Compositions and methods for detecting viral pathogens in a sample

Abstract

The present disclosure relates to amplification primers, hybridization assay probes, compositions comprising such primers and probes, and related reagents, kits, and methods that can be used to analyze samples for the presence of target nucleic acids of SARS-CoV-2, influenza virus type A, influenza virus type B, respiratory syncytial virus type A, and / or respiratory syncytial virus type B. The present disclosure provides compositions (including kits and reagents) and methods for the in vitro diagnostic analysis of influenza virus type A (Flu A), influenza virus type B (Flu B), SARS-CoV-2, respiratory syncytial virus type A (RSV A), or respiratory syncytial virus type B (RSV B) nucleic acids in a sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 6 / 374,422, filed September 2, 2022, which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing in the file DIA.0184.02_SeqList_ST26.xml is 296 kilobytes in size, created on August 31, 2023, and is incorporated herein by reference. [Background technology]

[0003] background The present disclosure relates to the field of biotechnology. More specifically, the present disclosure relates to compositions (including kits and reagents) and methods for analyzing samples to detect viral pathogens, particularly influenza virus, coronavirus, and respiratory syncytial virus.

[0004] Influenza is an acute respiratory illness in humans caused by infection with influenza (flu) viruses (mainly types A and B). Influenza virus type A is further categorized into subtypes based on two major surface protein antigens, hemagglutinin (H) and neuraminidase (N). Influenza virus type B is not categorized into subtypes. Influenza viruses are RNA viruses in the family Orthomyxoviridae. Influenza types A and B (Flu A and Flu B, respectively) are each separate genera containing one species and many subspecies.

[0005] Influenza epidemics occur annually worldwide. Both influenza A and B types circulate in the population, although influenza A usually predominates. These annual epidemics are due in part to antigenic variations in the virus's H and N surface proteins. Influenza is transmitted primarily through droplets (coughing or sneezing). Symptoms occur on average 1-2 days after exposure and include fever, chills, headache, fatigue, cough, and a head cold. Gastrointestinal symptoms (e.g., nausea, vomiting, and diarrhea) may occur primarily in children. Complications resulting from influenza include pneumonia, which can cause increased morbidity and mortality in children, the elderly, and immunocompromised populations. In the United States, influenza is estimated to result in more than 200,000 hospitalizations and up to 36,000 deaths annually. Influenza epidemics, or pandemics, occur rarely. In the 20th century, three influenza pandemics occurred in 1918, 1958, and 1968, each causing millions of deaths worldwide. Influenza can also affect other animals, including pigs, horses, and birds.

[0006] 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, as well as betacoronaviruses OC43, HKU1, SARS-CoV and SARS-CoV-2 (coronaviruses that cause severe acute respiratory syndrome, or SARS), and MERS-CoV (coronavirus that causes Middle East respiratory syndrome, or MERS). SARS-CoV-2 can cause a severe lower respiratory tract infection (COVID-19) and has been declared a global emergency by the World Health Organization (Huang et al., Lancet (2020) v395, issue 10223, p. 497).

[0007] Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and children. Like influenza, RSV is an RNA virus. It is a member of the Paramyxoviridae family within the Orthopneumovirus genus. There are two subtypes of RSV, A and B, which are distinguished based on antigenic and surface protein variations. Most annual epidemics involve a mixture of RSV A and RSV B, but one subtype may predominate during a season. RSV infection can cause severe respiratory illness in all age groups but is more prevalent in children, the elderly, and immunocompromised populations. RSV can infect up to 80% of children under the age of 1 year. Bronchiolitis and pneumonia are major clinical complications in infants and children, resulting in an estimated 51,000 to 82,000 hospitalizations per year in the United States. RSV infection is also a significant cause of severe respiratory illness and substantial mortality in the elderly, with estimated annual costs of hospitalization for RSV pneumonia ranging from $150 million to $680 million. Given the morbidity, mortality, and economic costs associated with influenza, coronavirus, and RSV infections, there is a clear need for improved detection of these pathogens. The present disclosure addresses this and other needs. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Huang et al., Lancet (2020) v395, issue 10223, p.497 Summary of the Invention [Means for solving the problem]

[0009] Abstract The present disclosure provides compositions (including kits and reagents) and methods for in vitro diagnostic analysis of influenza virus type A (Flu A), influenza virus type B (Flu B), SARS-CoV-2, respiratory syncytial virus type A (RSV A), or respiratory syncytial virus type B (RSV B) nucleic acid in a sample. Preferably, the in vitro diagnostic analysis utilizes polymerase chain reaction (PCR) or an isothermal amplification reaction, although other in vitro assay methodologies are contemplated for use with the compositions of the present disclosure. A particularly useful in vitro assay for use with target nucleic acids of Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B is a reverse transcription PCR (RT-PCR) assay, since these target nucleic acids are RNA viruses. A particularly useful and convenient in vitro assay for use with target nucleic acids of Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B is a real-time RT-PCR assay. A particularly useful and convenient in vitro assay for use with Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is the transcription-mediated amplification (TMA) reaction. A particularly useful and convenient in vitro assay for use with Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is the BiPhasic TMA reaction.

[0010] In one aspect, the sample is a biological sample. In one aspect, the biological sample is a clinical sample. In another aspect, the sample is a swab sample, for example, from a nasopharyngeal (NP) swab specimen obtained from a patient. In some embodiments, the compositions and methods can be used to aid in the differential diagnosis of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B infections. A negative result does not exclude such infections. Conversely, a positive result does not exclude bacterial infection or co-infection with other viruses. To obtain a final diagnosis of respiratory virus infection, the use of additional laboratory tests and clinical findings may also be considered.

[0011] One aspect provides hybridization assay probes useful for detecting target nucleic acid sequences of Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B. Preferably, such probe molecular species comprise a target hybridizing sequence that is substantially complementary to a probe target nucleic acid sequence in the viral genome targeted for detection or an amplicon generated therefrom. In a preferred embodiment, the probe target nucleic acid sequence consists of about 17 to about 100 contiguous bases contained within the targeted viral genome (or an amplicon generated therefrom).

[0012] In some preferred embodiments, the Flu A probe comprises a sequence that is preferably one of SEQ ID NOs: 6 to 22. In particularly preferred embodiments, two probes (each independently selected from SEQ ID NOs: 6 to 22) are used in tandem to target two different regions of the Flu A genome or amplification products generated therefrom.

[0013] In some preferred embodiments, the Flu B probe sequence is preferably one of SEQ ID NOs: 30-57 and 59-66.

[0014] In some preferred embodiments, the SARS-CoV-2 probe sequence is preferably one of SEQ ID NOs: 118, 128, 130, 133, 136, 139, 142, 145, 154, 193, 242, 246, 258, 261, and 265. In particularly preferred embodiments, two probes (each independently selected from SEQ ID NOs: 118, 128, 130, 133, 136, 139, 142, 145, 154, 193, 242, 246, 258, 261, and 265) are used in tandem to target two different regions of the SARS-CoV-2 genome or amplification products generated therefrom.

[0015] In some preferred embodiments, the RSV A probe sequence is preferably one of SEQ ID NOs: 71, 75-78, 80-87, 89-91, 97, and 98.

[0016] In some preferred embodiments, the RSV B probe sequence is preferably one of SEQ ID NOs: 102, 103, and 107-114.

[0017] Preferably, the probe species are labeled, and optionally, distinguishably labeled, so that any one probe species can be distinguished from other probe species in a multiplex detection assay. Distinguishable labeling can be achieved using two or more detectable labels, for example, a chemiluminescent moiety, a fluorophore moiety, and both a fluorophore moiety and a quencher moiety.

[0018] Another aspect of the present disclosure relates to nucleic acid molecules that are amplification primers for use in the in vitro amplification of Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B target nucleic acid sequences.

[0019] A related aspect of the present disclosure relates to such primer pairs that can be used to amplify desired amplicons containing target nucleic acid sequences. These primers include one or more of the following primer pairs: a first Flu A primer pair, a second Flu A primer pair that can be used to amplify a region of the Flu A target nucleic acid that differs from the region of the Flu A target nucleic acid that can be amplified using the first Flu A primer pair, a Flu B primer pair, a first SARS-CoV-2 primer pair, a second SARS-CoV-2 primer pair that can be used to amplify a region of the SARS-CoV-2 target nucleic acid that is derived from the region of the SARS-CoV-2 target nucleic acid that can be amplified using the first SARS-CoV-2 primer pair, an RSV A primer pair, and an RSV B primer pair. These primer pairs include first and second primers that can be used to generate corresponding amplicons for Flu A, Flu B, SARS-CoV-2, RSV A, and / or RSV B if a viral pathogen is present in the biological sample being tested.

[0020] Generally, a primer pair includes a first primer that includes a priming nucleotide sequence that is substantially complementary to a first target nucleic acid sequence in the viral genome, a portion of which is to be amplified. Preferably, the first and second target nucleic acid sequences are spaced apart in the target nucleic acid by at least 10 nucleotides, preferably about 50 to 1,000 nucleotides, each of which preferably consists of about 17 to about 100 contiguous bases in the viral genome to be detected. In some embodiments, one or more of the primers in one or more primer pairs further include a primer upstream region having a non-target hybridizing sequence, a nucleotide sequence that is not complementary to the target nucleotide sequence of the primer.

[0021] A preferred first primer for generating the first Flu A amplicon has a priming nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 23. A preferred second primer for generating the first Flu A amplicon has a priming nucleotide sequence of SEQ ID NO: 25 or SEQ ID NO: 28.

[0022] A preferred first primer for generating a second Flu A amplicon has a priming nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 24. A preferred second primer for generating a second Flu A amplicon has a priming nucleotide sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

[0023] A preferred first primer for generating a Flu B amplicon has a priming nucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 67. A preferred second primer for generating a Flu B amplicon has a priming nucleotide sequence of SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.

[0024] A preferred first primer for generating a SARS-CoV-2 amplicon has a priming sequence of SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:126, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:176, SEQ ID NO:181, SEQ ID NO:240, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:262, or SEQ ID NO:263. A preferred second primer for generating a SARS-CoV-2 amplicon has a priming nucleotide sequence of SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:170, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:247, SEQ ID NO:254, SEQ ID NO:257, or SEQ ID NO:264.

[0025] A preferred first primer for generating an RSV A amplicon has a priming nucleotide sequence of SEQ ID NO: 79 or SEQ ID NO: 88. A preferred second primer for generating an RSV A amplicon has a priming nucleotide sequence of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, or SEQ ID NO: 96.

[0026] A preferred first primer for generating an RSV B amplicon has a priming nucleotide sequence of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 106. A preferred second primer for generating an RSV B amplicon has a priming nucleotide sequence of SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 115.

[0027] In some preferred embodiments, the probes and / or primers contain one or more methylated cytosine bases.

