more modified nucleotides may be the same or different. In some embodiments, a thymidine nucleotide may be substituted for a uridine nucleotide. In some embodiments, all thymidine nucleotides may be substituted for uridine nucleotides. In some embodiments, 5-methyl-2-deoxycytosine bases may be used to increase duplex stability (compared to the corresponding unmethylated amplification oligonucleotide) by increasing the Tm by about 0.5°-1.3°C for each 5'methyl-2'deoxycytosine incorporated into the oligonucleotide.

[0247] A "target nucleic acid" is a nucleic acid containing a target sequence to be amplified and / or detected. The target nucleic acid may be DNA or RNA, and may be single-stranded or double-stranded. The target nucleic acid may be, but is not limited to, a genomic nucleic acid or a transcribed nucleic acid, such as mRNA. In the case of a single-stranded target nucleic acid, such as a single-stranded RNA virus or mRNA, the target nucleic acid includes its complement. The target nucleic acid may also be a nucleic acid derived from a genomic nucleic acid or a transcribed nucleic acid. The target nucleic acid (including its complement, if appropriate) contains a sequence that hybridizes to a capture oligonucleotide, primer, and / or probe used to amplify and / or detect the target nucleic acid. The target nucleic acid may contain other sequences in addition to the target nucleic acid that may not be amplified.

[0248] A "target sequence" or "target nucleic acid sequence" is a specific nucleotide sequence of a target nucleic acid to be amplified and / or detected. The target sequence, including its complement, contains a sequence that hybridizes to the primers and probes used to amplify and / or detect the target nucleic acid during an amplification process (e.g., PCR, TMA). Unless the context clearly indicates otherwise, if the target nucleic acid is originally single-stranded, the term "target sequence" will also refer to a sequence complementary to the "target sequence," if present in the target nucleic acid; if the target nucleic acid is originally double-stranded, the term "target sequence" will refer to both the sense (+) strand and the antisense (-) strand.

[0249] A "target-hybridizing sequence," "target-hybridizing region," or "target-specific sequence" is a sequence in an oligomer that hybridizes to a region in a target nucleic acid sequence. A target-hybridizing region is a consecutive sequence of nucleotides that hybridizes to a consecutive complementary sequence of nucleotides in a target nucleic acid sequence. A target-hybridizing sequence is configured to specifically hybridize with a target nucleic acid. A target-hybridizing sequence is configured to specifically hybridize with a target nucleic acid. A target-hybridizing sequence can be, but is not necessarily, 100% complementary to the portion of the target nucleic acid sequence to which it is configured to hybridize. A target-hybridizing sequence can also contain insertions, deletions, and / or substitutions of nucleotide residues compared to the target sequence, provided that the target-hybridizing sequence specifically hybridizes with the target nucleic acid. A primer or probe can contain both target-specific and non-target-specific sequences. A target-specific sequence (or target-hybridizing sequence or target-hybridizing region) is a portion of an oligonucleotide that is configured to hybridize to a target nucleic acid. Reference to an oligonucleotide (e.g., a primer or probe) that includes a target-hybridizing sequence consisting of SEQ ID NO: X indicates that the portion of the oligonucleotide that is complementary to the target nucleic acid consists solely of the indicated SEQ ID NO. The oligonucleotide may contain other non-target-hybridizing sequences or other components (e.g., labels), but the target-hybridizing sequence consists of the sequence of the indicated SEQ ID NO.

[0250] "Non-target-specific sequence" or "sequence that does not hybridize to the target" refers to a region of an oligomer sequence that does not stably hybridize to the target sequence under standard hybridization conditions. Oligomers with non-target-specific sequences include, but are not limited to, promoter primers, promoter donors, target capture oligonucleotides, torches, and molecular beacons.

[0251] "Targeting a sequence" is used in reference to a region of a SARS-CoV-2 nucleic acid to which an oligonucleotide hybridizes in a manner that allows for amplification and / or detection as described herein. In some embodiments, the oligonucleotide is complementary to the targeted SARS-CoV-2 nucleic acid sequence and contains no mismatches. In some embodiments, the oligonucleotide is complementary to the targeted SARS-CoV-2 nucleic acid sequence but contains 1, 2, 3, 4, or 5 mismatches.

[0252] The term "configured to" refers to the actual arrangement of the polynucleotide sequence of a sequence that hybridizes to the target of the referenced oligonucleotide. For example, an amplification oligomer that is configured to generate a specified amplicon from a target sequence has a polynucleotide sequence that hybridizes to the target nucleic acid and can be used in an amplification reaction to generate the amplicon. Also, by way of example, an oligonucleotide that is configured to specifically hybridize to a target sequence has a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0253] The term "configured to specifically hybridize to" indicates that the target-hybridizing region of an amplification oligonucleotide, detection probe, or other oligonucleotide is designed to have a polynucleotide sequence capable of targeting the sequence of a referenced SARS-CoV-2 target sequence. The oligonucleotide is designed to function as a component of an assay for amplification and / or detection of a SARS-CoV-2 target nucleic acid in a sample, and is therefore designed to target SARS-CoV-2 in the presence of other nucleic acids commonly found in the sample being tested. "Specifically hybridizing to" does not mean hybridizing exclusively to, as some small level of hybridization to non-target nucleic acids may occur, as is understood in the art. Rather, "specifically hybridizing to" means that the oligonucleotide is configured to function in the assay to primarily hybridize to the target, allowing for accurate amplification and / or detection of the target nucleic acid in the sample.

[0254] An "amplification oligonucleotide," "amplification oligomer," or "primer" is an oligonucleotide that hybridizes to a target nucleic acid and participates in a nucleic acid amplification reaction, e.g., serves as a primer. An amplification oligomer may have a 3' end that is extended by polymerization as part of a nucleic acid amplification reaction. Alternatively, an amplification oligomer may have a 3' end that is not extended by polymerization but provides a component that facilitates nucleic acid amplification, e.g., a promoter sequence linked 5' to the sequence that hybridizes to the amplification oligomer's target. Such an amplification oligomer is referred to as a promoter-provider. An amplification oligomer that provides both a 3' target-hybridizing region that is extendable by polymerization and a 5' promoter sequence is referred to as a promoter-primer. Amplification oligomers can be modified, as needed, to include a 5' non-target-hybridizing region, e.g., a tag, a promoter (as noted), or other sequence used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0255] "Nucleic acid amplification" refers to any in vitro procedure that generates multiple copies of a target nucleic acid sequence, or its complementary sequence, or a fragment thereof (i.e., an amplified sequence that contains less than the entire target nucleic acid). Examples of nucleic acid amplification procedures include transcription-related methods, such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), etc. (e.g., U.S. Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), and polymerase chain reaction (PCR) (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159).

[0256] "Transcription-mediated amplification" uses DNA polymerase (e.g., reverse transcriptase), RNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, primers (including promoter-primers), and optionally other oligonucleotides, to generate multiple RNA transcripts from a nucleic acid template (see U.S. Patent Nos. 5,399,491 and 5,554,516 to Kacian et al., U.S. Patent No. 5,444,445 to Burg ... (These are described in detail in U.S. Patent No. 37,990, Gingeras et al., PCT No. WO88 / 01302 and WO88 / 10315, Malek et al., U.S. Patent No. 5,130,238, Urdea et al., U.S. Patent No. 4,868,105 and U.S. Patent No. 5,124,246, McDonough et al., PCT No. WO94 / 03472, and Ryder et al., PCT No. WO95 / 03430, each of which is incorporated herein by reference.) The method using TMA has been described in detail before (U.S. Patent No. 5,399,491 and U.S. Patent No. 5,554,516, each of which is incorporated herein by reference).TMA can be substantially isothermal amplification.TMA can also be carried out as a two-phase amplification reaction.

[0257] The term "substantially isothermal amplification" refers to an amplification reaction carried out at a substantially constant temperature. The isothermal portion of the reaction is preceded or followed by one or more steps at various temperatures, such as an initial denaturation step and a final heat inactivation or cooling step. It will be understood that this definition, rather than excluding small variations in temperature, is used to differentiate isothermal amplification techniques from other amplification techniques known in the art that essentially rely on "temperature cycling" to generate amplified products.

[0258] An "amplicon" or "amplification product" is a nucleic acid molecule produced in a nucleic acid amplification reaction that is derived from (amplified from) a target nucleic acid. The amplicon or amplification product contains the target nucleic acid sequence, which may be in the same or opposite orientation as the target nucleic acid.

[0259] "Relative Fluorescence Units" ("RFU") are a unit of measurement for fluorescence intensity. RFUs vary depending on the characteristics of the detection means used in the measurement and can be used as a measure to compare relative intensities between a sample and a control.

[0260] A "detector probe oligomer," "detector probe," or "probe" is an oligomer that specifically hybridizes to a target sequence comprising an amplified product under conditions that promote nucleic acid hybridization for the detection of a target nucleic acid. Detection can be either direct (i.e., a probe hybridized directly to the target) or indirect (i.e., a probe hybridized to an intermediate structure that links the probe to the target). The target sequence of a probe generally refers to the particular sequence within a larger sequence to which the probe specifically hybridizes. A detector probe can contain target-specific and non-target-specific sequences. Such non-target-specific sequences can include sequences that will impart a desired secondary or tertiary structure, e.g., a hairpin structure, that can be used to facilitate detection and / or amplification. A probe can have a detectable label. A detectable target can be linked to the probe directly or indirectly.

[0261] A "molecular torch" or "torch" is a type of probe that can be used to indicate whether an amplicon is present in a sample. Molecular torches contain distinct self-complementary regions. When exposed to a target, the two (fully or partially complementary) self-complementary regions of the molecular torch melt, thereby allowing individual nucleotides (including the target binding domain) to hybridize to complementary consecutive nucleotides on the target nucleic acid sequence. Molecular torches are designed so that the target binding domain favors hybridization to the target nucleic acid sequence over the target closing domain (self-complementary region). The target binding domain and target closing domain of a molecular torch contain interactive labels (e.g., a fluorescent dye and a quencher, a FRET pair), resulting in a different signal being generated when the molecular torch self-hybridizes as opposed to hybridizing to the target nucleic acid sequence (thereby enabling detection of a probe:target duplex in a test sample in the presence of unhybridized probe). Methods for synthesizing labels, attaching labels to nucleic acids, and detecting signals from labels are well known in the art (see, e.g., Sambrook et al., supra, at Chapter 10, and U.S. Pat. No. 6,229,133). Patents Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333, and European Patent Application No. 0747706).

[0262] "Stringent hybridization conditions" or "stringent conditions" are conditions that allow an oligomer to hybridize preferentially to a target sequence and not to nucleic acids derived from closely related non-target nucleic acids (i.e., conditions that allow an oligomer to hybridize to its target sequence in such a way that it forms a stable oligomer:target hybrid but does not form a sufficient number of stable oligomer:non-target hybrids, allowing amplification and / or detection of the target nucleic acid but not of non-target organisms). The definition of stringent hybridization conditions remains the same, but the actual reaction environment that can be used for stringent hybridization can vary depending on factors such as the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and the sequence of non-target nucleic acids that may be present in the test sample, and the target sequence. Hybridization conditions include the temperature and composition of the hybridization reagent or solution. Stringent hybridization conditions can be easily determined by those skilled in the art.

[0263] A "label" or "detectable label" is a moiety or compound directly or indirectly attached to a probe that is detected or provides a detectable signal. Direct linkages may use covalent or non-covalent interactions (e.g., hydrogen bonding, hydrophobic or ionic interactions, and chelate or coordinate complex formation), while indirect linkages may use bridging moieties or linkers (e.g., via antibodies or additional oligonucleotides that amplify the detectable signal. Any detectable moiety can be used, such as radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles (e.g., latex or metal beads) that impart a detectable color, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds, e.g., acridinium ester ("AE") compounds), and fluorescent compounds (i.e., fluorophores). Fluorophores may be used in combination with a quencher molecule that absorbs light emitted by the fluorophore when in close proximity to the fluorophore. Detectably labeled probes include, but are not limited to, TaqMan™ probes, AE-labeled probes, molecular torches, and molecular beacons.