[0028] Another related aspect of the present disclosure relates to compositions comprising such probes, primers, and primer pairs. Such compositions include dry or liquid compositions. Dry compositions include lyophilized reagents including one or more of the primers and probes.

[0029] Another aspect of the present disclosure relates to kits containing primers and / or probes. Such kits may also contain salts, enzymes, dNTPs, rNTPs, other substrates, and / or instructions for use of such materials. The primers, probes, salts, enzymes, dNTPs, rNTPs, and / or other substrates of the kits may be in dry or aqueous form.

[0030] Another aspect of the present disclosure relates to reagents comprising primers and / or probes. Such reagents may also comprise salts, enzymes, dNTPs, rNTPs, and / or other substrates. The primers, probes, salts, enzymes, dNTPs, rNTPs, and / or other substrates of the reagents may be in dry or aqueous form.

[0031] Yet another aspect of the disclosure relates to methods of using such primers and probes to analyze a sample to determine whether the sample contains one or more of a Flu A target nucleic acid, a Flu B target nucleic acid, a SARS-CoV-2 target nucleic acid, a RSV A target nucleic acid, and a RSV B target nucleic acid.

[0032] Embodiment 1: A composition or kit for the analysis of one or more of a plurality of pathogen-associated target nucleic acid species that may be present in a biological sample, the composition or kit comprising: (a) a first SARS-CoV-2 primer pair for generating a first SARS-CoV-2 amplicon from the biological sample, the primer pair comprising (i) a first SARS-CoV-2 primer and (ii) a second SARS-CoV-2 primer; and / or (b) a second SARS-CoV-2 primer pair for generating a second SARS-CoV-2 amplicon from the biological sample, the primer pair comprising (i) a third SARS-CoV-2 primer and (ii) a fourth SARS-CoV-2 primer; and / or (c) a first Flu A amplicon for generating a first Flu A amplicon from the biological sample. and / or (d) a second Flu A primer pair for generating a second Flu A amplicon from the biological sample, the primer pair comprising (i) a third Flu A primer and (ii) a fourth Flu A primer; and / or (e) a Flu B primer pair for generating a Flu B amplicon from the biological sample, the primer pair comprising (i) a first Flu B primer and (ii) a second Flu B primer; and / or (f) a RSV A primer pair for generating a RSV A amplicon from the biological sample, the primer pair comprising (i) a first RSV A primer and (ii) a second RSV A primer;And / or (g) a RSV B primer pair for generating a RSV B amplicon from the biological sample, the primer pair comprising (i) a first RSV B primer and (ii) a second RSV B primer; wherein (a)(i) primer comprises a target hybridizing sequence consisting of a nucleotide sequence of 18 to 35 nucleotides in length, the nucleotide sequence being within SEQ ID NO: 123 and including SEQ ID NO: 122, wherein (a)(i) primer and (a)(ii) primer generate an amplicon comprising SEQ ID NO: 124. A composition or kit;

[0033] Embodiment 2: The composition or kit of embodiment 1, wherein the primer (a)(i) is 20-23 nucleotides in length. Embodiment 3: The composition or kit of embodiment 1 or embodiment 2, wherein the primer (a)(ii) is 20-30 nucleotides in length. Embodiment 4: The composition or kit of embodiment 3, wherein the primer (a)(ii) is 22-25 nucleotides in length. Embodiment 5: The composition or kit of embodiment 1, 2, or 3, wherein the primer (a)(i) is 22-23 nucleotides in length. Embodiment 6: The composition or kit of embodiment 5, wherein the primer (a)(i) is SEQ ID NO: 116 or SEQ ID NO: 119 or SEQ ID NO: 121. Embodiment 7: The composition or kit of embodiment 1, 2, 5, or 6, wherein the primer (a)(i) comprises at least one nucleotide analogue. Embodiment 8: The composition or kit of embodiment 1, 2, 5, 6, or 7, wherein the primer (a)(i) comprises at least one nucleotide analogue that is 5-Me-C. Embodiment 9: The composition or kit of embodiment 1, 2, or 3, wherein the primer (a)(ii) is 24 nucleotides in length and comprises at least one nucleotide analogue. Embodiment 10: The composition or kit of embodiment 1, 2, 3, or 9, wherein the primer (a)(ii) is SEQ ID NO: 117. Embodiment 11: The composition or kit of embodiment 1, 2, 3, 9, or 10, wherein the primer (a)(ii) comprises at least one nucleotide analogue that is 5-Me-C.

[0034] Embodiment 12: The composition or kit of any one of Embodiments 1 to 11, wherein the composition or kit further comprises a detection probe oligomer.Embodiment 13: The composition of Embodiment 1, wherein the primer (a)(i) is 18 to 35 nucleotides in length contained within SEQ ID NO: 125 and comprises SEQ ID NO: 122, and the detection probe oligomer is 25 to 40 nucleotides in length and comprises a target-hybridizing sequence consisting of a sequence contained within SEQ ID NO: 125.Embodiment 14: The composition or kit of Embodiment 12 or Embodiment 13, wherein the detection probe oligomer is SEQ ID NO: 118.

[0035] Embodiment 15: The third primer of 1(b)(i) and the fourth primer of 1(b)(ii) are each independently selected from the group consisting of SEQ ID NOs: 120, 126, 127, 129, 131, 132, 134, 135, 137, 138, 140, 141, 143, 144, 146, 147, 148, 149, 170, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 230, 233, 239, 240, 241, 247, 248, 254, 256, 257, 262, 263, 264, 266, 267, 268, 272, 273, 276, and 283. Embodiment 16: The composition or kit of any one of embodiments 1 to 15, wherein the first primer of 1(c)(i), the second primer of 1(c)(ii), the third primer of 1(d(i), and the fourth primer of 1(d)(ii) are each independently selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 23, 24, 25, 26, 27, 28, 305, 306, 307, 308, 309, 312, and 313. Embodiment 17: The composition or kit of any one of embodiments 1 to 16, wherein the first primer of 1(e)(i) and the second primer of 1(e)(ii) are each independently selected from the group consisting of SEQ ID NOs: 29, 58, 67, 68, 69, 70, and 314. Embodiment 18: Embodiment 19: The composition or kit of any of embodiments 1 to 18, wherein the first primer of 1(f)(i) and the second primer of 1(f)(ii) are each independently selected from the group consisting of SEQ ID NOs: 72, 73, 74, 79, 88, 92, 93, 94, 95, and 96. Embodiment 20: The composition or kit of any of embodiments 13 to 19, wherein each primer individually comprises 0 to 20 nucleotide analogues.Embodiment 21: The composition or kit of any of embodiments 13 to 20, wherein each primer individually comprises 0 to 20 5-Me-C nucleotide analogs.

[0036] Embodiment 22: The composition or kit of embodiment 15, further comprising a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the second SARS-CoV-2 primer pair. Embodiment 23: The composition or kit of embodiment 22, wherein the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 128, 130, 133, 136, 139, 142, 145, 154, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 242, 246, 258, 261, 265, 277, 278, 279, 284, 285, 286, 287, 288, 289, 290, 304, 159, 160, 161, 162, 163, 164, 165, 166, 167, and 168.

[0037] Embodiment 24: The composition or kit of embodiment 16, wherein the composition or kit further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the first Flu A primer pair.Embodiment 25: The composition or kit of embodiment 16, wherein the composition or kit further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the second Flu A primer pair.Embodiment 26: The composition or kit of embodiment 24 or 25, wherein the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 269, 280, 281, 282, 301, and 302.

[0038] Embodiment 27: The composition or kit of embodiment 17, further comprising a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the Flu B primer pair.Embodiment 28: The composition or kit of embodiment 27, wherein the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 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, 59, 60, 61, 62, 63, 64, 65, 66, 282, and 303.

[0039] Embodiment 29: The composition or kit of embodiment 18, wherein the composition or kit further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to the amplicon generated by the RSV A primer pair.Embodiment 30: The composition or kit of embodiment 29, wherein the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 71, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 97, and 98.

[0040] Embodiment 31: The composition or kit of embodiment 19, wherein the composition or kit further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the RSV B primer pair.Embodiment 32: The composition or kit of embodiment 31, wherein the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 102, 103, 107, 108, 109, 110, 111, 112, 113, and 114.

[0041] Embodiment 33: The composition or kit of any one of embodiments 12 to 14 and 22 to 32, wherein each detection probe oligomer comprises a nucleotide sequence that is 0% to 100% nucleotide analogues.Embodiment 34: The composition or kit of any one of embodiments 12 to 14 and 22 to 33, wherein each detection probe oligomer comprises a nucleotide sequence that is 0% to 100% 5-Me-C nucleotide analogues.Embodiment 35: The composition or kit of any one of embodiments 12 to 14 and 22 to 34, wherein each detection probe oligomer comprises a 2' O-methyl backbone.Embodiment 36: The composition or kit of any one of embodiments 12 to 14 and 22 to 35, wherein at least one detection probe further comprises a detectable label.Embodiment 37: The composition or kit of any one of embodiments 12 to 14 and 22 to 36, wherein at least one detection probe further comprises a donor / acceptor label pair.

[0042] Embodiment 38: The composition or kit of any one of embodiments 1 to 37, wherein the composition or kit further comprises a target capture oligonucleotide. Embodiment 39: The composition or kit of embodiment 38, wherein the target capture oligonucleotide is selected from the group consisting of SEQ ID NOs: 228, 229, 299, 300, 310, and 311. Embodiment 40: The composition or kit of any one of embodiments 1 to 37, wherein the composition or kit further comprises two or more target capture oligonucleotides, each of the target capture oligonucleotides independently selected from the group consisting of SEQ ID NOs: 228, 229, 299, 300, 310, and 311.

[0043] Embodiment 41: The composition of any one of embodiments 1 to 40, wherein the composition is an aqueous formulation.Embodiment 42: The composition of embodiment 41, wherein the aqueous formulation further comprises reagents for amplification and / or detection and / or target capture reactions.Embodiment 43: The composition of any one of embodiments 1 to 40, wherein the composition is a dry formulation.Embodiment 44: The composition of embodiment 43, wherein the dry formulation further comprises reagents for amplification and / or detection and / or target capture reactions.Embodiment 45: A kit comprising the composition of any one of embodiments 41 to 44.

[0044] Embodiment 46: A kit or composition for the detection of amplicons generated in a nucleic acid amplification reaction of a biological sample suspected of containing SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B, wherein the kit or composition comprises: (a) with respect to an amplicon generated with a SARS-CoV-2 primer pair, the probe molecule species comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 118, 128, 130, 133, 136, 139, 142, 145, 154, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 242, 246, 258, 261, 265, 277, 278, 279, 284, 285, 286, 287, 288, 289, 290, 304, 159, 160, 161, 162, 163, 164, 165, 166, 167, and 168; and / or (b) Flu For amplicons generated with the A primer pair, the probe molecule species comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 269, 280, 281, 282, 301, and 302; and / or (c) Flu For amplicons generated with the B primer pair, the probe molecule species comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 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, 59, 60, 61, 62, 63, 64, 65, 66, 282, and 303; and / or (d) for amplicons generated with the RSV A primer pair, the probe molecule species comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 71, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 97, and 98;and / or (e) for amplicons generated with the RSV B primer pair, the probe molecule species comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 102, 103, 107, 108, 109, 110, 111, 112, 113, and 114. Embodiment 47: The composition or kit of embodiment 46, wherein each detection probe oligomer comprises a nucleotide sequence that is 0% to 100% nucleotide analogues. Embodiment 48: The composition or kit of embodiment 46 or 47, wherein each detection probe oligomer comprises a nucleotide sequence that is 0% to 100% 5-Me-C nucleotide analogues. Embodiment 49: The composition or kit of embodiment 46, 47, or 48, wherein each detection probe oligomer comprises a 2'-O-methyl backbone. Embodiment 50: The composition or kit of any one of embodiments 46 to 49, wherein at least one detection probe further comprises a detectable label. Embodiment 51: The composition or kit of any one of embodiments 46 to 50, wherein at least one detection probe further comprises a donor / acceptor label pair.

[0045] Embodiment 52: The composition of any one of embodiments 46 to 51, wherein the composition is an aqueous formulation.Embodiment 53: The composition of any one of embodiments 46 to 51, wherein the composition is a dry formulation.Embodiment 54: A kit comprising the composition of embodiment 52 or 53.

[0046] Embodiment 55: A reaction mixture comprising the composition of embodiment 41, further comprising one or more of a reverse transcriptase, a DNA polymerase, a buffer, and dNTPs.Embodiment 56: A reaction mixture made by rehydrating the composition of embodiment 43, further comprising one or more of a reverse transcriptase, a DNA polymerase, a buffer, and dNTPs.Embodiment 57: A reaction mixture comprising the composition of embodiment 52.

[0047] Embodiment 58: A method for determining whether a biological sample contains SARS-CoV-2, influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus A (RSV A), and / or respiratory syncytial virus B (RSV B), comprising: (a) contacting a target nucleic acid molecule from the biological sample with one or more of the detection probe oligonucleotides of embodiment 50 or 51 under stringent hybridization conditions; (b) detecting the presence or absence of a detectable label, wherein the presence indicates that a probe:target nucleic acid duplex was formed in step (a); (c) determining whether the biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B based on whether the presence of the detectable label was detected in step (b). Embodiment 59: The method of embodiment 58, wherein the detection probe oligonucleotides are distinguishably labeled, allowing for the determination of the presence or absence of each of SARS-CoV-2, Flu A, Flu B, and RSV A and RSV B in step (c).Embodiment 60: The method of embodiment 58 or 59, wherein the target nucleic acid that forms the probe:target nucleic acid duplex in step (b) is an amplicon generated using a primer pair.

[0048] Embodiment 61: A method for determining whether a biological sample contains SARS-CoV-2, influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus A (RSV A), and / or respiratory syncytial virus B (RSV B), the method comprising the steps of: (a) contacting target nucleic acid molecules from the biological sample with one or more primer pairs of any one of embodiments 1 to 11 to form an amplification reaction mixture; (b) performing a nucleic acid amplification reaction to generate amplicons from any target nucleic acid molecules in the amplification reaction mixture of step (a); and (c) determining whether amplicons are generated in step (b), thereby determining whether the biological sample contains SARS-CoV-2, Flu A, Flu B, and RSV A and RSV B. Embodiment 62: The method of embodiment 61, wherein prior to step (a), a target capture reaction is performed to extract target nucleic acid molecules from the biological sample. Embodiment 63: The method of embodiment 61 or embodiment 62, wherein steps (b) and (c) are performed simultaneously.Embodiment 64: The method of embodiment 61, 62, or 63, wherein the nucleic acid amplification reaction is a PCR reaction.Embodiment 65: The method of any one of embodiments 61 to 64, wherein the nucleic acid amplification reaction is a real-time nucleic acid amplification reaction.

[0049] Embodiment 66: The method of any one of embodiments 61 to 65, wherein the detecting step (c) is carried out using one or more detection probe oligonucleotides.Embodiment 67: The method of embodiment 66, wherein the detection probe oligonucleotides are labeled with a donor / acceptor label pair.Embodiment 68: The method of embodiment 66 or embodiment 67, wherein the detection probe oligonucleotides are differentially labeled to allow determination of which of SARS-CoV-2, Flu A, Flu B, and RSV A and RSV B are present in the sample.

[0050] Embodiment 69: The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen.Embodiment 70: The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen that is a nasopharyngeal specimen.Embodiment 71: The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen that is a bronchoalveolar specimen.Embodiment 72: The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen that is a lower respiratory tract specimen.Embodiment 73: The method of any one of embodiments 61 to 72, wherein the biological sample is collected into a sample transport medium.Embodiment 74: The method of embodiment 73, wherein the sample transport medium comprises one or more of a buffer, a chelating agent, an anionic detergent, and a degradative enzyme.

[0051] Embodiment 75: A system for performing one or more steps of the method according to any one of embodiments 61 to 74. Embodiment 76: A system according to embodiment 75, wherein the system is an automated system. Embodiment 77: A system for performing one or more steps of the method according to any one of embodiments 58 to 60. Embodiment 78: A system according to embodiment 77, wherein the system is an automated system.

[0052] Embodiment 78: A composition or kit or method for the analysis of one or more of a plurality of pathogen-associated target nucleic acid species that may be present in a biological sample, comprising: (a) a first SARS-CoV-2 primer pair for generating a first SARS-CoV-2 amplicon from said biological sample, said primer pair comprising: (i) a first SARS-CoV-2 primer and (ii) a second SARS-CoV-2 primer, wherein the primer in 1(a)(i) comprises a target hybridizing sequence consisting of a nucleotide sequence that is 18 to 35 contiguous nucleotides in length contained within and including SEQ ID NO: 123. wherein primer 1(a)(i) and primer 1(a)(ii) comprise SEQ ID NO: 124 and generate an amplicon having a length of 57 to 89 contiguous nucleotides; and optionally, (b) a second SARS-CoV-2 primer pair for generating a second SARS-CoV-2 amplicon from said biological sample, said primer pair comprising (i) a third SARS-CoV-2 primer and (ii) a fourth SARS-CoV-2 primer; and / or (c) a first SARS-CoV-2 primer pair for generating a first Flu amplicon from said biological sample. a first Flu A primer pair for generating an A amplicon, the primer pair comprising (i) a first Flu A primer and (ii) a second Flu A primer; and / or (d) a second Flu A primer pair for generating a second Flu A amplicon from the biological sample, the primer pair comprising (i) a third Flu A primer and (ii) a fourth Flu A primer; and / or (e) a Flu B primer pair for generating a Flu B amplicon from the biological sample, the primer pair comprising (i) a first Flu B primer and (ii) a second Flu B primer;and / or (f) a RSV A primer pair for generating a RSV A amplicon from the biological sample, the primer pair comprising (i) a first RSV A primer and (ii) a second RSV A primer; and / or (g) a RSV B primer pair for generating a RSV B amplicon from the biological sample, the primer pair comprising (i) a first RSV B primer and (ii) a second RSV B primer. Embodiment 79: The composition, kit, or method of embodiment 78, wherein the primer of (a)(ii) comprises a target hybridizing sequence consisting of a nucleotide sequence having a length of 22 to 25 nucleotides. Embodiment 80: The composition, kit, or method of embodiment 78, wherein the target hybridizing sequence of the primer of (a)(i) is SEQ ID NO: 116, SEQ ID NO: 119, or SEQ ID NO: 121. Embodiment 81: The composition or kit or method of embodiment 78, 79 or 80, wherein the primer (a)(i) comprises at least one nucleotide analogue; the primer (a)(i) comprises at least one nucleotide analogue that is 5-Me-C; the primer (a)(ii) is 24 nucleotides in length and comprises at least one nucleotide analogue; the primer (a)(ii) comprises at least one nucleotide analogue that is 5-Me-C; or a combination thereof. Embodiment 82: The composition or kit or method of embodiment 78-81, wherein the target hybridizing sequence of the primer (a)(ii) is SEQ ID NO: 117.

[0053] Embodiment 83: The composition or kit or method of any of embodiments 78 to 82, wherein the composition or kit further comprises a detection probe oligomer. Embodiment 84: The composition or kit or method of embodiment 83, wherein the composition or kit further comprises a detection probe oligomer comprising a target hybridizing sequence that is 25 to 40 contiguous nucleotides in length and consists of a nucleotide sequence contained within SEQ ID NO: 124. Embodiment 85: The composition or kit or method of embodiment 84, wherein the target hybridizing sequence of the detection probe oligomer is 25 to 27 contiguous nucleotides in length and is contained within SEQ ID NO: 125. Embodiment 86: The composition or kit or method of embodiment 83, 84, or 85, wherein the target hybridizing sequence of the detection probe oligomer is SEQ ID NO: 118.

[0054] Embodiment 87: The method further comprises one or more of the second SARS-CoV-2 primer pair, the first Flu A primer pair, the second Flu A primer pair, the Flu B primer pair, the RSV A primer pair, and the RSV B primer pair, wherein for the second SARS-CoV-2 primer pair of (a) 1(b), the target hybridizing sequences of 1(b)(i) and the target hybridizing sequences of 1(b)(ii) are SEQ ID NOs: 146 and 170; SEQ ID NOs: 126 and 127; SEQ ID NOs: 146 and 127; SEQ ID NOs: 263 and 264; SEQ ID NOs: 131 and 132; SEQ ID NOs: 181 and 129; SEQ ID NOs: 131 and 129; SEQ ID NOs: 262 and 257; SEQ ID NOs: 140 and 141; SEQ ID NOs: 134 and 135; SEQ ID NOs: 140 and 135; SEQ ID NOs: 143 and 144; (b) for the first Flu A primer pair in 1(c), the target hybridizing sequence of 1(c)(i) and the target hybridizing sequence of 1(c)(ii) are selected from the group consisting of SEQ ID NOs: 5 and 26; SEQ ID NOs: 5 and 27; SEQ ID NOs: 5 and 266; and SEQ ID NOs: 5 and 267; (c) for the second Flu A primer pair in 1(d), the target hybridizing sequence of 1(d)(i) and the target hybridizing sequence of 1(d)(ii) are selected from the group consisting of SEQ ID NOs: 23 and 25; (d) for the Flu B primer pair of 1(e), the target hybridizing sequence of 1(e)(i) and the target hybridizing sequence of 1(e)(ii) are selected from the group consisting of SEQ ID NOs: 67 and 58; SEQ ID NOs: 67 and 68; and SEQ ID NOs: 67 and 70;The composition or kit or method of any one of embodiments 78-86, wherein (e) for the RSV A primer pair of 1(f), the target hybridizing sequence of 1(f)(i) and the target hybridizing sequence of 1(f)(ii) are selected from the group consisting of SEQ ID NOs: 79 and 72; SEQ ID NOs: 79 and 73; SEQ ID NOs: 79 and 74; SEQ ID NOs: 79 and 92; SEQ ID NOs: 79 and 93; SEQ ID NOs: 79 and 94; SEQ ID NOs: 79 and 95; and SEQ ID NOs: 79 and 96; and (f) for the RSV B primer pair of 1(g), the target hybridizing sequence of 1(g)(i) and the target hybridizing sequence of 1(g)(ii) are selected from the group consisting of SEQ ID NOs: 99 and 104; SEQ ID NOs: 99 and 105; SEQ ID NOs: 100 and 115; SEQ ID NOs: 101 and 115; and SEQ ID NOs: 106 and 115.