[0264] A "quencher" is a molecule that absorbs light. Quenchers are commonly used in combination with luminescent labels, such as fluorophores, to absorb emitted light when in close proximity to the fluorophore. Quenchers are well known in the art and include, but are not limited to, Black Hole Quencher™ (or BHQ™, BHQ-1™, or BHQ-2™), Blackberry Quencher, Dabcyl, QSY, and Tamra™ compounds, to name a few.

[0265] A "non-extendable" oligomer contains a blocking moiety at or near its 3' end to prevent extension. The blocking group near the 3' end, in some embodiments, is within 5 residues of the 3' end and is large enough to restrict polymerase binding to the oligomer. In other embodiments, the blocking group is covalently attached to the 3' end. Suitable blocking groups include, for example, alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, cordycepin, 3'-deoxynucleotides, 3'-phosphorylated nucleotides, inverted nucleotides, proteins, peptides, and labels, such as fluorophores or quenchers.

[0266] Reference to a "sequence of SEQ ID NO: X" refers to a sequence of nucleobases, nucleotides, and / or nucleotide analogs linked together to form a biopolymer. Reference to a sequence by SEQ ID NO: does not imply the identity of the backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or any nucleobase modifications (e.g., methylation of cytosine residues ("5MeC")), unless the context clearly dictates otherwise. In some cases, the SEQ ID NO: sequence is followed by the statement "comprising [x to y] nucleotide analogs." It is understood that nucleotide analogs can be substitutions within the SEQ ID NO: sequence, additions to the SEQ ID NO: sequence, or both. Unless the context clearly dictates otherwise, reference to a sequence by SEQ ID NO: includes reference to its complementary sequence (e.g., reference to the sequence 5'-ttagc-3' includes reference to the sequence 5'-gctaa-3').

[0267] "Separating" or "purifying" refers to the removal of one or more components or a mixture, e.g., a sample, from one or more other components in the mixture. Sample components include nucleic acids, including target nucleic acids, cellular fragments, proteins, carbohydrates, lipids, and other compounds. Separating or purifying does not imply any degree of purification. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target nucleic acid or amplified product is separated or removed from other components in the mixture.

[0268] "Specificity" in the context of amplification and / or detection systems refers to the characteristic of the system that describes its ability to distinguish between target and non-target sequences, depending on the sequence and assay conditions.In the context of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of by-products (e.g., signal-to-noise ratio).In the context of detection, specificity generally refers to the ratio of the signal generated from target nucleic acid to the signal generated from non-target nucleic acid.

[0269] "Sensitivity" refers to the precision with which a nucleic acid amplification reaction can be detected or quantified. The sensitivity of an amplification reaction is generally a measure of the minimum copy number of a target nucleic acid that can be easily detected in an amplification system, and depends, for example, on the detection assay used and the specificity of the amplification reaction, for example, the ratio of specific amplicons to by-products. Detailed Description

[0270] Provided herein are compositions (including amplification oligomer combinations), kits, formulations, and methods for amplifying and / or detecting SARS-CoV-2 nucleic acids from a sample. In some embodiments, the sample is a biological sample. The compositions, kits, and methods provide oligonucleotides and oligonucleotide sequences targeting the ORF1ab gene sequence of SARS-CoV-2, or their complementary sequences. The described oligonucleotides include amplification oligonucleotides, which can include primers, promoter primers, blocked oligonucleotides, and promoter-donor oligonucleotides, the functions of which have been previously described (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 5,399,491, 5,554,516, 5,824,518, and 7,374,885, each of which is incorporated herein by reference). The described oligonucleotides also include probes for detecting amplified sequences of SARS-CoV-2 and target capture oligonucleotides for capturing SARS-CoV-2 target nucleic acids.

[0271] The method provides sensitive and specific detection of SARS-CoV-2 nucleic acids. The method includes performing nucleic acid amplification of a SARS-CoV-2 target region and detecting the amplified product, e.g., by specifically hybridizing the amplified product with a nucleic acid detection probe that provides a signal indicative of the presence of SARS-CoV-2 in the sample. The amplification step includes contacting the sample with one or more amplification oligomers specific for a target sequence in the SARS-CoV-2 target nucleic acid to generate an amplified product if SARS-CoV-2 nucleic acid is present in the sample. Amplification involves synthesizing additional copies of the target sequence or its complement by using at least one nucleic acid polymerase and at least one amplification oligomer to generate copies from a template strand (e.g., by using the template strand to extend the sequence from a primer). In some embodiments, detecting the amplified product comprises a hybridization step that involves contacting the amplified product with at least one detector probe oligomer specific to the sequence amplified by the selected amplification oligomer, e.g., a sequence contained in the target sequence to which the amplification oligomer hybridizes and the target sequences flanking it.

[0272] Amplification oligomer combinations and probes for amplifying and detecting SARS-CoV-2 are described. In some embodiments, the amplification oligomer combination includes at least two primers configured to amplify a target nucleic acid sequence. In some embodiments, the amplification oligomer combination includes a first primer (e.g., an NT7 primer or a forward primer) and a second primer (e.g., a promoter primer, a T7 primer, or a reverse primer). It will be understood by those skilled in the art that at least one primer contains a sequence that hybridizes to the target in a sense orientation and at least one primer contains a sequence that hybridizes to the target in an antisense orientation relative to the target nucleic acid. The primers are configured so that the antisense primer is located downstream of the sense primer, and the sense primer is located downstream of the antisense primer (i.e., at least two primers are located on either side of the target region to be amplified and are configured to prime polymerization in the direction of the other primer, thereby amplifying the region between the two primers). In some embodiments, compositions, formulations, reaction mixtures and kits are described that include combinations of amplification oligomers and corresponding probes for amplifying and detecting at least two regions of SARS-CoV-2.

[0273] Amplification and / or detection methods according to the present disclosure may further include obtaining a sample for subsequent steps of the method. In some embodiments, "obtaining" a sample to be used includes, for example, receiving the sample at a laboratory or other location where one or more steps of the method are performed, and / or retrieving the sample from a location within the facility where one or more steps of the method are performed (e.g., from a repository or other depository).

[0274] In some embodiments, the compositions, formulations, kits, and methods provide exponential amplification of a SARS-CoV-2 target sequence in an in vitro amplification reaction that utilizes at least two amplification oligomers flanking the target region to be amplified. The amplification reaction can be cyclic or isothermal.

[0275] In some embodiments, the composition is configured to specifically hybridize to a SARS-CoV-2 nucleic acid that has minimal cross-reactivity to one or more non-SARS-CoV-2 pathogens. In some embodiments, the composition is configured to specifically hybridize to a SARS-CoV-2 nucleic acid that has minimal cross-reactivity to one or more non-SARS-CoV-2 pathogens listed in Table 5 (see Example 4 below). In other, non-mutually exclusive embodiments, the composition is configured to specifically hybridize to a SARS-CoV-2 nucleic acid that has minimal cross-reactivity to one or more coronavirus pathogens selected from OC43, HKU1, NL63, and 229E. In some embodiments, the composition is part of a multiplex system further including components and methods for detecting one or more pathogens that are not SARS-CoV-2 (e.g., one or more non-SARS-CoV-2 pathogens, such as one or more of the pathogens listed in Table 5).

[0276] In some aspects, methods are provided for utilizing the oligomers or combinations of oligomers described herein. Any method disclosed herein should be further understood as a disclosure of the corresponding use of the materials involved in the method for the purposes of the method. Any method disclosed herein should also be understood as a disclosure of the oligonucleotides and compositions used in the method. Any of the oligomers containing sequences that hybridize to a target of SARS-CoV-2, and any combinations (e.g., kits and compositions) comprising such oligomers, should be further understood as disclosed for use in detecting or quantifying SARS-CoV-2, and for use in preparing compositions for detecting or quantifying SARS-CoV-2.

[0277] Broadly speaking, the method may include one or more of the following components: target capture, in which SARS-CoV-2 nucleic acid (e.g., from a sample such as a clinical sample), if present, is annealed to a target capture oligonucleotide; isolation, e.g., washing, to remove material not associated with the target capture oligonucleotide; amplification; and amplicon detection, e.g., amplicon quantification, which may be performed in real time with amplification. In some embodiments, the method includes each of the foregoing steps. In some embodiments, the amplification includes exponential amplification, optionally with a preceding linear amplification step (e.g., two-phase amplification). In some embodiments, the amplification includes exponential amplification and amplicon detection. In some embodiments, the method includes any two of the components listed above. In some embodiments, the method includes any two components listed adjacently above, e.g., washing and amplification, or amplification and detection.

[0278] In some embodiments, amplifying the SARS-CoV-2 target sequence utilizes an in vitro amplification reaction that uses at least two amplification oligomers flanking the target region to be amplified. Oligomer combinations particularly suitable for amplifying SARS-CoV-2 target regions are described herein. In some embodiments, the oligomer combination includes at least two amplification oligomers for amplifying a first target region of a SARS-CoV-2 target nucleic acid corresponding to a region from about nucleotide 1141 to about nucleotide 1235 of SEQ ID NO:1. The at least two amplification oligomers for amplifying the first target region of a SARS-CoV-2 target nucleic acid can include a first SARS-CoV-2-specific amplification oligomer and a second SARS-CoV-2-specific amplification oligomer. In some embodiments, the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, the sequence that hybridizes to this target being 18-27 contiguous nucleotides in length, contained in SEQ ID NO:26, and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; and the second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target, the sequence that hybridizes to this target being 18-23 contiguous nucleotides in length, contained in SEQ ID NO:85, and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:86, SEQ ID NO:87, or SEQ ID NO:88. In some embodiments, the oligomer combination includes at least two amplification oligomers for amplifying a second target region of a SARS-CoV-2 target nucleic acid corresponding to a region from about nucleotide 4642 to about nucleotide 4744 of SEQ ID NO: 1. The minimum two amplification oligomers for amplifying the second target region of a SARS-CoV-2 target nucleic acid can include a first SARS-CoV-2-specific amplification oligomer and a second SARS-CoV-2-specific amplification oligomer.In some such embodiments, the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, the sequence that hybridizes to this target being 20-23 contiguous nucleotides in length and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; and the second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target being 16-27 contiguous nucleotides in length and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:112, or SEQ ID NO:113. In some embodiments, both the first SARS-CoV-2 target region and the second SARS-CoV-2 target region are amplified in a multiplex format. In some embodiments, one or both of the first SARS-CoV-2 target region and the second SARS-CoV-2 target region and the internal control target region are amplified in a multiplex format. Exemplary amplification oligomers for amplifying the first and / or second SARS-CoV-2 target region are listed in Table 39 below, and specific combinations of first and second amplification oligomers for each of the first and second target regions are provided herein (see the Embodiments section above, and Examples 2-13 below). SARS-CoV-2 Region 1 Primers and Probes

[0279] In some embodiments, the first SARS-CoV-2 Region 1-specific primer (amplification oligomer, e.g., forward primer, non-T7 primer, or NT7 primer) comprises a target-hybridizing region 18-27 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO:26, or its RNA equivalent, or a DNA / RNA chimeric form thereof, and contains the nucleotide sequence of SEQ ID NO:27 or 28, or its RNA equivalent, or a DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 1-specific primer comprises a target-hybridizing region 18-27 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO:26, or its RNA equivalent, or a DNA / RNA chimeric form thereof, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or 28, or its RNA equivalent, or a DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 1-specific primer comprises the nucleotide sequence of SEQ ID NO: 12, 18, 19, 20, or 21, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 1-specific primer consists essentially of the nucleotide sequence of SEQ ID NO: 12, 18, 19, 20, or 21, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 1-specific primer consists of the nucleotide sequence of SEQ ID NO: 12, 18, 19, 20, or 21, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 1-specific primer comprises a nucleotide sequence having up to 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 12, 18, 19, 20, or 21, or an RNA equivalent or DNA / RNA chimeric form thereof.In some embodiments, the first SARS-CoV-2 Region 1-specific primer comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 12, 18, 19, 20, or 21, or an RNA equivalent or DNA / RNA chimeric form thereof. The first SARS-CoV-2 Region 1-specific primer may have one or more modified nucleotides or nucleotide analogs. In some embodiments, the first SARS-CoV-2 Region 1-specific primer is an NT7 primer.

[0280] In some embodiments, the second SARS-CoV-2 Region 1-specific primer (reverse primer, promoter primer, or T7 primer) comprises a target-hybridizing region 18 to 23 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO: 85, or its RNA equivalent, or a DNA / RNA chimeric form thereof, and contains the nucleotide sequence of SEQ ID NO: 86, 87, or 88, or its RNA equivalent, or a DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 1-specific primer comprises a target-hybridizing region 18 to 23 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO: 85, or its RNA equivalent, or a DNA / RNA chimeric form thereof, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, 87, 88, or its RNA equivalent, or a DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 1-specific primer comprises the nucleotide sequence of SEQ ID NO: 13, 71, 72, 73, 74, or 75, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 1-specific primer consists essentially of the nucleotide sequence of SEQ ID NO: 13, 71, 72, 73, 74, or 75, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 1-specific primer consists of the nucleotide sequence of SEQ ID NO: 13, 71, 72, 73, 74, or 75, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 1-specific primer comprises a nucleotide sequence having at most 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 13, 71, 72, 73, 74, or 75, or an RNA equivalent or DNA / RNA chimeric form thereof.In some embodiments, the second SARS-CoV-2 Region 1-specific primer comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 13, 71, 72, 73, 74, or 75, or an RNA equivalent or DNA / RNA chimeric form thereof. The second SARS-CoV-2 Region 1-specific primer may have one or more modified nucleotides or nucleotide analogs. In some embodiments, two different second SARS-CoV-2 Region 1 primers are used in the TMA reaction.

[0281] In some embodiments, the second SARS-CoV-2 Region 1 primer comprises a SARS-CoV-2 Region 1 promoter primer. In some embodiments, the SARS-CoV-2 Region 1 promoter primer comprises the nucleotide sequence of SEQ ID NO: 57, 58, 59, 60, 61, or 62, or an RNA equivalent or a DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 1 promoter primer consists essentially of the nucleotide sequence of SEQ ID NO: 57, 58, 59, 60, 61, or 62, or an RNA equivalent or a DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 1 promoter primer consists of the nucleotide sequence of SEQ ID NO: 57, 58, 59, 60, 61, or 62, or an RNA equivalent or a DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 1 promoter primer comprises a nucleotide sequence having at most 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 57, 58, 59, 60, 61, or 62, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 1 promoter primer comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 57, 58, 59, 60, 61, or 62, or an RNA equivalent or DNA / RNA chimeric form thereof. The SARS-CoV-2 Region 1 promoter primer may have one or more modified nucleotides or nucleotide analogs. In some embodiments, two different SARS-CoV-2 Region 1 promoter primers are used in the TMA reaction.

[0282] In some embodiments, the SARS-CoV-2 Region 1 probe comprises a target-hybridizing region of 19-27 nucleobases in length, the target-hybridizing region contained in SEQ ID NO: 50 or its RNA equivalent, DNA / RNA chimeric form, and / or complement, and contains the nucleotide sequence of SEQ ID NO: 51, 52, 53, or 114, or its RNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 1 probe comprises a target-hybridizing region of 19-27 nucleobases in length, the target-hybridizing region contained in SEQ ID NO: 50 or its RNA equivalent, DNA / RNA chimeric form, and / or complement, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 51, 52, 53, or 114, or its RNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 1 probe comprises the nucleotide sequence of SEQ ID NO: 14, 32-41, 103, 104, 107 or 108, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 Region 1 probe consists essentially of the nucleotide sequence of SEQ ID NO: 14, 32-41, 103, 104, 107 or 108, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 Region 1 probe consists of the nucleotide sequence of SEQ ID NO: 14, 32-41, 103, 104, 107 or 108, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 Region 1 probe comprises a nucleotide sequence having up to 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 14, 32-41, 103, 104, 107, or 108, or its DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement.In some embodiments, a SARS-CoV-2 Region 1 probe comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NOs: 14, 32-41, 103, 104, 107, or 108, or its DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement. A SARS-CoV-2 Region 1 probe may have one or more modified nucleotides or nucleotide analogs.

[0283] In some embodiments, the SARS-CoV-2 Region 1 probe comprises a SARS-CoV-2 Region 1 Torch. In some embodiments, the SARS-CoV-2 Region 1 Torch comprises the nucleotide sequence of SEQ ID NO: 103 or 104, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 1 Torch consists essentially of the nucleotide sequence of SEQ ID NO: 103 or 104, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 1 Torch consists of the nucleotide sequence of SEQ ID NO: 103 or 104, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 1 Torch comprises a nucleotide sequence having at most 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 103 or 104, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 1 Torch comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 103 or 104, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. The SARS-CoV-2 Region 1 Torch may have one or more modified nucleotides or nucleotide analogs.

[0284] The SARS-CoV-2 first amplification oligomer combination comprises at least one first primer and at least one second primer described above. In some embodiments, the SARS-CoV-2 Region 1 first amplification oligomer combination comprises at least one first primer and at least two different second primers. SARS-CoV-2 Region 2 Primers and Probes

[0285] In some embodiments, the first SARS-CoV-2 Region 2-specific primer ((amplification oligomer, e.g., forward primer, non-T7 primer, or NT7 primer)) comprises a target-hybridizing region 20-23 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO:29 or its RNA equivalent or DNA / RNA chimeric form, and contains the nucleotide sequence of SEQ ID NO:30, 31, or 111, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 2-specific primer comprises a target-hybridizing region 20-23 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO:29 or its RNA equivalent or DNA / RNA chimeric form, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, 31, or 111, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 2-specific primer comprises the nucleotide sequence of SEQ ID NO: 15, 22, 23, 24, or 25, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 2-specific primer consists essentially of the nucleotide sequence of SEQ ID NO: 15, 22, 23, 24, or 25, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 2-specific primer consists of the nucleotide sequence of SEQ ID NO: 15, 22, 23, 24, or 25, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the first SARS-CoV-2 Region 2-specific primer comprises a nucleotide sequence having up to 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 15, 22, 23, 24, or 25, or an RNA equivalent or DNA / RNA chimeric form thereof.In some embodiments, the first SARS-CoV-2 Region 2-specific primer comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 15, 22, 23, 24, or 25, or an RNA equivalent or DNA / RNA chimeric form thereof. The first SARS-CoV-2 Region 2-specific primer may have one or more modified nucleotides or nucleotide analogs. In some embodiments, the SARS-CoV-2 Region 2 first primer is an NT7 primer.

[0286] In some embodiments, the second SARS-CoV-2 Region 2-specific primer (reverse primer, promoter primer, or T7 primer) comprises a target-hybridizing region 16 to 27 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO: 89 or its RNA equivalent or DNA / RNA chimeric form, and contains the nucleotide sequence of SEQ ID NO: 90, 1112, or 113, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 2-specific primer comprises a target-hybridizing region 16 to 27 nucleobases in length, wherein the target-hybridizing region is contained in SEQ ID NO: 89 or its RNA equivalent or DNA / RNA chimeric form, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, 1112, or 113, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 2-specific primer comprises the nucleotide sequence of SEQ ID NO: 16, 76, 77, 79, 80, 81, 82, 83, or 84, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 2-specific primer consists essentially of the nucleotide sequence of SEQ ID NO: 16, 76, 77, 79, 80, 81, 82, 83, or 84, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 Region 2-specific primer consists of the nucleotide sequence of SEQ ID NO: 16, 76, 77, 79, 80, 81, 82, 83, or 84, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the second SARS-CoV-2 region 2-specific primer comprises a nucleotide sequence having up to 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 16, 76, 77, 79, 80, 81, 82, 83, or 84, or an RNA equivalent or a DNA / RNA chimeric form thereof.In some embodiments, the second SARS-CoV-2 Region 2-specific primer comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 16, 76, 77, 79, 80, 81, 82, 83, or 84, or an RNA equivalent or DNA / RNA chimeric form thereof. The second SARS-CoV-2 Region 2-specific primer may have one or more modified nucleotides or nucleotide analogs. In some embodiments, two different second SARS-CoV-2 Region 2-specific primers are used in the TMA reaction.

[0287] In some embodiments, the second SARS-CoV-2 Region 2-specific primer comprises a SARS-CoV-2 Region 2 promoter primer. In some embodiments, the SARS-CoV-2 Region 2 promoter primer comprises the nucleotide sequence of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, or 78, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 2 promoter primer consists essentially of the nucleotide sequence of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, or 78, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 2 promoter primer consists of the nucleotide sequence of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, or 78, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 2 promoter primer comprises a nucleotide sequence having at most 0, 1, 2, 3, 4, or 5 mismatches to the nucleotide sequence of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, or 78, or an RNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 Region 2 promoter primer comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, or 78, or an RNA equivalent or DNA / RNA chimeric form thereof. The SARS-CoV-2 Region 2 promoter primer may have one or more modified nucleotides or nucleotide analogs. In some embodiments, two different SARS-CoV-2 Region 2 promoter primers are used in the TMA reaction.

[0288] In some embodiments, the SARS-CoV-2 Region 2 probe comprises a target-hybridizing region 25-29 nucleobases in length, the target-hybridizing region contained in SEQ ID NO: 54 or its RNA equivalent, DNA / RNA chimeric form, and / or complement, and contains the nucleotide sequence of SEQ ID NO: 55, 56, or 115, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 2 probe comprises a target-hybridizing region 25-29 nucleobases in length, the target-hybridizing region contained in SEQ ID NO: 54 or its RNA equivalent, DNA / RNA chimeric form, and / or complement, and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 55, 56, or 115, or its RNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 2 probe comprises the nucleotide sequence of SEQ ID NO: 17, 43-48, 105, 106, 109 or 110, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 Region 2 probe consists essentially of the nucleotide sequence of SEQ ID NO: 17, 43-48, 105, 106, 109 or 110, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 Region 2 probe consists of the nucleotide sequence of SEQ ID NO: 17, 43-48, 105, 106, 109 or 110, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 Region 2 probe comprises a nucleotide sequence having up to 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 17, 43-48, 105, 106, 109, or 110, or its DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement.In some embodiments, a SARS-CoV-2 Region 2 probe comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NOs: 17, 43-48, 105, 106, 109, or 110, or a DNA or RNA equivalent, DNA / RNA chimeric form, and / or complement thereof. A SARS-CoV-2 Region 2 probe may have one or more modified nucleotides or nucleotide analogs.

[0289] In some embodiments, the SARS-CoV-2 Region 2 probe comprises a SARS-CoV-2 Region 2 Torch. In some embodiments, the SARS-CoV-2 Region 2 Torch comprises the nucleotide sequence of SEQ ID NO: 105 or 106, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 2 Torch consists essentially of the nucleotide sequence of SEQ ID NO: 105 or 106, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 2 Torch consists of the nucleotide sequence of SEQ ID NO: 105 or 106, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 2 Torch comprises a nucleotide sequence having at most 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 105 or 106, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. In some embodiments, the SARS-CoV-2 Region 2 Torch comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 105 or 106, or its DNA equivalent, DNA / RNA chimeric form, and / or complement. The SARS-CoV-2 Region 2 Torch may have one or more modified nucleotides or nucleotide analogs.

[0290] The primer set for SARS-CoV-2 region 2 comprises at least one first primer and at least one second primer described above. In some embodiments, the primer set for SARS-CoV-2 region 2 comprises at least one first primer and at least two different second primers.

[0291] In some embodiments, the method further includes a step of purifying the SARS-CoV-2 target nucleic acid in the sample from other components, e.g., before amplification, e.g., before, simultaneously with, or after the capture step. Such purification may include methods to separate and / or concentrate organisms contained in the sample from other sample components, or methods to remove or degrade non-nucleic acid sample components, e.g., proteins, carbohydrates, salts, lipids, etc. In certain embodiments, the target nucleic acid is specifically or nonspecifically captured and separated from other sample components. Nonspecific target capture methods may include selective precipitation of nucleic acids from a substantially aqueous mixture, attachment of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture containing SARS-CoV-2 nucleic acids and other sample components.