[0055] Embodiment 88: The composition or kit or method of embodiment 84, wherein each primer individually comprises 0 to 20 nucleotide analogues.Embodiment 89: The composition or kit or method of embodiment 88, wherein at least one of the nucleotide analogues is a 5-Me-C analogue.

[0056] Embodiment 90: The composition or kit or method of embodiment 78, wherein each primer individually comprises 0 to 20 nucleotide analogues.Embodiment 91: The composition or kit or method of embodiment 90, wherein at least one of the nucleotide analogues is a 5-Me-C analogue.

[0057] Embodiment 92: The composition or kit or method as described above further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the second SARS-CoV-2 primer pair, wherein the target hybridizing sequence of 1(b)(i), the target hybridizing sequence of 1(b)(ii), and the target hybridizing sequence of the second SARS-CoV-2 region detection probe oligomer are selected from the group consisting of SEQ ID NOs: 146, 170, and 154; SEQ ID NOs: 126, 127, and 128; SEQ ID NOs: 146, 127, and 154; SEQ ID NOs: 146, 127, 154, and 128; SEQ ID NOs: 263, 264, and 265; SEQ ID NOs: 131, 132, and 133; SEQ ID NOs: 181, 129, and 130; SEQ ID NOs: 131, 129, and 133; SEQ ID NOs: 131, 129, and 130; SEQ ID NOs: 131, 129, 133, and 130; SEQ ID NOs: 262, 257, and 261; SEQ ID NOs: 140, 141, and 142; SEQ ID NOs: 134, 135, and 136; SEQ ID NOs: 140, 135, and 142; SEQ ID NOs: 140, 135, and 136; SEQ ID NOs: 140, 135, 142, and 136; SEQ ID NOs: 143, 144, and 145; SEQ ID NOs: 176, 239, and 193; SEQ ID NOs: 143, 239, and 145; SEQ ID NOs: 143, 239, 145, and 193; SEQ ID NOs: 137, 138, and 139; SEQ ID NOs: 176, 138, and 193; SEQ ID NOs: 176, 138, and 139; SEQ ID NOs: 176, 138, 193, and 139; SEQ ID NOs: 256, 254, and 258; SEQ ID NOs: 137, 254, and 139; SEQ ID NOs: 137, 254, and 258; SEQ ID NOs: 137, 254, 139, and 258; SEQ ID NOs: 240, 241, and 242; SEQ ID NOs: 256, 241, and 258; SEQ ID NOs: 256, 241, 258, and 242; SEQ ID NOs: 248, 247, and 246; SEQ ID NOs: 240, 247, and 242; SEQ ID NOs: 240, 247, and 246;and SEQ ID NOs: 240, 247, 242, and 246.

[0058] Embodiment 93: The composition or kit or method of embodiment 87 or 92, further comprising a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon produced by the first Flu A primer pair, wherein the target hybridizing sequence of 1(c)(i), the target hybridizing sequence of 1(c)(ii), and the target hybridizing sequence of the detection probe oligomer are selected from the group consisting of SEQ ID NOs: 5, 26, and 20; SEQ ID NOs: 5, 27, and 20; SEQ ID NOs: 5, 266, and 20; and SEQ ID NOs: 5, 267, and 20. Embodiment 94: The composition or kit or method as described above further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the second Flu A primer pair, wherein the target hybridizing sequence of 1(d)(i), the target hybridizing sequence of 1(d)(ii), and the target hybridizing sequences of the detection probe oligomer are selected from the group consisting of SEQ ID NOs:23, 25, and 9; SEQ ID NOs:23, 25, and 14; SEQ ID NOs:23, 25, 9, and 14; SEQ ID NOs:23, 6, and 9; SEQ ID NOs:23, 6, and 14; SEQ ID NOs:23, 6, 9, and 14; SEQ ID NOs:23, 28, and 9; SEQ ID NOs:23, 28, and 14; SEQ ID NOs:23, 28, 9, and 14; SEQ ID NOs:23, 25, and 8; SEQ ID NOs:23, 25, and 13; SEQ ID NOs:23, 25, 8, and 13; SEQ ID NOs:23, 6, and 8; The composition or kit or method of embodiment 87, 92, or 93, wherein the sequences are selected from the group consisting of: SEQ ID NOs: 23, 6, and 13; SEQ ID NOs: 23, 6, 8, and 13; SEQ ID NOs: 23, 28, and 8; SEQ ID NOs: 23, 28, and 13; or SEQ ID NOs: 23, 28, 8, and 13.Embodiment 95: The composition or kit or method of embodiment 87, 92, 93, or 94, wherein the composition or kit or method further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon produced by the Flu B primer pair, and wherein the target-hybridizing sequence of the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 59, 63, 64, 65, and 66. Embodiment 96: The composition, kit, or method of embodiment 87, 92, 93, 94, or 95, further comprising a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the RSV A primer pair, wherein the target hybridizing sequence of the detection probe oligomer is selected from the group consisting of SEQ ID NOs: 71, 75, 77, 78, 81, 83, 89, 90, 91, 97, and 98. Embodiment 97: The composition or kit or method further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the RSV B primer pair, wherein the target hybridizing sequence of 1(g)(i), the target hybridizing sequence of 1(g)(ii), and the target hybridizing sequence of the detection probe oligomer are selected from the group consisting of SEQ ID NOs:100, 115, and 102; SEQ ID NOs:101, 115, and 102; SEQ ID NOs:106, 115, and 102; SEQ ID NOs:100, 115, and 108; SEQ ID NOs:101, 115, and 108; SEQ ID NOs:106, 115, and 108; SEQ ID NOs:100, 115, and 110; SEQ ID NOs:101, 115, and 110; SEQ ID NOs:106, 115, and 110; SEQ ID NOs:100, 115, and 112; The composition or kit or method of embodiment 87, 92, 93, 94, 95, or 96, wherein the nucleic acid sequence is selected from the group consisting of: SEQ ID NOs: 101, 115, and 112; SEQ ID NOs: 106, 115, and 112; SEQ ID NOs: 100, 115, and 114; SEQ ID NOs: 101, 115, and 114; and SEQ ID NOs: 106, 115, and 114.

[0059] Embodiment 98: The composition or kit or method of any one of embodiments 83, 84, 85, 86, 89, 90, 91, 92, or 93, wherein individually each detection probe oligomer comprises a nucleotide sequence that is 0% to 100% nucleotide analogues, wherein each detection probe oligomer comprises a nucleotide sequence that is 0% to 100% 5-Me-C nucleotide analogues, or each detection probe oligomer comprises a nucleotide sequence that comprises at least one nucleotide analogue, wherein at least one nucleotide analogue is 5-Me-C. Embodiment 99: The composition or kit or method of any one of embodiments 83, 84, 85, 86, 89, 90, 91, 92, 93, or 98, wherein each detection probe oligomer comprises a 2' O-methyl backbone. Embodiment 100: The composition or kit or method of any one of embodiments 83, 84, 85, 86, 92, 93, 94, 95, 96, 97, 98, or 99, wherein at least one detection probe further comprises a detectable label. Embodiment 101: The composition or kit or method of any one of embodiments 83 to 86 and 15 to 22, wherein at least one detection probe further comprises a donor / acceptor label pair.

[0060] Embodiment 102: The composition according to any one of embodiments 78 to 101, wherein the composition is an aqueous formulation.Embodiment 103: The composition according to any one of embodiments 78 to 101, wherein the composition is a dry formulation.Embodiment 104: The composition according to embodiment 102 or embodiment 103, further comprising reagents for an amplification reaction, reagents for a detection reaction, reagents for a target capture reaction, or a combination thereof.Embodiment 105: A kit comprising the composition according to any one of embodiments 102 to 104.

[0061] Embodiment 106: (a) hybridizing, under stringent hybridization conditions, a target nucleic acid molecule from said biological sample with SEQ ID NOs: 9, 14, 20, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 59, 63, 64, 65, 66, 71, 75, 77, 78, 81, 83, 89, 90, 91, 97, 98, 102, 103, 108, 110, 112, 114, 118, 128, 130, 133, 136, 139, 142, 145, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 20 104. The method of any one of embodiments 92-101 for determining whether a biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B, comprising: (a) contacting a biological sample with one or more of detection probe oligonucleotides selected from the group consisting of: (a) 4, 193, 242, 246, 258, and 265; (b) detecting the presence or absence of a detectable label, the presence of which indicates that a probe:target nucleic acid duplex was formed in step (a); and (c) determining that the biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B based on whether the presence of the detectable label was detected in step (b). Embodiment 107. The method of embodiment 106, wherein the detection probe oligonucleotides are distinguishably labeled, allowing for the determination of the presence or absence of each of SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B in step (c). Embodiment 108: The method of embodiment 106 or 107, wherein the target nucleic acid that forms the probe:target nucleic acid duplex in step (b) is an amplicon generated using a primer pair.

[0062] Embodiment 109: A method for determining whether a biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B, the method comprising the steps of: (a) contacting target nucleic acid molecules from the biological sample with one or more primer pairs of any one of embodiments 78 to 91 to form an amplification reaction mixture; (b) performing a nucleic acid amplification reaction to generate amplicons from any target nucleic acid molecules in the amplification reaction mixture of step (a); and (c) determining whether amplicons are generated in step (b), thereby determining whether the biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B. Embodiment 110: The method of embodiment 109, wherein steps (b) and (c) are performed simultaneously. Embodiment 111: The method of embodiment 109 or 110, wherein the nucleic acid amplification reaction is a PCR reaction and / or a real-time nucleic acid amplification reaction. Embodiment 112: The method of any one of embodiments 109 to 111, wherein the detecting step (c) is carried out using one or more detection probe oligonucleotides.Embodiment 113: The method of embodiment 112, wherein the detection probe oligonucleotides are labeled with a donor / acceptor label pair.Embodiment 114: The method of embodiment 113, wherein the detection probe oligonucleotides are differentially labeled to allow determination of whether SARS-CoV-2, Flu A, Flu B, RSV A, and / or RSV B are present in the sample.

[0063] Embodiment 115: The method of any one of embodiments 109 to 114, wherein the biological sample comprises a clinical specimen that is a nasopharyngeal specimen.Embodiment 116: The method of any one of embodiments 109 to 115, wherein the biological sample comprises a clinical specimen that is a lower respiratory tract specimen.Embodiment 117: The method of any one of embodiments 109 to 116, wherein the biological sample is collected into a sample transport medium.Embodiment 118: The method of embodiment 117, wherein the sample transport medium comprises one or more of a buffer, a chelating agent, an anionic detergent, and a degradative enzyme.

[0064] Embodiment 119: A SARS-CoV-2 primer comprising a target hybridizing sequence that is 18 to 35 nucleotides in length, wherein the target nucleic acid sequence comprises the sequence of SEQ ID NO: 122 and is contained within SEQ ID NO: 123. Embodiment 120: A SARS-CoV-2 primer comprising a target hybridizing sequence that is 18 to 35 nucleotides in length, wherein the target hybridizing sequence hybridizes to SEQ ID NO: 124. Embodiment 121: An amplicon comprising a nucleotide sequence that is 89 to 100 contiguous nucleotides in length and that is generated using a primer pair comprising the primer of embodiment 119 and the primer of embodiment 120. Embodiment 122: The amplicon of embodiment 121, wherein the amplicon is a double-stranded amplicon that further comprises a reverse complementary nucleic acid strand. Embodiment 123: A SARS CoV-2 detection probe oligomer comprising a target hybridizing sequence having a length of 18 to 27 nucleotides, said target hybridizing sequence being included in SEQ ID NOs: 124 and 125. Embodiment 124: An amplicon comprising a nucleotide sequence having a length of 80 to 90 nucleotides, said sequence being included in SEQ ID NO: 150. Embodiment 125: The amplicon of embodiment 124, wherein the amplicon comprises the 5'-terminal residue of SEQ ID NO: 150 or the 3'-terminal residue of SEQ ID NO: 150, or the complement of the 5'-terminal residue of SEQ ID NO: 150 or the complement of the 3'-terminal residue of SEQ ID NO: 150. Embodiment 126: The amplicon of embodiment 125, wherein the amplicon is a double-stranded amplicon further comprising an antisense nucleic acid strand. Embodiment 127: The amplicon of any one of embodiments 124 to 126, wherein the amplicon sequence comprises SEQ ID NO: 181 or its reverse complement. Embodiment 128: A forward primer and probe combination, wherein (i) the forward primer is 13 to 28 nucleotides in length, is contained within SEQ ID NO: 169, and comprises a target hybridizing sequence comprising SEQ ID NO: 153, and (ii) the detection probe is 17 to 40 nucleotides in length, is contained within SEQ ID NO: 169, and comprises a target hybridizing sequence comprising SEQ ID NO: 157.Embodiment 129: A forward and reverse primer set, wherein the forward primer is 13 to 28 nucleotides in length and comprises a target hybridizing sequence contained within SEQ ID NO: 151 and comprising SEQ ID NO: 153, and wherein the reverse primer is 15 to 35 nucleotides in length and comprises a target hybridizing sequence contained within SEQ ID NO: 171 and comprising SEQ ID NO: 172. Embodiment 130: An amplicon 75 to 95 nucleotides in length and generated using a SARS-CoV-2 forward primer 13 to 28 nucleotides in length and comprising a target hybridizing sequence contained within SEQ ID NO: 169 and comprising SEQ ID NO: 153, and a SARS-CoV-2 reverse primer 15 to 35 nucleotides in length and comprising a target hybridizing sequence that hybridizes to SEQ ID NO: 173 and comprises SEQ ID NO: 172. Embodiment 131: An amplicon 68 to 103 nucleotides in length and comprising SEQ ID NO: 188. Embodiment 132: The amplicon of embodiment 131, wherein the amplicon sequence comprises SEQ ID NO: 187. Embodiment 133: The amplicon of embodiment 131, wherein the amplicon sequence comprises SEQ ID NO: 186. Embodiment 134: The amplicon of any of embodiments 131 to 133, wherein the amplicon sequence further comprises SEQ ID NO: 231. Embodiment 135: A SARS-CoV-2 primer pair, wherein the forward primer is 15 to 35 nucleotides in length and the reverse primer is 15 to 35 nucleotides in length, and wherein the primer pair generates the amplicon of any one of embodiments 131 to 134. Embodiment 136: An amplicon 115 to 137 nucleotides in length and comprises SEQ ID NO: 253. Embodiment 137. The amplicon of embodiment 136, wherein the amplicon sequence comprises SEQ ID NO: 249. Embodiment 138: The amplicon of embodiment 136, wherein the sequence comprises SEQ ID NOs: 249 and 250.Embodiment 139: A detection probe for detecting the amplicon of any one of embodiments 136 to 138, which is 15 to 40 nucleotides in length and comprises a target hybridizing sequence that is comprised within SEQ ID NO: 249, not comprised within SEQ ID NO: 251, and hybridizes to SEQ ID NO: 250. Embodiment 240: A combination comprising a SARS-CoV-2 reverse primer and a SARS-CoV-2 detection probe for amplifying and detecting an amplicon from any one of embodiments 136 to 138, wherein said reverse primer is 15 to 21 nucleotides in length and comprises a target hybridizing sequence that is comprised within SEQ ID NO: 250, not comprised within SEQ ID NO: 252, and hybridizes to SEQ ID NO: 249, and wherein said detection probe is 15 to 40 nucleotides in length and comprises a target hybridizing sequence that is comprised within SEQ ID NO: 249, not comprised within SEQ ID NO: 251, and hybridizes to SEQ ID NO: 250. Embodiment 141: A kit comprising any one of the primers according to embodiments 119, 120, 128, 130, 135, or 140. Embodiment 142: A kit comprising any one of the detection probes of embodiments 123, 128, 139, or 140. Embodiment 143: A kit comprising at least one primer from embodiment 141 and at least one detection probe from embodiment 142. Embodiment 144: A kit comprising a primer pair for generating from a SARS-CoV-2 target nucleic acid and an amplicon that is at least 90% identical to the amplicon of embodiments 121 to 127, 130 to 134, 136, 137, or 138. Embodiment 145: A kit comprising a detection probe for detecting a SARS-CoV-2 amplicon that is at least 90% identical to the amplicon of embodiments 121 to 127, 130 to 134, 136, 137, or 138.

[0065] The foregoing and other objects, features, and advantages of the above compositions and methods will be apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "an oligonucleotide" includes a plurality of oligonucleotides, etc. The conjunction "or" shall be construed in the inclusive sense, i.e., as equivalent to "and / or," unless an inclusive sense is irrational in the context.

[0067] It is recognized that "about" necessarily precedes temperatures, concentrations, times, and the like discussed in this disclosure, so that small and insubstantial deviations are within the scope of the present teachings herein. In general, the term "about" indicates insubstantial variations in the amounts of components of a composition that do not have any significant effect on the activity or stability of the composition. All ranges should be interpreted as including the endpoints, absent an exclusionary expression such as "excluding the endpoints"; thus, for example, "within 10 to 15" includes the values ​​10 and 15, and all whole and partial values ​​therebetween (where applicable).

[0068] Unless specifically noted, embodiments herein that describe "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the described components; embodiments herein that describe "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the described components; and embodiments herein that describe "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the described components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional components, compositions, or method steps that do not essentially alter the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics include the ability to detect a target nucleic acid present in a sample with a specificity that distinguishes the target nucleic acid from other known respiratory pathogens. Any component, composition, or method step that has a substantial effect on the basic and novel characteristics of the present disclosure falls outside the scope of this term.

[0069] The term "complement" refers to a nucleic acid molecule that contains a contiguous nucleotide sequence that is complementary (with respect to standard nucleotides, A:T, A:U, C:G) to a contiguous nucleic acid sequence of another nucleic acid molecule. Two nucleic acid sequences are "fully complementary" if their respective contiguous nucleic acid sequences are at least 70% complementary. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0070] "Substantially homologous," "substantially corresponding," or "substantially corresponds" means that a nucleic acid molecule comprises a contiguous nucleic acid sequence that is at least 70% homologous to a contiguous nucleic acid sequence of another nucleic acid molecule.

[0071] A "sample" or "biological sample" is any tissue or polynucleotide-containing material obtained from a human, animal, or environmental sample and may contain a target nucleic acid. Biological samples include peripheral blood, mucus, plasma, serum, saliva, cerebrospinal fluid, urine, bone marrow, or other bodily fluids, biopsied tissue, or other substances of biological origin, as well as solutions or compositions containing substances of biological origin, such as bronchial lavage fluid. Samples can be obtained from several sources, including clinical sources where the sample is collected to determine the presence or absence of a target nucleic acid in the sample and then provide a diagnosis to the patient. Samples can be chemically and / or mechanically treated to disrupt tissue or cellular structures, thereby releasing intracellular components into solution.

[0072] "Nucleotide" includes both nucleotides and nucleosides, including deoxyribonucleotides (e.g., dATP, dCTP, dGTP, dTTP), ribonucleotides (e.g., rATP, rCTP, rGTP, rUTP), and analogs thereof. A nucleotide comprises a purine or pyrimidine base glycosidically linked to a ribose or deoxyribose sugar, and a phosphate group linked to the ribose or deoxyribose sugar. A nucleoside comprises a purine or pyrimidine base glycosidically linked to a ribose or deoxyribose sugar, but lacks the phosphate residue present in a nucleotide. As used herein, nucleotides and nucleosides refer to monomers of DNA or RNA, respectively. See, for example, Kornberg and Baker, DNA Replication, 2nd Edition (Freeman, San Francisco, 1992).

[0073] In reference to a compound, the term "analog" refers to a compound that has a similar structure to that of another compound, but differs from it in that one or more different atoms, functional groups, or substructures have been removed or replaced with one or more other atoms, functional groups, or substructures. In the context of nucleotides or nucleosides, an analog refers to a compound that can be incorporated into nucleic acid molecules (e.g., primers, probes, and / or amplification products) in the same way as the analog nucleotide / side. Nucleotide / side analogs are generally added to synthetic oligonucleotides (e.g., primers and probes) using phosphoramidite chemical techniques and devices. Nucleotide / side analogs are generally added to amplification products by including the analog in the reaction mixture, where an appropriate polymerase (e.g., DNA polymerase) incorporates the analog into the amplification product. Nucleotide / side (hereinafter "nucleotide") analogs include synthetic nucleotides with modified base moieties and / or modified sugar moieties and / or modified phosphate groups. See, e.g., Scheit, Nucleotide Analogues (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990). Such analogs include synthetic nucleotides designed to enhance binding properties, reduce complexity, increase specificity, etc.