[0292] Target capture is generally performed in a solution-phase mixture containing one or more target capture oligonucleotides (TCOs) that hybridize to SARS-CoV-2 target sequences under hybridization conditions. In some embodiments, the TCOs comprise an immobilized capture probe binding region. In some embodiments involving TCOs with immobilized capture probe binding regions, the SARS-CoV-2 target:TCO complex is captured by adjusting the hybridization conditions so that the immobilized capture probe binding region hybridizes to the immobilized probe. In some embodiments, the immobilized probe is attached to a particulate solid support, such as a paramagnetic bead. Selective and non-selective target capture methods are also described, for example, in U.S. Pat. No. 6,110,678 and International Patent Application Publication No. WO 2008 / 016988 (each incorporated herein by reference). Exemplary SARS-CoV-2-specific target capture oligonucleotides are listed in Table 39 below and further illustrated in the Examples section above, as well as in Examples 10 and 11 below.

[0293] Capture can be followed by isolation, where, for example, the complex on the solid support is separated from other sample components. Isolation can be accomplished by any suitable technique, for example, by washing the support associated with the SARS-CoV-2 target sequence one or more times (e.g., two or three times) to remove other sample components and / or unbound oligomers. In embodiments using a particulate solid support, e.g., paramagnetic beads, the particles associated with the SARS-CoV-2 target can be suspended in a wash solution and recovered from the wash solution, in some embodiments, by using magnetic attraction. To limit the number of handling steps, the SARS-CoV-2 target nucleic acid can be amplified by simply mixing the target sequence in the complex on the support with amplification oligomers and proceeding with the amplification step.

[0294] Sample preparation can also include pooling multiple samples into a single pooled batch. Preferably, for pooling, aliquots of each sample are pooled into a larger batch. The larger batch of pooled samples can be from a plurality of samples, the plurality of samples being from 2 to about 200 individual samples. SARS-CoV-2 target capture oligonucleotides

[0295] In some embodiments, the SARS-CoV-2 target capture oligonucleotide comprises a target-hybridizing region 21-24 nucleobases in length, wherein the target-hybridizing region comprises the nucleotide sequence of SEQ ID NO: 97, 98, 99, 100, 101, or 104, or a DNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 target capture oligonucleotide comprises a target-hybridizing region 21-24 nucleobases in length, wherein the target-hybridizing region comprises a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 97, 98, 99, 100, 101, or 104, or a DNA equivalent, DNA / RNA chimeric form, and / or complement thereof. In some embodiments, the SARS-CoV-2 target capture oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 97, 98, 99, 100, 101, or 104, or a DNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the region of the SARS-CoV-2 target capture oligonucleotide that hybridizes to its target consists essentially of the nucleotide sequence of SEQ ID NO: 97, 98, 99, 100, 101, or 104, or a DNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the region of the SARS-CoV-2 target capture oligonucleotide that hybridizes to its target consists of the nucleotide sequence of SEQ ID NO: 97, 98, 99, 100, 101, or 104, or a DNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the SARS-CoV-2 target capture oligonucleotide comprises a nucleotide sequence having up to 0, 1, 2, 3, 4, or 5 mismatches with the nucleotide sequence of SEQ ID NO: 228, 97, 98, 99, 100, 101, or 104, or a DNA equivalent or DNA / RNA chimeric form thereof.In some embodiments, the SARS-CoV-2 target capture oligonucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 97, 98, 99, 100, 101, or 104, or a DNA equivalent or DNA / RNA chimeric form thereof. The SARS-CoV-2 target capture oligonucleotide may have one or more modified nucleotides or nucleotide analogs.

[0296] In some embodiments, the SARS-CoV-2 region 1 target capture oligonucleotide is T n A mIn some embodiments, the influenza target capture oligonucleotide is linked to the sequence: n is an integer between 0 and 3, and m is an integer between 14 and 50. In some embodiments, n is 3 and m is 30. In some embodiments, the influenza target capture oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 91, 92, 93, 94, 95, or 96, or a DNA equivalent or a DNA / RNA chimeric form thereof. In some embodiments, the influenza target capture oligonucleotide consists essentially of the nucleotide sequence of SEQ ID NO: 91, 92, 93, 94, 95, or 96, or a DNA equivalent or a DNA / RNA chimeric form thereof. In some embodiments, the influenza target capture oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 91, 92, 93, 94, 95, or 96, or a DNA equivalent or a DNA / RNA chimeric form thereof. In some embodiments, the influenza target capture oligonucleotide comprises a nucleotide sequence having at most 0, 1, 2, 3, 4, or 5 mismatches to the nucleotide sequence of SEQ ID NO: 91, 92, 93, 94, 95, or 96, or a DNA equivalent or DNA / RNA chimeric form thereof. In some embodiments, the influenza target capture oligonucleotide comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% homology to the nucleotide sequence of SEQ ID NO: 91, 92, 93, 94, 95, or 96, or a DNA equivalent or DNA / RNA chimeric form thereof. The SARS-CoV-2 region 1 promoter primer may have one or more modified nucleotides or nucleotide analogs.

[0297] In some embodiments, the composition, formulation, or kit contains one or more positive controls. The positive control can be a nucleic acid containing a target sequence of SARS-CoV-2 region 1, SARS-CoV-2 region 2, SARS-CoV-2 region 3, influenza A region 1, influenza A region 2, influenza B region 1, or influenza B region 2. The positive control can be, but is not limited to, DNA, RNA, a plasmid, or an in vitro transcript.

[0298] The detection step can be performed using any of a variety of known techniques for detecting signals specifically associated with the amplified target sequence, such as by hybridizing the amplification product with a labeled detector probe and detecting a signal from the labeled probe (including, in some embodiments, from a label released from the probe after hybridization). In some embodiments, the labeled probe includes a second moiety, e.g., a quencher or other moiety that interacts with the label, such as a fluorophore as discussed above. The detection step can also provide additional information about the amplified sequence, such as all or part of its nucleic acid sequence. Detection can be performed after the amplification reaction is complete, or can be performed simultaneously with the amplification of the target region, e.g., in real time. In some embodiments, the amplified product is detected near or at the end of the amplification step. In embodiments, a linear detector probe is used to obtain a signal indicating hybridization of the probe to the amplified product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is performed by chemiluminescence using a luminometer. (See, e.g., International Patent Application Publication No. WO 89 / 002476, incorporated herein by reference.) In some embodiments, detection is performed in real time. In some embodiments, the detector probe is a hairpin probe. The hairpin probe can be, but is not limited to, a molecular beacon, a molecular torch, or a hybridization switch probe (e.g., a dual-labeled hairpin probe containing both a fluorescent label and a quenching moiety) that is labeled with a reporter moiety that is detected when the probe binds to the amplified product. In some embodiments, the hairpin probe is used for real-time detection. In some embodiments, the detector probe is a linear oligomer, such as an oligomer labeled with both a fluorophore and a quenching moiety (e.g., a TaqMan probe).Such probe can comprise the sequence that hybridizes to target and the sequence that does not hybridize to target.Various forms of such probe have been previously described (see, for example, U.S. Patent Nos. 5,210,015, 5,487,972, 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274 and 6,361,945, and U.S. Patent Application Publication Nos. 20060068417A1 and 20060194240A1, each of which is incorporated herein by reference).In some embodiments, hairpin probe is used for real-time detection.In some embodiments, linear oligomer is used for real-time detection. Exemplary SARS-CoV-2-specific detector probe oligomers are listed below in Table 39 and further illustrated (e.g., including their use in combination with first and second SARS-CoV-2-specific amplification oligomers for detection of SARS-CoV-2 target nucleic acids) in the Examples section above and in Examples 2-13 below.

[0299] In some embodiments, detection is performed at time intervals. Detection can be performed by measuring fluorescence at regular time intervals. The time intervals can be, but are not limited to, 1 to 60 seconds, 1 to 120 seconds, 1 to 180 seconds, 1 to 240 seconds, or 1 to 300 seconds. In some embodiments, the time intervals are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds. For detection performed at regular time intervals, each interval is referred to as a cycle. Detection can be performed over 20 to 240 cycles, 30 to 210 cycles, 40 to 180 cycles, 50 to 150 cycles, or 60 to 120 cycles. For example, detection can be performed every 30 seconds for 120 cycles over 60 minutes. Detection can be performed at the start or end of a cycle. Detection can also be performed continuously. Multiplexed amplification and / or detection

[0300] A "multiplex" amplification reaction, e.g., an isothermal amplification reaction, is characterized in that two or more different amplification products or amplicons are generated by using two or more combinations of amplification oligomers in the same amplification reaction. A multiplex amplification reaction contains two or more combinations of amplification oligomers (e.g., two or more sets of a first (NT7) primer and a second (promoter) primer) to amplify two target sequences. A multiplex amplification system includes a composition, formulation, reaction mix, or kit for performing a multiplex amplification and / or detection reaction. In addition to the combination of amplification oligomers, the multiplex amplification system may further include two or more probes and / or two or more TCOs for detecting the corresponding amplicons.

[0301] In some embodiments, a multiplex amplification system for amplifying and / or detecting the presence or absence of SARS-CoV-2 comprises a combination of two or more amplification oligomers selected from the group consisting of: (a) a first amplification oligomer combination for amplifying a target sequence within a first region (region 1) of SARS-CoV-2; (b) a second amplification oligomer combination for amplifying a target sequence within a second region (region 2) of SARS-CoV-2; (c) a control amplification oligomer combination for amplifying a target sequence in a control target nucleic acid; and (d) A combination of one or more amplification oligomers for amplifying another target sequence in one or more non-SARS-CoV-2 microorganisms (e.g., non-SARS-CoV-2 pathogens). Any of the above multiplex amplification systems may further include probes for detecting the amplicons generated by the amplification oligomer combination and / or one or more TCOs for capturing the SARS-CoV-2 target nucleic acid or the internal control target nucleic acid.

[0302] Probes utilized in multiplex amplification systems can be labeled to distinguish any one probe species (or probes for detecting one species) from other probe species (or probes for detecting other species) in a multiplex detection assay. In some embodiments, probes for detecting one or both SARS-CoV-2 amplicons can be distinguished from probes for detecting an internal control amplicon. In a multiplex amplification reaction having a combination of amplification oligomers for amplifying two different regions of a SARS-CoV-2 target nucleic acid, the probes for detecting the SARS-CoV-2 amplicon can utilize the same or different labels. Labels can be, but are not limited to, fluorophores and fluorophore / quencher combinations (i.e., FRET hybridization probes as described in Matthews and Kricka, Analytical Biochemistry, vol. 169 (1988), pp: 1-25).

[0303] Compositions, formulations, kits, and methods for detecting SARS-CoV-2 nucleic acids can optionally include oligonucleotides for amplification and / or detection of a non-SARS-CoV-2 internal control (IC) nucleic acid. The control is amplified and detected in the same or parallel assay reaction mixture by using amplification and detection oligomers specific to the IC sequence. The IC nucleic acid sequence can be, for example, a DNA plasmid, an RNA template sequence (e.g., an in vitro transcript), or a synthetic nucleic acid spiked into the sample. Alternatively, the IC nucleic acid sequence can be a cellular component, which can be derived from an exogenous cellular source or endogenous to the specimen. In these cases, the internal control nucleic acid is co-amplified with the SARS-CoV-2 nucleic acid in the amplification reaction mixture. The internal control amplification product and the SARS-CoV-2 target sequence amplification product can be detected independently.

[0304] In certain embodiments, amplification and detection of a signal from an amplified IC sequence demonstrates that the assay reagents, equipment, conditions, and execution of the assay steps were functional and properly used in the assay if no signal was obtained for the intended target SARS-CoV-2 nucleic acid (e.g., a sample that tested negative for SARS-CoV-2). If a quantitative result is desired, the IC can also be used as an internal calibrator for the assay; i.e., the signal obtained from IC amplification and detection is used to set parameters used in an algorithm for quantifying the amount of SARS-CoV-2 nucleic acid in a sample based on the signal obtained for the amplified SARS-CoV-2 target sequence. ICs are also useful for monitoring the integrity of one or more steps in an assay. Primers and probes for the IC target sequence can be constructed and synthesized using any known method, provided that the primers and probes function for amplification of the IC target sequence and detection of the amplified IC sequence using substantially the same assay conditions as those used to amplify and detect the SARS-CoV-2 target sequence. In some embodiments that include a target capture-based purification step, a target capture probe specific for the IC target is included in the assay for the target capture step, and thus the IC is treated in the assay in a manner similar to that for the SARS-CoV-2 analyte of interest in all of the assay steps.

[0305] Formulations and kits for determining the presence or absence of SARS-CoV-2 in a sample are described. In some embodiments, the formulation or kit includes at least one primer set (amplification oligomer combination) for amplifying a target sequence in SARS-CoV-2. In some embodiments, the formulation or kit includes at least two primer sets (amplification oligomer combinations) for amplifying two different target sequences in SARS-CoV-2. Any of the formulations or compositions described herein can be provided as an aqueous solution. In some embodiments, the formulation is an aqueous formulation including (1) at least two SARS-CoV-2-specific amplification oligomers for amplification of SARS-CoV-2 target regions described herein and (2) an organic buffer. In some embodiments, the formulation is an aqueous formulation including (1) at least four SARS-CoV-2-specific amplification oligomers for amplification of two different SARS-CoV-2 target regions described herein and (2) an organic buffer. In some embodiments, the formulation or kit further includes a probe for detecting amplicons generated by the amplification oligomers. Kits or aqueous formulations for amplifying SARS-CoV-2 nucleic acids may include one or more additional components, such as a DNA polymerase enzyme, reverse transcriptase, or detector probe oligomer. In some embodiments, the formulation is an aqueous formulation comprising (1) a SARS-CoV-2-specific detector probe oligomer described herein and (2) an organic buffer. Kits or aqueous formulations containing one or more detector probe oligomers may include one or more additional components, such as a surfactant, a DNA polymerase enzyme, a reverse transcriptase, or at least one amplification oligomer. Particularly suitable surfactants include, for example, polyethylene glycol mono[4-(1,1,3,3-tetramethylbutyl)phenyl] ether and polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, or polysorbate 60).In some embodiments, the surfactant in the aqueous detection probe formulation is a non-linear surfactant (i.e., a surfactant having a branched chain structure), such as a polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate 20, polysorbate 40, or polysorbate 60) or digitonin. The above-described kit or aqueous formulation for amplifying or detecting SARS-CoV-2 nucleic acids may further include a bulking agent, such as trehalose, raffinose, or a combination thereof. In some embodiments, the above-described aqueous formulation contains an inorganic salt, such as magnesium, potassium, or sodium; in some such variations, the concentration of the inorganic salt is 4 mM or less. A particularly suitable organic buffer for the above-described aqueous formulation is Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol). In some embodiments, the kit may further contain one or more of a target capture reagent (TCR), an amplification (AMP) reagent, and a promoter reagent. The kit may further contain one or more of a buffer, an enzyme reagent, an RNA polymerase, dNTPs, NTPs, a sample transport medium, a target capture wash solution, a target enhancer reagent, and a reconstitution reagent.

[0306] Any of the aqueous formulations described can be aliquoted and dried (e.g., lyophilized) into, for example, vials, ampoules, multiwell plates, or other containers according to procedures known in the art. The dried formulations can be used for long-term storage. The dried product generally appears as a powder or cake. The container is then generally sealed. In some embodiments, aliquots of the dried formulation are transferred to wells of a multiwell plate, which is then sealed. Methods for preparing such dried formulations from aqueous formulations, and dried formulations prepared by such methods, are further aspects of the present disclosure. In some embodiments, dried formulations are provided that allow for reconstitution into the aqueous formulations described herein. Dry formulations for amplification or detection of SARS-CoV-2 nucleic acids generally contain a bulking agent, such as trehalose, raffinose, or a combination thereof, in addition to one or more amplification oligomers and / or detection probes described herein. In some embodiments, the formulation further comprises an inorganic salt. In some embodiments, the percentage of inorganic salt mass to dry formulation mass is 0.249% or less, 0.222% or less, or 0.195% or less. Methods of preparing aqueous formulations from the lyophilized formulations described herein are also encompassed by the present disclosure; such methods generally include dissolving the dried formulation in a suitable diluent (e.g., an organic buffer or water) to obtain a reconstituted formulation.

[0307] Also provided are reaction mixtures for determining the presence or absence of a SARS-CoV-2 target nucleic acid in a sample. Reaction mixtures according to the present disclosure include one or both of: (1) a combination of oligomers described herein for amplifying a SARS-CoV-2 target nucleic acid; and (2) one or more detector probe oligomers described herein for determining the presence or absence of a SARS-CoV-2 amplification product. The reaction mixture may further include one or more additional components, such as a TCO or nonspecific capture probe, such as a poly-(k) capture probe as described in US 2013 / 0209992, which is incorporated herein by reference. In the case of an amplification reaction mixture, the reaction mixture generally includes other reagents suitable for in vitro amplification, such as a buffer, a salt solution, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP, and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or a reverse transcriptase and / or an RNA polymerase), and generally includes test sample components that may or may not contain a SARS-CoV-2 target nucleic acid. The reaction mixture may include amplification oligomers for only one target region of the SARS-CoV-2 genome, or may include amplification oligomers for multiple SARS-CoV-2 target regions. Additionally, in the case of a reaction mixture that includes a detector probe with a combination of amplification oligomers, the selection of amplification oligomers and detector probe oligomers for the reaction mixture is related by a common target region (i.e., the reaction mixture includes a probe that binds to a sequence that can be amplified by the combination of amplification oligomers in the reaction mixture). In some embodiments, the reaction mixture comprises the aqueous formulation described above. In some embodiments, the reaction mixture is reconstituted with water, a reconstitution reagent, or an organic buffer from the dry formulation described above. In some embodiments, the reaction mixture comprises a TCO and a primer. In some embodiments, the reaction mixture comprises a TCO, a primer set, and a probe.

[0308] Also provided herein are kits for carrying out the methods described herein. Kits according to the present disclosure include one or both of: (1) a combination of oligomers described herein for amplifying a SARS-CoV-2 target nucleic acid; and (2) one or more detector probe oligomers described herein for determining the presence or absence of a SARS-CoV-2 amplification product. The kits may further include several optional components, such as a TCO or capture probe, e.g., a poly-(k) capture probe as described in US2013 / 0209992. Other reagents that may be present in the kit include reagents suitable for performing in vitro amplification, such as buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP; and / or ATP, CTP, GTP, and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or a reverse transcriptase and / or RNA polymerase). The oligomers described herein can be packaged in a variety of different embodiments, and one of skill in the art will understand that the present disclosure encompasses many different kit configurations. For example, a kit may include amplification oligomers for only one target region of the SARS-CoV-2 genome, or may include amplification oligomers for multiple SARS-CoV-2 target regions. Additionally, in kits that include a detector probe along with a combination of amplification oligomers, the selection of amplification oligomers and detector probe oligomers for the kit are linked by a common target region (i.e., the kit includes a probe that binds to a sequence amplifiable by the combination of amplification oligomers in the kit). In some embodiments, the kit further includes a set of instructions for practicing a method according to the present disclosure, which instructions may be associated with a package insert and / or packaging for the kit or its components.

[0309] The compositions, kits, formulations, reaction mixtures, and methods are further illustrated by the following non-limiting examples. [Example]

[0310] Example 1 Real-time PCR amplification and detection of coronaviruses using different combinations of primers and probes This example describes a screening experiment testing primer and probe combinations for real-time PCR amplification and detection of coronaviruses. Reactions are generally set up and performed as presented herein and as follows.

[0311] Several primer and probe mixtures (PPR mixes) were prepared in microcentrifuge tubes containing forward primer, reverse primer, and dual-labeled hairpin detector probe. The internal control PPR mix contained 0.625 μM of each primer and 0.5 μM of probe, while the coronavirus PPR mix contained 1.25 μM of each primer and probe. These PPR mixes also contained 150 mM KCl, 10 mM MgCl2, and were brought to a final volume with 10 mM TRIS. The IC detector probe was labeled with Quasar 705 and Black Hole Quencher 2, and each coronavirus ("CoV") detector probe was labeled with FAM and Black Hole Quencher 1 (all available from BioSearch Technologies, Inc., Novato, CA, or Glen Research, Inc., Sterling, VA).

[0312] An equal volume of the internal reference PPR mix (275 μL) was added to each coronavirus mixture (275 μL) to obtain a 1.25× PPR mix (total volume 550 μL). Amplification and detection reactions were prepared by combining 20 μL of each PPR mix and 5 μL of target nucleic acid (e.g., synthetic target nucleic acid or purified virus) eluate with a 1.25× master mix containing dNTPs, dUTP, Taq polymerase, reverse transcriptase, and RNase inhibitor. Each amplification and detection mix was then overlaid with oil. Synthetic coronavirus target nucleic acid was prepared from a stock concentration to obtain a concentration of 1,000 copies (200 copies / μL) in 5 μL aliquots per reaction. Dilutions were made into sample transport medium (containing lithium lauryl sulfate (LLS), EDTA, and sodium phosphate). Amplification and detection reactions were set up in 12 reactions per condition: six positive reactions for the target nucleic acid and six negative reactions for the target nucleic acid. Negative reactions contained sample transport medium without coronavirus target nucleic acid. Reactions were performed in real time with temperature cycling using the Panther Fusion system (available from Hologic, Inc., Marlborough MA). MyAccess on the Panther Fusion system Data were analyzed using the JMP Tool and JMP Chart Analysis.

[0313] The resulting amplification curves are evaluated for the difference between the Ct and RFU signal of the positive samples and the background RFU of the negative samples.

[0314] Example 2 Linearity and Sensitivity Testing of SARS-CoV-2 Primer and Probe Mixes Individual and combined linearity and sensitivity tests were performed on in vitro transcripts (IVT suspended in sample transport medium (STM) and supplemented with 1,000 HeLa cells / mL). These experiments were performed in two stages. In the first stage, 1,000 copies / reaction of IVT (SEQ ID NOs: 2 and 3) were tested with individual primer and probe sets (see Table 1) and combined oligo sets to gather information about the individual and combined performance characteristics of these primer and probe combinations. In the second stage, the linearity and sensitivity of the combined oligo sets were tested on 1 x 10 7 Seven concentration levels of IVT (SEQ ID NOs: 2 and 3) were tested, ranging from (1E+7) copies / reaction to 10 copies / reaction.

[0315] Two SARS-CoV-2 primer and probe (PPR) mixes were prepared as generally described above in Example 1. These SARS-CoV-2 PPR mixes contained the indicated primers and probes containing analog nucleotides and varying fluorophore / quencher configurations (see Table 1). 705 (Q705); CalRed 610, Black Hole Quencher 1 (BHQ-1); and Black Hole Quencher 2 (BHQ-2) are available from Biosearch Technologies, Inc., Novato, CA. 5MeC is available from Sigma-Aldrich Corp., St. Louis, MO. Propyne dU is available from Glen Research, Sterling, VA. Positive reactions were set up as follows: (i) SARS-CoV-2 PPR Mix 1 and IC PPR Mix; (ii) SARS-CoV-2 PPR Mix 5 and IC PPR Mix; and (iii) SARS-CoV-2 PPR Mix 1, SARS-CoV-2 PPR Mix 5, and IC PPR Mix. Negative reactions contained only IC PPR Mix. Positive and negative reactions were each performed in multiples of 6 for Stage 1 and in multiples of 12 for Stage 2. [Table 1]

[0316] Table 2 shows a summary of the results for the individual and combined primer / probe mixes. IC reactions showed 100% detection in all conditions tested. No issues were observed with the combined oligo sets. Linearity and sensitivity data for the combined PPR mixes listed in (iii) above are summarized in Table 3 and Figure 1. These results show that 100% detection of both IVTs was observed down to 100 cp / rxn. PCR efficiency was high with R values ​​>0.99 and slopes of -3.33. IC showed 100% detection at all dilutions. [Table 2] [Table 3]