[0074] "DNA" refers to deoxyribonucleic acid, and a DNA oligonucleotide (e.g., a primer or probe) refers to a polymer of deoxyribonucleotides linked by phosphodiester bonds. DNA oligonucleotides can be single-stranded (ssDNA) or double-stranded (dsDNA) and can contain both single-stranded and double-stranded (or "duplex") regions. "RNA" refers to ribonucleic acid, and an RNA oligonucleotide (e.g., a probe) refers to a polymer of ribonucleotides linked by phosphodiester bonds. RNA oligonucleotides can be single-stranded (ssRNA) or double-stranded (dsRNA) and can contain both single-stranded and double-stranded (or "duplex") regions. Single-stranded DNA (or regions thereof) and ssRNA can hybridize to form a double-stranded complex (or region) if sufficiently complementary. "RNA equivalent," "DNA equivalent," "RNA equivalent base," and "DNA equivalent base" refer to RNA and DNA molecules that have similar complementary base-pair hybridization properties. RNA and DNA equivalents have different sugar moieties (ribose vs. deoxyribose) and may differ, for example, by the presence of uracil in RNA and thymine in DNA. The differences between DNA and RNA equivalents do not contribute to differences in homology (or sequence identity), because the equivalents have the same degree of homology to a particular sequence.

[0075] The term "oligonucleotide" (or "oligomer" or "oligo") refers to a polymeric compound containing two or more linked RNA nucleotides, DNA nucleotides, RNA nucleotide analogs, DNA nucleotide analogs, or combinations thereof. An oligonucleotide may contain other molecules that may be present in the linked sequence of nucleotides and do not interfere with hybridization of the polynucleotide with a second molecule having a complementary sequence. For example, an oligonucleotide may contain two or more linked nucleotides on one side of a linker molecule and two or more linked nucleotides on a second side of the linker molecule, often in the configuration of a molecular torch. Oligonucleotides are preferably polymeric chains of 10 to 200 contiguous nucleotides and are preferably synthesized using any of a variety of well-known enzymatic or chemical methods. Whenever an oligonucleotide (or other nucleic acid) is represented by a string of letters, the nucleotides are understood to be in the 5'-3' orientation from left to right, and unless otherwise noted, "A" defines adenosine (dATP / rATP) or an analog thereof, "C" defines cytidine (dCTP / rCTP) or an analog thereof, "G" defines guanosine (dGTP / rGTP) or an analog thereof, "U" defines uracil (rUTP) or an analog thereof, and "T" defines thymidine (dTTP) or an analog thereof. Oligonucleotides of the present disclosure contain the four naturally occurring nucleotides and often contain unnatural nucleotide analogs. Oligonucleotides disclosed herein include, but are not limited to, amplification oligonucleotides (e.g., primers and promoter primers), detection probes (e.g., linear probes, TaqMan probes, AE-labeled probes, molecular torches, and molecular beacons), target capture oligomers, amplicons, amplification products, and in vitro transcripts. The "backbone" of an oligonucleotide may be composed of a variety of linkages known in the art, including one or more sugar-phosphodiester linkages, peptide-nucleic acid linkages (PNAs), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.The sugar moiety of an oligonucleotide can be either ribose or deoxyribose, or similar compounds with known substitutions (such as 2'-O-methylribose) and 2' halide substitutions (such as 2'-O-Me or 2'-F). Nucleotide analogs in an oligonucleotide sequence can include inosine or "I", 5-Me-dC, isoguanine, other derivatives of purine or pyrimidine bases, or abasic residues (e.g., nucleoside residues). See, for example, The Biochemistry of the Nucleic Acids, pages 5-36, Adams et al., 11th Edition, 1992; and PCT Publication No. WO 93 / 13121.

[0076] A "probe" is an oligonucleotide that specifically hybridizes to a target nucleic acid sequence in a nucleic acid, preferably amplified nucleic acid, under conditions that promote hybridization, forming a detectable hybrid. The probe oligonucleotide contains one or more of a contiguous nucleotide sequence, a target-hybridizing sequence, a non-target-hybridizing sequence, a detectable label, a linker, and a nucleotide analog. The probe preferably has an oligonucleotide length of about 10 to 100 contiguous nucleotides and includes RNA, DNA, analogs, modified backbones, and combinations thereof. The probe may contain a target-specific sequence and, optionally, other sequences that are non-target-hybridizing sequences (e.g., sequences that do not hybridize to the nucleic acid to be detected). Such non-target-hybridizing sequences may include sequences that contribute to the three-dimensional structure of the probe, as is common in molecular torches and molecular beacons, for example. See, for example, U.S. Patent Nos. 5,118,801, 5,312,728, 5,925,517, and 6,361,945. Probes can contain a detectable label attached either to the end of the probe or to the interior of the probe. A detectable label refers to one or more atoms that can be specifically detected to indicate the presence of a substance to which the one or more atoms are attached. Labels include dyes, particles, chromophores (e.g., atoms or molecules that impart a detectable color), combination fluorescent energy transfer labels, electrophores, redox-active moieties (e.g., transition metals), enzymes, haptens, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent moieties), fluorophores, mass labels, and radioactive labels. Labels and related detection methods are well known.See, e.g., Styer and Haugland, (1967), Proc. Natl. Acad. Sci. USA 98:719; U.S. Patent Nos. 6,627,748; 6,150,097; 6,004,745; 5,948,899; 5,656,207; 5,658,737; 5,591,578; 5,491,063; 5,283,174; and 5,201,015.

[0077] The term "fluorophore" refers to the fluorescent compound that can re-emit light when excited by light.Fluorophores include, for example, fluorescent lanthanide complexes (including those of europium and terbium), fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, Cy3, Cy5, CalFluor Red™, CalFluor Orange™, stilbene, Quasar dyes (for example, Quasar 570, Quasar 670, Quasar 705), Lucifer Yellow, Cascade Blue™, Texas Red, Alexa dyes, phycoerythrin, Bodipy and others known in the art. See, e.g., Haugland, Molecular Probes Handbook (Eugene, OR), 6th ed.; The Synthegen catalog (Houston, TX); Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press New York (1999), and WO 98 / 59066.

[0078] The term "quencher" is used to refer to a molecule that absorbs light. Quenchers are commonly used in combination with light-emitting labels, such as fluorophores, to absorb the emitted light when in close proximity to the fluorophore. Quenchers are well known in the art and include, for example, Black Hole Quencher™ (or BHQ™, BHQ-1™, or BHQ-2™), Blackberry® Quencher, Dabcyl, QSY, and Tamra™ compounds, to name a few.

[0079] Linear probe, molecular torch and beacon are preferably labeled with an interactive pair of detectable labels.Preferably, the detectable labels that are members of an interactive pair of detectable labels interact with each other through FRET or non-FRET energy transfer mechanism, and are often referred to as "donor / acceptor pair".Here, donor first absorbs energy, then transfers energy, and acceptor is the part to which this energy is subsequently transferred.When the two labels of a donor / acceptor pair are held close enough that the energy emitted by one label can be accepted or absorbed by the second label, the two labels are said to be in an "energy transfer relationship" with each other.This is, for example, when molecular beacon or molecular torch is kept in a "closed" state by forming a stem duplex, and the fluorescence emission from the fluorophore attached to one arm of the probe is quenched by the quencher moiety on the opposite arm. This is also the case, for example, when a linear probe is labeled with a fluorophore and a quencher at a certain distance along the linear probe, and the fluorescence emission from the bound fluorophore is quenched by the bound quencher. In these cases, the spatial separation of the fluorophore and quencher molecules (for example, by "opening" a molecular torch or beacon, or by hydrolysis of the linear probe molecule) is required. Examples of donor / acceptor pairs include pairs of fluorophores (e.g., fluorescein, IAEDANS, EDANS, coumarin, BODIPY FL, BODIPY, Lucifer Yellow, eosin, erythrosine, tetramethylrhodamine, CalOrange, CalRed, Quasar, Texas Red, CY5, or CY3) and quenchers (e.g., tetramethylrhodamine, fluorescein, DABCYL, BHQ-1, BHQ-2, QSY7, or BBQ).Labels are available from LGC Biosearch Technologies (Petaluma, CA); Glen Research (Sterling, VA); Integrated DNA Technologies (Skokie, IL); Thermo Fisher (Waltham, MA); and others.

[0080] The synthesis technique and the method of binding label to nucleic acid and detecting label are well known in the art.See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; U.S. Patent No. 5,658,737, U.S. Patent No. 5,656,207, U.S. Patent No. 5,547,842, U.S. Patent No. 5,283,174 and U.S. Patent No. 4,581,333; and published European Patent Application No. 0 747 706).

[0081] An "amplification primer" or "primer" is an optionally modified oligonucleotide that can hybridize to a target nucleic acid sequence or its complement and participate in a nucleic acid amplification reaction. A primer oligonucleotide comprises one or more of a contiguous nucleotide sequence, a target-hybridizing sequence, a non-target-hybridizing sequence, a linker, and a nucleotide analog. A primer preferably has an oligonucleotide length of about 10 to 100 contiguous nucleotides. The target nucleic acid sequence of a primer generally refers to both a sequence (or its complement) contained within the genetic information of the organism to be detected and a sequence (or its complement) contained within the amplified nucleic acid molecule that specifically hybridizes to at least a portion of the primer oligonucleotide using standard hydrogen bonding. A primer hybridizes to a target nucleic acid sequence and typically has a 3' end that can be extended by a DNA polymerase to incorporate nucleotides complementary to the target nucleic acid sequence and generate its double-stranded portion (e.g., a promoter provider is a type of primer that does not have an extendable 3' end; see, e.g., U.S. Pat. No. 7,939,260). Primers include, but are not limited to, non-T7 primers, promoter primers, promoter providers, and T7 promoter primers.

[0082] "Capture oligonucleotide" refers to at least one nucleic acid oligonucleotide that allows the connection of a target nucleic acid and an immobilized oligonucleotide due to base pair hybridization (preferably resulting in an immobilized probe:capture oligonucleotide:target nucleic acid complex). A capture oligonucleotide preferably contains two binding regions: a target nucleic acid binding region and an immobilized probe binding region (usually contiguous on the same oligonucleotide), but the capture oligonucleotide may contain a target nucleic acid binding region and an immobilized probe binding region present on two different oligonucleotides connected together by one or more linkers. The target hybridization region of a capture probe can be specific to the target nucleic acid (e.g., sufficiently complementary to the target nucleic acid sequence) or non-specific to the target nucleic acid. Target capture systems including capture oligonucleotides are described in U.S. Patent No. 6,110,678, WO 2021 / 097358, and 9,051,601.

[0083] "Immobilized probe" or "immobilized nucleic acid" refers to a nucleic acid that directly or indirectly connects a capture oligonucleotide to an immobilized support. An immobilized probe is an oligonucleotide attached to a solid support that facilitates separation of the bound target nucleic acid from unbound material in a sample.