[0317] Example 3 Virus sensitivity experiments in the presence of HeLa cells To demonstrate the sensitivity and linearity of the combined PPR mixes containing SARS-CoV-2 PPR Mix 1, SARS-CoV-2 PPR Mix 5, and each of the IC PPR Mixes from Table 1, specific viral strains were tested. Only the IC PPR Mix gave a negative reaction. The viral strain was SARS-associated coronavirus 2, isolate USA-WA1 / 2020 (NR-52281 obtained through BEI Resources, NIAID, NIH). The virus stock concentration was 2.8 x 10 after 6 days at 37°C and 5% CO2. 5 TCID 50 The stock virus was spiked into STM containing 10,000 HeLa cells / mL, yielding 14–1.4 × 10 -4 TCID 50 The virus was diluted to six working concentrations ranging from 1.4 x 10 / mL (Table 4). Positive and negative reactions were performed in triplicate. The results of the viral linearity and sensitivity tests are shown in Table 4, with the corresponding linearity plots shown in Figure 2. These results indicate that 100% detection (3 / 3 replicates) was achieved for the virus strains at 1.4 x 10 / mL. -3 TCID 50 IC was detected under all test conditions. [Table 4]

[0318] Example 4 Specificity and interference experiments (panel and specific MERS / SARS tests) The cross-reactivity of the combined PPR mix was evaluated with 53 microorganisms commonly found during respiratory tract infections. These microorganisms were grouped into 16 panels, as listed in Table 5, and tested using a combined PPR mix containing SARS-CoV-2 PPR Mix 1, SARS-CoV-2 PPR Mix 5, and IC PPR Mix from Table 1. Stock concentrations of each organism were spiked into sample transport medium to form final-concentration microbial samples. The final concentrations of each organism were then pooled into one of 16 different panels, as listed in Table 5. 360 μL of each panel was extracted, and 5 μL of the 360 ​​μL eluate was used in the final PCR reaction. One extraction and three PCR replicates were performed for each panel. IVT (SEQ ID NOs: 2 and 3) at a concentration of 10,000 copies / reaction was used as a positive control. All panels tested were negative for SARS-CoV-2 when using the combined PPR mix. When the combined PPR mix was used, the positive control reactions were positive and the negative control reactions were negative. Specificity results are summarized in Table 6. Interference was also assessed concurrently with the specificity experiments. Reactivity for the corresponding IVT at a concentration of 10,000 copies / reaction was assessed in the presence of the panels listed in Table 5. The results are summarized in Table 6. IVT is detected by all panels tested. None of the microorganisms tested was detected by the SARS-CoV-2 PPR mix (i.e., the SARS-CoV-2 PPR mix did not cross-react with any of the challenge organisms). Similarly, none of the microorganisms interfered with the detection of the SARS-CoV-2 target nucleic acid by the PPR mix. [Table 5-1] [Table 5-2] [Table 5-3] [Table 6]

[0319] Example 5 Isothermal amplification and real-time detection of SARS-CoV-2 using different combinations of primers and probes. This example describes a screening experiment testing several primer and probe combinations for real-time isothermal amplification and detection of SARS-CoV-2. Reactions were generally set up and performed as presented herein and as follows.

[0320] Sample, lysis, and target capture reagents commonly used in isothermal assays include: Sample Transport Reagent: 110 mM lithium lauryl sulfate (LLS), 15 mM NaH2PO4, 15 mM Na2HPO4, 1 mM EDTA, 1 mM EGTA, pH 6.7; Lysis Buffer: 790 mM HEPES, 230 mM succinic acid, 10% (w / v) LLS, and 680 mM LiOH monohydrate; Target Capture Reagent (TCR): 250 mM HEPES, 1.88 M LiCl, 310 mM LiOH, 100 mM EDTA, pH 6.4, and 250 μg / ml paramagnetic particles (0.7-1.05 micrometer particles, Sera-Mag™ MG-CM) covalently attached to (dT)14 oligomers. Washing solution: 10 mM HEPES, 150 mM NaCl, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v / v) ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate, pH 7.5. Amplification reagents commonly used in isothermal assays include the following: Amplification reagent: 125 mM HEPES, 26.7 mM rATP, 33.3 mM rGTP, 5 mM each of rCTP and UTP, 1.33 mM each of dATP, dCTP, dGTP, and dTTP, 8% (w / v) trehalose, pH 7.7, to which primers and probes may be added. Real-time amplification reagent: 11.61 mM Tris base, 14.94 mM Tris-HCl, 28.5 mM MgCl, 23.30 mM KCl, 3.3% glycerol, 0.02% PRO CLIN 300, 0.05 mM zinc acetate dihydrate, 0.76 mM each of dATP, dCTP, dGTP, and dTTP, 6.50 mM each of ATP, CTP, and GTP, 7.50 mM UTP, to which primers and probes may be added. Enzyme reagents commonly used in isothermal assays include the following:Enzyme Reagent: 57.46 mM HEPES, 49.58 mM N-acetyl-L-cysteine, 0.98 mM EDTA free acid, 0.039 mM EDTA disodium dihydrate, 0.10 v / v TRITON® X-100, 49.61 mM KCl, 0.20 v / v glycerol, 0.03 w / v trehalose dihydrate, and approximately 90 U / µl MMLV reverse transcriptase (RT) per reaction and approximately 20 U / µl T7 RNA polymerase per reaction (in this case, 1 U RT incorporates 1 nmol dTTP using 200-400 µM oligo-dT primed polyA template for 10 min at 37°C, and 1 U T7 RNA polymerase incorporates 1 nmol ATP into RNA using a T7 promoter in a DNA template for 1 h at 37°C). Reagents commonly used in probe reactions for detecting AE labels include the following: Probe reagent for acridinium ester (AE)-labeled probes: (a) 100 mM Li succinate, 3% (w / v) LLS, 10 mM mercaptoethanesulfonate (MES), and 3% (w / v) polyvinylpyrrolidone, or (b) a solution of 100 mM Li succinate, 0.1% (w / v) LLS, and 10 mM MES. Hybridization reagent: (Type C) 100 mM succinic acid, 2% (w / v) LLS, 100 mM LiOH, 15 mM aldrithiol-2, 1.2 M LiCl, 20 mM EDTA, and 3.0% (v / v) ethanol, pH 4.7. Selection reagent: 600 mM boric acid, 182.5 mM, for hydrolysis of AE labels on unbound oligomers. NaOH, 1% (v / v) octoxynol (TRITON® X-100), pH 8.5-9.2. Detection reagents for AE labeling are Detection Reagent I: 1 mM nitric acid and 32 mM HO, and Detection Reagent II: 1.5 M NaOH. (See also U.S. Patent Nos. 5,283,174, 5,656,744, 5,658,737, 5,888,779, 6,110,678, and 10,196,674, which describe compositions and methods for target capture, isothermal amplification, and detection reactions.)

[0321] Oligo screening experiments for amplification oligos and dual-labeled hairpin detection probes were performed on an OEM platform (Stratagene Mx3000) using a real-time TMA format. Briefly, amplification reagents containing a T7 promoter primer / non-T7 primer / detection probe combination were added to a multiwell plate in a total volume of 30 μl per well. Then, 10 μl (for a total reaction volume of 40 μl) of in vitro transcribed target nucleic acid (SEQ ID NO: 2 and / or 3) was added to each well, at a concentration of 1 × 10 of target nucleic acid per reaction. 7 The samples were spiked with 100 μl of 10 ...

[0322] In a first series of experiments, 60 combinations of T7 promoter primers, non-T7 primers, and dual-labeled detector probe oligonucleotides were tested in real-time TMA amplification and detection reactions as described immediately above. Table 7 discloses the sequences and concentrations of the various T7 promoter primers tested. Table 8 discloses the sequences and concentrations of the various non-T7 primers tested. Figure 9 discloses the sequences and concentrations of the various dual-labeled hairpin detector probes (torches). Each of these reaction mixtures was tested alone, and the performance of each combination was recorded as TTime in Table 10 below. Combination conditions are presented in the order of T7 promoter primer, non-T7 primer, and torch using the Rxn component IDs. [Table 7] [Table 8] [Table 9] [Table 10-1] [Table 10-2]

[0323] In this experiment, combinations containing SEQ ID NO: 57 or SEQ ID NO: 58 consistently demonstrated faster TTime. Combinations using either SEQ ID NO: 57 or 58 as the T7 promoter donor and SEQ ID NO: 12, a non-T7 primer, demonstrated slower TTime compared to combinations containing these same T7 promoter primers. All combinations produced acceptable results.

[0324] Example 6 Analytical sensitivity of several primer and probe combinations In this next example, oligonucleotide combinations, each containing one of several T7 promoter primers (Table 11), a single non-T7 primer (Table 12), and a single Torch (Table 13), were tested against dilutions of target nucleic acid to determine their sensitivity. Reaction mixtures were prepared as generally described for Example 5 above, and reactions were performed in triplicate. Target nucleic acid was tested at dilutions ranging from 6.7 log copies down to 1.7 log copies. [Table 11] [Table 12] [Table 13]

[0325] Using the Rxn component ID format presented in and described for Table 10, the results of the combination conditions and tests are presented in Table 14 below. [Table 14]

[0326] In this experiment, all combinations demonstrated good sensitivity, with combinations 5b1 and 6b1 demonstrating good sensitivity down to 1.70 log copies of target nucleic acid per reaction. In another experiment (not shown), combination 6b1 was prepared and run as described in this example, but on a Panther instrument. In another experiment, combination 6b1 demonstrated sensitivity down to 5 copies of target nucleic acid per reaction.

[0327] In further sensitivity experiments, several combinations containing 0.2 pm / μL of SEQ ID NO:57 as the promoter primer, 0.15 pm / μL of SEQ ID NO:104 as the torch, and 0.4 pm / μL of one of SEQ ID NOs:12, 21, 18, or 20 as the non-T7 primer were further tested for sensitivity. Reactions were set up as generally described above, and each condition was run in triplicate. The combination condition index from Table 10 is used in the data table (Table 15 shows the average TTime of the triplicate reactions). [Table 15]

[0328] These data show that combinations 6a1, 6b1, 6c1 and 6e1 each have good sensitivity, with some demonstrating detection of as few as 1.7 logs of copies per reaction.

[0329] Example 7 Isothermal amplification and real-time detection of SARS-CoV-2 using different combinations of primers and probes. Sixty additional combinations of T7 promoter primers, non-T7 primers, and dual-labeled detector probe oligonucleotides were tested in real-time TMA amplification and detection reactions as described in Example 5. Table 16 discloses the sequences and concentrations of the various T7 promoter primers tested. Table 17 discloses the sequences and concentrations of the various non-T7 primers tested. And Figure 18 discloses the sequences and concentrations of the various dual-labeled hairpin detector probes (torches). Each of these reaction mixtures was tested alone, and the performance of each combination was recorded as TTime in Table 19. Combination conditions are presented in the order of T7 promoter primer, non-T7 primer, and torch using the Rxn component IDs. [Table 16] [Table 17] [Table 18]

[0330] Each of the T7 promoter primers, non-T7 primers, and torch components from Tables 16-18 above were combined and tested. The combination conditions are presented in Table 19 in the order of T7 promoter primer, non-T7 primer, torch using the Rxn component IDs. [Table 19-1] [Table 19-2]

[0331] In this experiment, combinations containing SEQ ID NO: 70, SEQ ID NO: 78, or SEQ ID NO: 24 consistently showed fast TTime. All combinations exhibiting TTime, except for combination condition 3c2, produced good results.

[0332] Example 8 Analytical sensitivity of several primer and probe combinations Combinations of oligonucleotides, each containing one of several T7 promoter primers (Table 20), a single non-T7 primer (Table 21), and a single detector probe (Table 22), were tested against dilutions of target nucleic acid to determine their sensitivity. Reaction mixtures were prepared generally as described for Example 5, and reactions were performed in triplicate. [Table 20] [Table 21] [Table 22]

[0333] The combined conditions and test results are presented in Table 23 using the Rxn component ID format presented in the description for Table 19. [Table 23]

[0334] In this experiment, combinations 1d1, 2d1, and 4d1 showed good sensitivity up to 3.7 log copies of target nucleic acid per reaction, and combinations 1d1 and 2d1 showed good sensitivity up to 2.7 log copies per reaction. Combination 6d1 was not reactive in this experiment.

[0335] In further sensitivity experiments, several combinations containing 0.2 pm / μL of SEQ ID NO: 78 as the promoter primer, 0.15 pm / μL of SEQ ID NO: 106 as the torch, and 0.4 pm / μL of one of SEQ ID NOs: 15, 23, 24, or 25 as the non-T7 primer were further tested for sensitivity. Reactions were set up as generally described immediately above, and each combination was run in triplicate. The combination condition index from Table 19 is used in the data table (Table 24: shows the average TTime for triplicate reactions). [Table 24]

[0336] These data show these combinations to have good sensitivity, with some showing detection of as few as 2.7 logs of copies per reaction.

[0337] Example 9 A multiplex formulation with primer-probe combinations targeting two regions of SARS-CoV-2 Several oligomer combinations were prepared in reaction mixtures to perform multiplex, real-time amplification and detection reactions targeting two regions of a coronavirus target nucleic acid. These reaction mixtures included T7 promoter primers having the sequences set forth in SEQ ID NO:57 and SEQ ID NO:70, non-T7 primers having the sequences set forth in SEQ ID NO:21 and SEQ ID NO:24, and detector probes having the sequences set forth in SEQ ID NO:104 (labeled with FAM as the fluorophore and Dabcyl as the quencher) and SEQ ID NO:106 (labeled with HEX as the fluorophore and Dabcyl as the quencher). In each of the various combinations, the T7 promoter primer of SEQ ID NO:70 was included in the reaction mixture at either 0.1 pm / μL, 0.2 pm / μL, or 0.3 pm / μL, while the other oligomeric components were provided at the same concentrations (0.2 pm / μL for SEQ ID NO:57, 0.4 pm / μL for SEQ ID NO:21, 0.4 pm / μL for SEQ ID NO:24, 0.15 pm / μL for SEQ ID NO:104, and 0.15 pm / μL for SEQ ID NO:106). Reactions were otherwise set up and run generally as described in Example 5. The results are shown in Table 25 and refer to combinations that differ by concentration of SEQ ID NO:70. [Table 25-1] [Table 25-2]

[0338] In this example, the multiplex combination with SEQ ID NO: 70 at 0.2 pm / μL demonstrated the most robust performance, detecting up to 1.7 log copies in the FAM channel and up to 3.7 log copies in the HEX channel. All combinations performed well and provided consistent TTime for triplicate conditions.

[0339] Example 10 Analytical sensitivity testing using target capture oligonucleotides and primer-probe combinations in singleplex and multiplex reactions. Two oligomer combinations were prepared to perform singleplex and multiplex target capture, amplification, and endpoint detection reactions targeting two regions of the coronavirus target nucleic acid. The two regions of the coronavirus target nucleic acid are represented by IVTs having the sequences shown in SEQ ID NOs: 2 and 3. A stock concentration of SEQ ID NO: 2 (2.4 x 10) was prepared using sample transport medium as a diluent. 15 copies / mL) and stock concentration of SEQ ID NO:3 (1.5 x 10 15 The IVTs (copies / mL) were serially diluted to 300, 100, 30, 10, 3, and 1 copy / mL, respectively. The negative control was sample transport medium without IVT. The target capture oligonucleotides, primer oligonucleotides, and probe oligonucleotides are shown in Table 26. The detection probe oligo was labeled with acridinium ester (AE). The negative control was added to five wells of a reaction plate. For singleplex reactions, each dilution of each IVT was also added to the reaction plate in multiples of 5. For multiplex reactions, combined dilutions of two IVTs were added to the reaction plate in multiples of 5. Each reaction was performed in a total volume of 100 μL. The target capture reaction was then performed, and the captured samples were eluted into separate wells for isothermal amplification and detection. The assay was performed using a Panther instrument from Hologic, Inc. The results are shown in Table 27. [Table 26] [Table 27]

[0340] The results of this experiment showed comparable sensitivity for both regions of the coronavirus target nucleic acid, with 100% positivity for the singleplex and multiplex conditions at 30 copies / mL, 80% positivity for the singleplex condition and 100% positivity for the multiplex condition at 10 copies / mL, and 60% positivity at 3 copies / mL for one of the singleplex conditions and 1 copy / mL for the multiplex condition. Negative reaction wells did not show false positives. The reaction curves showed good kinetics.

[0341] Example 11 Microbial Cross-Reactivity and Interference Testing The purpose of this experiment was to demonstrate that the primer and probe combinations in Table 28 do not cross-react with genetically related or commonly encountered microorganisms. In addition, this experiment demonstrated that the microorganisms evaluated do not interfere with the ability of the primer and probe combinations in Table 28 to detect SARS-CoV-2. Various microorganisms listed in Table 29 were detected in pooled clinical nasopharyngeal (NP) swabs processed at a 1:1.56 STM ratio, with a 0.03 TCID 50 Microorganisms were tested in the presence and absence of SARS-CoV-2 inactivated culture virus (BEI Resources, Manassas, VA; catalog number NR-52281) spiked at 1 / mL (3x LoD). Stock concentrations of the microorganisms listed in Table 29 were diluted with sample transport medium to achieve target concentrations (see Table 30). Assays were set up and performed as generally described in Example 10. [Table 28] [Table 29-1] [Table 29-2] [Table 30]

[0342] Assays were set up and performed as generally described in Example 10. Briefly, for the first region of the SARS-CoV-2 target nucleic acid, a target capture reagent was prepared containing a target capture oligonucleotide (0.666 mg / L SEQ ID NO:92), an amplification reagent was prepared containing a T7 primer (0.1 pmol / μL SEQ ID NO:57) and a non-T7 primer (0.2 pmol / μL SEQ ID NO:21), and a detection hybridization reagent containing an AE-labeled detection probe (5E3 RLU / μL SEQ ID NO:36). Similarly, for the second region of the SARS-CoV-2 target nucleic acid, a target capture oligonucleotide (0.666 mg / L SEQ ID NO:96), an amplification reagent was prepared containing a T7 primer (0.1 pmol / μL SEQ ID NO:78) and a non-T7 primer (0.2 pmol / μL SEQ ID NO:24), and a detection hybridization reagent containing an AE-labeled detection probe (5E3 RLU / μL SEQ ID NO:45). A nucleic acid internal control system was also prepared containing a target capture oligonucleotide (SEQ ID NO: 10 at 0.5 mg / L in the target capture reagent), a T7 primer (SEQ ID NO: 6 at 0.2 pmol / μL in the amplification reagent), a non-T7 primer (SEQ ID NO: 7 at 0.4 pmol / μL in the amplification reagent), and an AE-labeled detector probe oligonucleotide (SEQ ID NO: 8 at 4E3 GLU / μL). Target signals were distinguished from internal control signals using a flasher / glower detector probe, which rapidly quenches and has a higher intensity than the slower, longer-lasting, and lower-intensity glower signal of the internal control probe, as measured by the internal control probe's slower, longer-lasting, and lower-intensity glower signal. All samples were run in triplicate on a Panther instrument (Hologic, Inc.). The results are presented in Table 31. [Table 31]

[0343] This example showed negative results for all challenge microorganism reactions in the absence of SARS-CoV-2 target nucleic acid (all internal controls were positive in these reactions). This example also showed 100% positive results for detection of SARS-CoV-2 when assayed in the presence of these challenge organisms. Therefore, the primers and probes do not cross-react with genetically related and commonly encountered nasopharyngeal microorganisms. Furthermore, genetically related and commonly encountered nasopharyngeal microorganisms do not interfere with detection of SARS-CoV-2 target nucleic acid using the primers and probes.

[0344] Example 12 Clinical performance of PCR and isothermal assays The clinical performance of the PCR and isothermal assays was determined using a panel of residual clinical specimens. For the experiment, 105 residual clinical nasopharyngeal specimens were collected from US patients with signs and symptoms of respiratory infection. The PCR assay was set up and performed generally as described in Example 2. The isothermal assay was set up and performed generally as described in Example 11. The PCR primers and probes were SEQ ID NOS: 12-17 (PCR internal controls were SEQ ID NOS: 4, 5, and 9). The isothermal primers and probes were SEQ ID NOS: 21, 24, 36, 45, 57, and 78 (isothermal internal controls were SEQ ID NOS: 6, 7, 8, and 10). The assays were performed on a Panther Fusion instrument, with one replicate per residual clinical sample per assay. The results are shown in Table 32. [Table 32]

[0345] These assays demonstrated 99.0% overall agreement, 100% positive agreement, and 98.2% negative agreement for these 105 clinical residual samples.

[0346] Example 13 Clinical performance of PCR and isothermal assays in pooled clinical samples Each PCR and isothermal assay was tested for its ability to detect only one positive clinical sample in a pool of five samples. 325 individual nasopharyngeal (NP) clinical samples were collected using nasopharyngeal, nasal, or oropharyngeal swabs and placed in 3 ml of viral transport medium or universal transport medium according to the manufacturer's instructions. These clinical samples were assayed individually to determine the presence or absence of SARS-CoV-2 in each sample. Samples were pooled using the individual test results to create 45 positive pooled samples and 20 negative pooled samples. A positive pooled sample was one positive individual sample combined with four negative individual samples. A negative pooled sample was a combination of five negative individual samples. 100 μL of each of the five individual samples was mixed with 780 μL of sample transport medium to achieve a pooled sample volume of 1.280 mL. One replicate from each positive and negative pool was tested in the PCR assay, and one replicate from each positive and negative pool was tested in the isothermal assay. Assays were performed as generally described above using the oligonucleotides disclosed in Tables 1 and 26, as well as the internal control oligonucleotides in SEQ ID NOS: 4-10. Assays were performed using a Panther Fusion instrument. Results are presented as Ct for the PCR reaction and kRLU for the isothermal amplification assay and are summarized in Tables 33-34. [Table 33] [Table 34]

[0347] Pooled results were analyzed against expected results to calculate the concordance rate. Results were analyzed to demonstrate assay performance within populations with different low-positive prevalence rates. Low-positive prevalence rates were determined by collecting 31,000 individual test results and determining the Ct distribution for these results. Ct results were divided into quartiles, and based on the Ct distribution, samples were determined to be low-positive if they had a Ct value higher than 34 (11.1% of all positive samples). Tables 35 and 36 show the positive concordance rate (PPA) and negative concordance rate (NPA) for the isothermal amplification assay and PCR assay, respectively, in a population with a low-positive prevalence rate of 11.1% (5 / 45). The isothermal amplification assay had a PPA of 100% (92.1-100), and the PCR assay had a PPA of 95.6% (85.2-98.8). Both assays had an NPA of 100% (83.9-100). [Table 35] [Table 36]

[0348] Tables 37 and 38 show the PPA and NPA of the isothermal amplification assay and PCR assay, respectively, in a population with a low positive prevalence of 25% (5 / 20). The isothermal assay had a PPA of 100% (83.9-100), and the PCR assay had a PPA of 90.0% (69.9-97.2). Both assays had an NPA of 100% (83.9-100). [Table 37] [Table 38]

[0349] Both the isothermal and PCR assays demonstrated greater than 90% PPA relative to expected results for pools from individual samples obtained in populations with low positive prevalence as high as 25%. In addition, both assays demonstrated at least 99% NPA. [Table 39-1] [Table 39-2] [Table 39-3] [Table 39-4] [Table 39-5] [Table 39-6]