[0084] The term "solid support" refers to any suitable medium present in a solid phase to which an immobilized probe or other agent can be attached or immobilized, either covalently or noncovalently.

[0085] "Separating" or "purifying" or "isolating" means that one or more components of the biological sample are removed from one or more other components of the sample. Sample components generally comprise nucleic acids in an aqueous liquid phase; components may also include other substances (e.g., proteins, carbohydrates, lipids, and labeled probes). Preferably, the separating, isolating, or purifying step removes at least about 70%, more preferably at least about 90%, and even more preferably at least about 95% of the other components present in the sample.

[0086] "Amplicon" refers to a DNA and / or RNA polynucleotide that is the product of a nucleic acid amplification or replication process, which can be formed using a variety of methods, including polymerase chain reaction (PCR) and transcription-associated amplification (e.g., TMA).

[0087] The term "multiplex," when used to describe a PCR or TMA amplification reaction, is characterized in that two or more distinct amplification products, or amplicons, are produced by means of using two or more pairs of amplification primers in the same amplification reaction.

[0088] "Target nucleic acid" or "target" refers to a nucleic acid that comprises a target nucleic acid sequence. As described herein, target nucleic acids include Flu A nucleic acids, Flu B nucleic acids, SARS-CoV-2 nucleic acids, RSV A nucleic acids, and RSV B nucleic acids. "Target nucleic acid sequence" (also referred to as "target nucleotide sequence," "target sequence," "target region," or "target nucleic acid molecule") refers to a specific deoxyribonucleotide or ribonucleotide molecule or nucleotide sequence that comprises all or a portion of the nucleotide sequence of a single-stranded nucleic acid molecule, and complementary deoxyribonucleotide or ribonucleotide sequences thereto.

[0089] "Transcription-associated amplification" refers to any type of nucleic acid amplification that uses an RNA polymerase to generate multiple RNA transcripts from a nucleic acid template. One example of a transcription-associated amplification method, referred to as "transcription-mediated amplification" (TMA), generally uses an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-template complementary oligonucleotide, which may optionally include one or more similar oligonucleotides. Variations of TMAs are well known in the art and are described, for example, in U.S. Patent Nos. 5,437,990; 5,399,491; 5,554,516; 5,130,238; 4,868,105; and 5,124,246; published PCT application numbers WO 93 / 22461, WO 88 / 01302, WO 88 / 10315, WO 94 / 03472, and WO 95 / 03430.

[0090] Sample, lysis, and target capture reagents commonly used with amplification assays include the following: "Sample transport medium" or "STM" is a phosphate-buffered solution (pH 6.7) containing a chelating agent (EDTA, EGTA, and / or the trisodium salt of methylglycine diacetic acid (Trilon M, NaMGDA, BASF)), and lithium lauryl sulfate (LLS). Sample transport medium formulations may further include one or more of the following: Good's buffer; a stabilizer (e.g., propylene glycol, polyol, sugar, sugar alcohol, lactic acid, boric acid, or a boric acid derivative); sodium xylene sulfonate; a surfactant; trisodium citrate; and one or more degradative enzymes (e.g., amylase, lipase, and protease).

[0091] The "target capture reagent" or "TCR" is a HEPES-buffered solution (pH 6.4) containing lithium chloride and EDTA along with magnetic solid support particles (1 micron SERA-MAG™ MG-CM particles, Seradyn, Inc. Indianapolis, IN) to which (dT)14-immobilized probe molecules are covalently attached. The TCR contains multiple oligos, which may include one or more target capture oligos (TCOs) and, optionally, one or more promoter primers. For multiplex BiPhasic TMA amplification reactions, the TCR contains a TCO and a promoter primer configured to hybridize to each target nucleic acid to be amplified and / or detected. See, e.g., WO 2014 / 036369 A1. For multiplex PCR amplification reactions, the TCR may contain a TCO configured to hybridize to each target nucleic acid to be amplified and / or detected. Alternatively, for multiplex amplification reactions, the TCR contains a "wobble probe TCO" (see, e.g., U.S. Patent No. 9,051,601). It hybridizes non-specifically to several target nucleic acids to be amplified and / or detected. In some embodiments, the target nucleic acids to be amplified and / or detected are selected from the group consisting of influenza A, influenza B, SARS-CoV-2, respiratory syncytial virus A, and respiratory syncytial virus B.

[0092] An "amplification reagent," "AMP reagent," or "AR" is a Tris-buffered solution (pH 7-8) containing magnesium chloride, potassium chloride, four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), four ribonucleotide triphosphates (rNTPs: ATP, CTP, GTP, and UTP), and one or more primers. For multiplex amplification, the AMP reagent includes one or more primers configured to hybridize to each target nucleic acid to be amplified and / or detected. In some embodiments, the target nucleic acid to be amplified and / or detected is selected from the group consisting of Flu A, Flu B, SARS-CoV-2, RSV A, RSV B, and combinations thereof. In some embodiments, the AMP reagent further includes a reverse transcriptase, an RNA polymerase, salts, and cofactors. The reverse transcriptase can be, but is not limited to, MMLV reverse transcriptase. The RNA polymerase can be, but is not limited to, T7 RNA polymerase.

[0093] A "promoter reagent" or "PR" is a Tris-buffered solution containing magnesium chloride, potassium chloride, four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), four ribonucleotide triphosphates (rNTPs: ATP, CTP, GTP, and UTP), one or more promoter primers, and one or more probes. The promoter primers in the promoter reagent target the same target nucleic acid as the promoter primers in the TCR. The promoter primers may have the same sequence as the promoter primers in the TCR or may have a different sequence from the promoter primers in the TCR. For multiplex amplification, the promoter reagent includes a promoter primer configured to hybridize to each target nucleic acid to be amplified and / or detected. In some embodiments, for multiplex amplification, the promoter reagent includes a probe configured to hybridize to each target nucleic acid to be detected. In some embodiments, the target nucleic acids to be amplified and / or detected are selected from the group consisting of Flu A, Flu B, SARS-CoV-2, RSV A, RSV B, and combinations thereof. In some embodiments, the promoter reagent further comprises a reverse transcriptase, an RNA polymerase, salts, and cofactors. The reverse transcriptase may be, but is not limited to, MMLV reverse transcriptase. The RNA polymerase may be, but is not limited to, T7 RNA polymerase. The enzyme reagent used in the amplification or pre-amplification reaction mixture is a HEPES-buffered solution (pH 6.5-8) containing MMLV reverse transcriptase, T7 RNA polymerase, salts, and cofactors.

[0094] Described herein are compositions (including kits and reagents) and methods for selectively detecting the nucleic acids of various viral pathogens in a sample, specifically influenza A (Flu A), influenza B (Flu B), SARS-CoV-2, respiratory syncytial virus A (RSV A), and respiratory syncytial virus B (RSV B). These compositions and methods can be used, for example, in diagnostic applications, to screen clinical samples, nasopharyngeal samples, bronchoalveolar samples, blood donations and blood products, or other tissues that may contain one or more of these pathogenic organisms.

[0095] As will be appreciated, any primer and probe sequences specific for Flu A, Flu B, SARS-CoV-2, RSV A, RSV B and / or other pathogenic viral targets can be used as primers or probes in any suitable primer / probe-based in vitro nucleic acid amplification method adapted for amplification of the intended target nucleic acid.

[0096] The amplification primers are useful as components of simplex or multiplex amplification reactions, in which amplicon species can be generated from the target-specific primers in the reaction mixture. Multiplex amplification reactions include primer pairs for amplifying two or more of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B, or further include primers for Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B and one or more additional targets (e.g., human metapneumovirus, rhinovirus, adenovirus, parainfluenza virus, non-SARS-CoV-2 coronavirus, and / or Bordetella).

[0097] Amplification methods useful in the context of the present disclosure include polymerase chain reaction (PCR); transcription-mediated amplification (TMA); nucleic acid sequence-based amplification (NASBA); strand displacement amplification (SDA); and amplification methods using self-replicating polynucleotide molecules and replicating enzymes (e.g., MDV-1 RNA and Q-beta enzyme). Methods for carrying out each of these various amplification techniques can be found in U.S. Patent Nos. 4,965,188, 5,399,491, 5,455,166, and 5,472,840; published European patent application EP 0 525 882; and Lizardi et al., BioTechnology 6:1197 (1988). In a particularly preferred embodiment, Flu A, Flu B, RSV A, and RSV B nucleic acid sequences are amplified using real-time RT PCR or BiPhasic TMA.

[0098] Due to the lack of sequence conservation among respiratory virus lineages and to accommodate mismatches / mutations between primers or probes and their corresponding target nucleic acid sequences in viral target nucleic acids, degenerate bases and non-Watson-Crick (NWC) base pairings can be included in primer or probe oligonucleotides in some preferred embodiments. An NWC position in an oligonucleotide refers to a position where the oligonucleotide has non-Watson-Crick pairing (e.g., GU, GT, or GA) (either G or U / T / A can be the base in the oligonucleotide) or is configured to hybridize to at least one target nucleic acid sequence using a nucleotide analog (e.g., G-inosine or G-5-methylC). In some embodiments, the NWC position is configured to hybridize via a wobble (GU or GT) or purine-purine (GA) pair. In some embodiments, when one or more degenerate bases are identified in the target nucleic acid sequence for a single primer or probe, multiple primer or probe species can be synthesized to include all base combinations.

[0099] Primers useful for carrying out amplification reactions can have different lengths to accommodate the presence of extraneous sequences that may not be involved in target binding and may not substantially affect the amplification or detection procedures.For example, promoter primers useful for carrying out amplification reactions according to the present disclosure have at least the minimum sequence that hybridizes with the desired target nucleic acid sequence and the promoter sequence located upstream of that minimum sequence.However, the insertion of a sequence between the target binding sequence and the promoter sequence can change the length of the primer without impairing its usefulness in amplification reactions.In addition, the length of the amplification primers and detection probes is a matter of choice, as long as the sequence of these oligonucleotides meets the minimum essential requirements for hybridizing with the desired complementary target sequence.

[0100] Hybridization assay probes useful for detecting Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B nucleic acid sequences comprise a sequence of bases substantially complementary to a selected target nucleic acid sequence (or an amplicon representing the corresponding region and its adjacent or surrounding regions) in the Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B genome. Such probes can optionally have additional bases outside of the targeted nucleic acid region, which may or may not be complementary to Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B nucleic acid.

[0101] Preferred probes are sufficiently homologous to the target nucleic acid to hybridize under stringent hybridization conditions corresponding to the designed amplification and detection reactions. For example, in PCR, extension and detection reactions are performed such that the oligonucleotides hybridize to their target nucleic acid sequences at a reaction temperature of approximately 60°C. Probes according to the present disclosure have sequences complementary to or corresponding to preselected target regions of the specific viral target nucleic acid targeted by the probe. Preferred probes have probe sequences ranging from 10 to 100 nucleotides in length that include a target hybridizing sequence of bases, along with any base sequence that is not complementary to the nucleic acid to be detected.