[0350] It will be appreciated from the foregoing that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the disclosure. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. In certain embodiments, for example, the following are provided: (Item 1) 1. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) a first amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target hybridizing sequence of 18 to 27 contiguous nucleotides in length, wherein the target hybridizing sequence is contained in SEQ ID NO:26 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (ii) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 18 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 85 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; a first amplification oligomer combination; (b) a second amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, (i) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 20 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:29 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (ii) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 16 to 27 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 89 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112, or SEQ ID NO: 113; a second amplification oligomer combination; A composition or kit comprising: (Item 2) 2. The composition or kit of item 1, wherein the first SARS-CoV-2-specific target-hybridizing sequence in (a)(i) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs; and / or the second SARS-CoV-2-specific amplification oligomer in (a)(ii) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs. (Item 3) the first SARS-CoV-2-specific target hybridizing sequence of (a)(i) contains the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an RNA equivalent or a DNA / RNA chimeric form thereof, and includes 0-7 nucleotide analogs; and / or the second SARS-CoV-2-specific target hybridizing sequence of (a)(ii) 3. The composition or kit of claim 2, wherein the sequence that hybridizes to a target specific for SARS-CoV-2 contains the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, or an RNA equivalent or a DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 4) 4. The composition or kit of any one of items 1 to 3, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (b)(i) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (b)(ii) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs. (Item 5) 5. The composition or kit of item 4, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (b)(i) contains the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (b)(ii) contains the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 6) 1. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising a combination of amplification oligomers comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid; (a) the first SARS-CoV-2-specific amplification oligomer comprises a target-hybridizing sequence of 18 to 27 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:26 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (b) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 18 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 85 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; Composition or kit. (Item 7) the sequence that hybridizes to the first SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0-7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75. 7. The composition or kit according to item 6, comprising nucleotide sequences differing by 5 nucleotides or more, or RNA equivalents or DNA / RNA chimeric forms thereof, and including 0 to 7 nucleotide analogs. (Item 8) 8. The composition or kit of item 7, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 9) 1. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, comprising a combination of amplification oligomers comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid; (a) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence that is 20 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:29 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (b) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 16 to 27 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 89 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112, or SEQ ID NO: 113; Composition or kit. (Item 10) 10. The composition or kit of item 9, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:16, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84, or an RNA equivalent or DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogs. (Item 11) 11. The composition or kit of item 10, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 12) S containing a sequence that hybridizes to the target of the SARS-CoV-2 specific detection probe A composition or kit comprising an ARS-CoV-2-specific detection probe oligomer, wherein the sequence that hybridizes to the target is (i) 19 to 27 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO:50 that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:114, or (ii) 25 to 29 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO:54 that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:115. (Item 13) 13. The composition or kit according to item 12, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detection probe contains a nucleotide sequence, a DNA equivalent, an RNA equivalent, or a DNA / RNA chimeric form thereof, which differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, and includes 0 to 7 nucleotide analogs. (Item 14) Item 15. The composition or kit according to Item 13, wherein the sequence hybridizing to the target of the SARS-CoV-2-specific detection probe comprises the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, its DNA equivalent, RNA equivalent, or DNA / RNA chimeric form, etc., and includes 0 to 7 nucleotide analogs. A formulation for amplifying SARS-CoV-2 nucleic acid in a sample, comprising: (a) a first amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 18 to 27 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:26 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (ii) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 18 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 85 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; a first amplification oligomer combination; (b) a second amplification oligomer combination comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, (i) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 20 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:29 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (ii) the second SARS-CoV-2-specific amplification oligomer is 16 to 27 a sequence of contiguous nucleotides that hybridizes to a second SARS-CoV-2-specific target, wherein the sequence that hybridizes to said target is contained in SEQ ID NO: 89 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112 or SEQ ID NO: 113; a second amplification oligomer combination; and (c) buffering agent; 1. A formulation comprising: (Item 16) 16. The formulation of item 15, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (a)(i) is SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (a)(ii) is SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues. (Item 17) 17. The formulation of item 15 or 16, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (b)(i) is SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (b)(ii) is SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues. (Item 18) 18. The formulation according to any one of items 15 to 17, which is a dry composition. (Item 19) A formulation for amplifying SARS-CoV-2 nucleic acid in a sample, comprising: (a) an amplification oligomer combination comprising a first and a second SARS-CoV-2-specific amplification oligomer capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, wherein the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 18 to 27 contiguous nucleotides in length, the target-hybridizing sequence being contained in SEQ ID NO:26 and having at most 0, 1, 2 or 3 nucleotides different from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; a combination of amplification oligomers, wherein the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target hybridizing sequence of 18 to 23 consecutive nucleotides in length, the target hybridizing sequence being contained in SEQ ID NO: 85 and containing a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87 or SEQ ID NO: 88; (b) buffering agent; 1. A formulation comprising: (Item 20) the sequence that hybridizes to the first SARS-CoV-2 specific target is SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0-7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2 specific target is SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75, or an RNA equivalent or a DNA / RNA chimeric form thereof. 20. The formulation according to item 19, comprising a 0-7 nucleotide analog. (Item 21) 21. The formulation according to item 19 or 20, which is a dry composition. (Item 22) A formulation for amplifying SARS-CoV-2 nucleic acid in a sample, comprising: (a) an amplification oligomer combination comprising a first and a second SARS-CoV-2-specific amplification oligomer capable of amplifying a target region of a SARS-CoV-2 target nucleic acid, wherein the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 20 to 23 contiguous nucleotides in length, the target-hybridizing sequence being contained in SEQ ID NO:29 and having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, a combination of amplification oligomers containing a nucleotide sequence that differs by 1, 2, 3, 4 or 5 nucleotides, wherein the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target hybridizing sequence that is 16 to 27 consecutive nucleotides in length, wherein the target hybridizing sequence is contained in SEQ ID NO: 89 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112 or SEQ ID NO: 113; (b) buffering agent; 1. A formulation comprising: (Item 23) 23. The formulation of item 22, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target is SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues, and / or the sequence that hybridizes to the second SARS-CoV-2-specific target is SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, comprising 0 to 7 nucleotide analogues. (Item 24) 24. The formulation according to item 22 or 23, which is a dry composition. (Item 25) A formulation for detecting SARS-CoV-2 in a sample, comprising: (a) a buffer; (b) a SARS-CoV-2-specific detection probe oligomer containing a sequence that hybridizes to the target of the SARS-CoV-2-specific detection probe; and the sequence that hybridizes to the target comprises: (i) is 19 to 27 contiguous nucleotides in length and contains a nucleotide sequence contained in SEQ ID NO: 50 that differs by at most 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 114; or (ii) a nucleotide sequence that is 25 to 29 consecutive nucleotides in length and that is contained in SEQ ID NO: 54 and differs from the nucleotide sequence of SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 115 by at most 0, 1, 2, 3, 4, or 5 nucleotides; formulation. (Item 26) The sequence hybridizing to the target of the SARS-CoV-2 specific detection probe is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110, and its DNA equivalent, RNA equivalent or DNA / RNA chimeric form, and those containing 0-7 nucleotide analogs. (Item 27) 27. The formulation according to item 25 or 26, which is a dry composition. (Item 28) 28. A kit comprising the dry composition of any one of items 18, 21, 24, and 27. (Item 29) 29. The kit of claim 28, further comprising a reconstitution reagent, wherein the dry composition is in a first vial within the kit and the reconstitution reagent is in a second vial within the kit. (Item 30) 28. A method for preparing an aqueous reaction mixture for determining the presence or absence of SARS-CoV-2 in a sample, comprising combining the dry composition of any one of embodiments 18, 21, 24 and 27 with a reconstitution reagent to produce an aqueous reaction mixture. (Item 31) 1. A method for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) contacting a sample with at least a first amplification oligomer combination and a second amplification oligomer combination, (i) the first amplification oligomer combination comprises first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a first target region of a SARS-CoV-2 target nucleic acid; (1) the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, the sequence being 18 to 27 consecutive nucleotides in length, and the sequence that hybridizes to the target is contained in SEQ ID NO:26 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (2) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 18 to 23 consecutive nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 85 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; (ii) the second amplification oligomer combination comprises first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a second target region of the SARS-CoV-2 target nucleic acid; (1) the first SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a first SARS-CoV-2-specific target, the sequence being 20 to 23 consecutive nucleotides in length, and the sequence that hybridizes to the target is contained in SEQ ID NO:29 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (2) The second SARS-CoV-2-specific amplification oligomer comprises a sequence that hybridizes to a second SARS-CoV-2-specific target, the sequence being 16 to 27 consecutive nucleotides in length, and the sequence that hybridizes to the target is contained in SEQ ID NO: 89 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112, or SEQ ID NO: 113; Steps and (b) incubating the sample under conditions suitable for in vitro nucleic acid amplification, wherein any SARS-CoV-2 target nucleic acid, if present in the sample, is amplified by one or more of the first or second SARS-CoV-2 target regions. used as a template to generate a plurality of amplicons; (c) detecting the presence or absence of said one or more amplicons, thereby determining the presence or absence of SARS-CoV-2 in said sample; A method comprising: (Item 32) 32. The method of claim 31, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (a)(i) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (a)(ii) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 33) 33. The method of claim 32, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (a)(i) contains the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (a)(ii) contains the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 34) 34. The method of any one of items 31 to 33, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (b)(i) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (b)(ii) contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84, or an RNA equivalent or a DNA / RNA chimeric form thereof, and comprises 0 to 7 nucleotide analogues. (Item 35) 35. The method of claim 34, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target in (b)(i) contains the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target in (b)(ii) contains the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 36) 1. A method for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) combining a sample with a combination of amplification oligomers comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid; contacting the substrate with a mating plate, (i) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 18 to 27 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:26 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:28; (ii) the second SARS-CoV-2-specific amplification oligomer comprises a second SARS-CoV-2-specific target-hybridizing sequence of 18 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO: 85 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO: 88; Steps and (b) incubating the sample under conditions suitable for in vitro nucleic acid amplification, wherein any SARS-CoV-2 target nucleic acid, if present in the sample, is used as a template to generate amplicons corresponding to the SARS-CoV-2 target region; (c) detecting the presence or absence of said amplicon, thereby determining the presence or absence of SARS-CoV-2 in said sample; A method comprising: (Item 37) 37. The method of claim 36, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0 to 7 nucleotide analogs. (Item 38) 38. The method of claim 37, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75, or an RNA equivalent or DNA / RNA chimeric form thereof, and includes 0 to 7 nucleotide analogs. (Item 39) 1. A method for determining the presence or absence of SARS-CoV-2 in a sample, comprising: (a) contacting the sample with a combination of amplification oligomers comprising first and second SARS-CoV-2-specific amplification oligomers capable of amplifying a target region of a SARS-CoV-2 target nucleic acid; (i) the first SARS-CoV-2-specific amplification oligomer comprises a first SARS-CoV-2-specific target-hybridizing sequence of 20 to 23 contiguous nucleotides in length, wherein the target-hybridizing sequence is contained in SEQ ID NO:29 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:111; (ii) the second SARS-CoV-2-specific amplification oligomer hybridizes to a second SARS-CoV-2-specific target having a length of 16 to 27 contiguous nucleotides; The sequence that hybridizes to the target is contained in SEQ ID NO: 89 and contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 90, SEQ ID NO: 112, or SEQ ID NO: 113. Steps and (b) incubating the sample under conditions suitable for in vitro nucleic acid amplification, wherein any SARS-CoV-2 target nucleic acid, if present in the sample, is used as a template to generate amplicons corresponding to the SARS-CoV-2 target region; (c) detecting the presence or absence of said amplicon, thereby determining the presence or absence of SARS-CoV-2 in said sample; A method comprising: (Item 40) 40. The method of claim 39, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains a nucleotide sequence that differs by at most 0, 1, 2, 3, 4, or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0 to 7 nucleotide analogs. (Item 41) 41. The method of claim 40, wherein the sequence that hybridizes to the first SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0 to 7 nucleotide analogs; and / or the sequence that hybridizes to the second SARS-CoV-2-specific target contains the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84, or an RNA equivalent or DNA / RNA chimeric form thereof, and contains 0 to 7 nucleotide analogs. (Item 42) 39. A multiplex method for determining the presence or absence of SARS-CoV-2 and at least one other pathogen in a sample, wherein the presence or absence of SARS-CoV-2 is determined using the method according to any one of items 31 to 38. (Item 43) 43. The multiplex method of claim 42, wherein the presence or absence of SARS-CoV-2 and one or more pathogens selected from the group consisting of influenza A, influenza B, respiratory syncytial virus A, respiratory syncytial virus B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, adenovirus, metapneumovirus, rhinovirus, coronavirus 229E, coronavirus NL63, coronavirus HKU1, coronavirus OC43, SARS-CoV (SARS), and MERS-CoV is determined. (Item 44) and detecting the presence or absence of the amplicon comprises contacting the sample with a SARS-CoV-2-specific detector probe oligomer comprising a sequence that hybridizes to a target of the SARS-CoV-2-specific detector probe, wherein the sequence that hybridizes to the target is (i) 19 to 27 contiguous nucleotides in length and is set forth in SEQ ID NO: 50. 42. The method of any one of items 31 to 41, wherein the nucleotide sequence is (i) comprised in SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 114 and differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 114; or (ii) comprised in SEQ ID NO: 54 and differs by at most 0, 1, 2, 3, 4 or 5 nucleotides from the nucleotide sequence of SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 115.

Claims

[Claim 1] The invention described in the specification.

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