[0102] Amplification of nucleic acids by transcription-mediated amplification (TMA) is a common technique in molecular biology and typically requires sample preparation, amplification, and product analysis. These steps are usually performed sequentially, although amplification and analysis can occur simultaneously. In an exemplary BiPhasic TMA reaction, a T7 (promoter) primer and a TCO are hybridized to the target sequence during target capture, followed by removal of excess T7 primer during a wash step prior to the first amplification reaction. In some embodiments, a TCO is hybridized to the target sequence during target capture. Excess TCO may also be removed during a wash step prior to the first amplification reaction. During the first amplification phase, AMP reagents and, optionally, enzyme reagents are introduced. In the presence of reverse transcriptase, the T7 primer hybridized to the captured target is extended to create a cDNA copy. A non-T7 primer subsequently hybridizes to the cDNA and is extended, filling in the promoter region of the T7 primer and creating an active double-stranded DNA template. T7 polymerase then generates multiple RNA transcripts from the template. The NT7 primer subsequently hybridizes to the RNA transcripts and is extended, generating promoterless cDNA copies of the target RNA template. The RNA strand is degraded by the RNase activity of the reverse transcriptase. Because free T7 primers are not available in the Phase 1 amplification mixture, the reaction does not proceed. The second phase begins with the addition of a promoter reagent, thus initiating the exponential amplification and detection of the cDNA pool generated in Phase 1.

[0103] Plate Setup: In some embodiments, four different plates are set up for use with two automated KingFisher devices. Reactions can also be performed in tubes or other containers. Plate 1 (TCR plate) contains the sample. Target capture reagent (e.g., 100 μL) is added to this plate. The TCO and T7 primer hybridize to the target nucleic acid (e.g., 400 μL sample). The TCO:target nucleic acid:T7 primer (pre-amplified hybrid) is captured using magnetic beads (probes immobilized on a solid support) and a magnet. For capture, Plate 1 is placed in a heat block and heated to 60-65°C (e.g., 62°C) for 20-30 minutes, followed by incubation at a lower temperature (e.g., 23°C) for 20 minutes to 2 hours. For single-phase TMA, the T7 primer may not be present in the TCR mixture. In some embodiments, the sample is combined with a TCR containing a TCO and a T7 primer for BiPhasic amplification. The sample and TCR mixture is then removed from the plate or tube, and the beads are washed twice with wash buffer. Plate 2 is a deep-well plate and holds 200-500 μL / well of wash buffer. The wash buffer contains detergent and alcohol, which is used to wash the captured pre-amplified hybrids. The captured hybrids are then transferred to Plate 3, which contains 200-500 μL / well of wash buffer and is used to provide a second wash of the captured pre-amplified hybrids. The captured hybrids are then transferred to Plate 4, which contains 50 μL / well of AMP reagent. Plate 4 is processed for real-time isothermal amplification and detection.

[0104] Biphasic Transcription-Mediated Amplification and Real-Time Detection. Phase 1 Amplification: Plate 4 is first incubated at approximately 42-44°C for 5-15 minutes (e.g., 43°C for 5 minutes). After the first incubation, 25 μL of enzyme reagent containing reverse transcriptase and T7 RNA polymerase is added to each well, and Plate 4 is incubated at approximately 42-44°C for approximately 5 minutes to generate a first amplification product. Phase 2 Amplification: 25 μL of promoter reagent containing T7 primer and probe oligonucleotide is added to the first amplification product, and the reaction is incubated at 42-43°C for 30-60 minutes to allow the formation of a second amplification product. The amplification product is detected in real time by recording the fluorescent signal from the probe at regular intervals.

[0105] Amplification of nucleic acids by polymerase chain reaction (PCR) is a common technique in molecular biology and typically requires sample preparation, amplification, and product analysis. These steps are usually performed sequentially, although amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs simultaneously with amplification in the same tube within the same instrument. This combined approach reduces sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions because samples do not need to be removed from their closed containers for further analysis. The concept of combining amplification and product analysis has become known as "real-time" PCR. See, for example, U.S. Patent Nos. 6,174,670 and 8,137,616. In real-time PCR, PCR product formation is monitored with each cycle of PCR. Amplification is typically measured in a thermocycler with additional devices for signal generation and detection from labels attached to probe oligonucleotide species during the amplification reaction. Many such devices for performing multiplexed diagnostic assays with three, four, or more distinguishably labeled hybridization probes in one reaction vessel are known in the art.

[0106] As is known, different formats exist for probe-based real-time detection of amplified DNA in multiplex assays. Common examples include the "TaqMan" probe system, molecular beacons, and molecular torches.

[0107] In the TaqMan probe format, single-stranded hybridization probes for a given target are labeled with a donor / acceptor pair of detectable labels. When the donor (e.g., a fluorophore moiety) is excited with light of the appropriate wavelength, the absorbed energy is transferred to the acceptor (e.g., a quencher moiety) according to the principle of FRET. During the annealing step of the PCR reaction cycle, the hybridization probe binds to the target DNA and is degraded by the 5'-3' exonuclease activity of Taq polymerase during the subsequent extension phase. As a result, the excited donor moiety and the acceptor moiety become spatially separated, thus allowing an unquenched signal (e.g., fluorescent emission) from the donor to be detected by the device. See, e.g., U.S. Patent No. 5,538,848.

[0108] Molecular beacons and molecular torch formats also typically include a hybridization probe labeled with a donor / acceptor pair (the donor and acceptor moieties are located at opposite ends of the probe). As a result of the secondary structure of the probe, which often involves hybridization of complementary regions at the ends of the probe, both the donor and acceptor moieties (e.g., fluorescent and quencher moieties) are in spatial proximity in solution. After hybridization of the target-hybridizing region of the probe to the desired target nucleic acid sequence, the donor and acceptor moieties are separated from each other, so that the emission can be measured after excitation of the donor moiety with light of an appropriate wavelength. See, for example, U.S. Patent No. 5,118,801.

[0109] In some preferred embodiments, real-time PCR is used to amplify and detect multiple target DNA sequences in multiplex assays.This method involves providing a composition or reaction mixture comprising nucleic acid from biological sample, probe, primer, and suitable polymerase activity for catalyzing amplification, subjecting this reaction mixture to a thermocycling protocol so that the amplification of multiple target sequences occurs, and monitoring the hybridization of each of the probe molecular species (for example, a pair of FRET hybridization probes) at least once after multiple amplification cycles.In the embodiment where the viral target nucleic acid to be detected is composed of one or more RNA molecules, this method typically involves first using reverse polymerase activity to convert RNA into DNA (for example, "complementary" DNA or "cDNA").

[0110] In such multiplexed embodiments, the composition or reaction mixture typically contains at least two, and preferably three to eight, detection probes. Preferably, each detection probe contains a donor / acceptor label pair. For multiplexed assays configured to distinguish each of the target nucleic acids from the others, the detection probes each contain a distinguishable donor / acceptor label pair. In addition, such compositions or reaction mixtures also contain several reagents, including one or more of the following: a buffer designed for PCR, dNTPs, a template-dependent DNA polymerase (preferably a thermostable DNA polymerase), and a reverse transcriptase.

[0111] During or after the amplification process is completed, the reaction is monitored to detect stable hybridization between one or more of the distinguishably labeled probe species present in the reaction and their corresponding target nucleic acid sequences (carried in amplicons generated using corresponding primer pairs for the particular viral (or other) pathogen to be detected). Based on whether the donor moiety from each of the different donor / acceptor pairs is detected, it can then be determined whether the biological sample contains Flu A, Flu B, SARS-CoV-2, RSV A, and / or RSV B and / or other pathogens targeted in a particular assay.

[0112] Certain preferred kits include one or more of the following: probes, primers, capture oligonucleotides, internal control oligonucleotides, other auxiliary oligonucleotides, buffers, dNTPs, DNA polymerase, reverse transcriptase, and instructions (or links to websites providing such instructions) for using the kit components.

[0113] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0114] General Reagents and Methods. Unless otherwise indicated, amplification was performed using a Panther Fusion instrument (Hologic, Inc.). Viral isolates used as amplification targets or controls were diluted in an appropriate medium, such as specimen transport medium (Hologic, Inc., catalog number PRD-04423 or PRD-04339); Micro Test M4 Media (Remel, Inc., catalog number R12500), Micro Test M5 Viral Transport Medium (Remel, Inc., catalog number R12515), Micro Test M6 Viral Transport Medium (Remel, Inc., catalog number R12530), Micro Test M4RT Viral Transport Medium (Remel, Inc., catalog number R12505), or Copan Universal Transport Medium (Copan Diagnostics, Inc., catalog number 330C). Nucleic acids were extracted from viral isolates using a nonspecific target capture procedure as described in U.S. Pat. No. 9,051,601.

[0115] PCR reaction mixtures were typically assembled as follows: 19.05 μL Supermix (Promega GoTaq® Supermix); 0.35 μL MMLV reverse transcriptase (35 U); 0.6 μL GoTaq MDX Hotstart Taq (3 U); 5 μL nucleic acid (primers, probe, and target in appropriate dilutions); = 25 μL total reaction volume. Promega, Madison, WI; New England Biolabs, Ipswich, MA; Sigma-Aldrich, St. Louis MO; Thermo Fisher, Waltham, MA, etc.

[0116] Described below are representative RT-PCR assays based on TaqMan reagent chemistry that provide for the detection of one or more target nucleic acids (e.g., Flu A, Flu B, SARS-CoV-2, RSV A, and / or RSV B) in a biological sample. The following description is provided for illustrative purposes only.

[0117] A typical process begins by collecting a sample from a subject (e.g., a nasopharyngeal swab). Unless the sample is to be assayed immediately, the sample is typically placed in a sealable container along with an appropriate volume of sample medium. Preferably, a Universal Internal Control (UIC) is also then added to the sample to monitor for inhibitors that may be present in the sample.

[0118] The nucleic acids in the sample are isolated using an automated Panther Fusion system (Hologic, Inc.) or other systems, such as the KingFisher Flex Magnetic Particle Processor (ThermoFisher), the MagNA Pure LC System (Roche) and MagNA Pure Total Nucleic Acid Isolation Kit (Roche; catalog number 03038505001), or the NucliSENS easyMAG System (bioMerieux) and Automated Magnetic Extraction Reagents (bioMerieux). The purified nucleic acids are then added to a PCR reaction mix along with a thermostable DNA polymerase and reverse transcriptase. The reaction mix contains oligonucleotide primer pairs and target-specific oligonucleotide probes for each of the target nucleic acids to be assayed, as well as dNTPs (dATP, dCTP, dGTP, dTTP (or dUTP)), MgCl2, stabilizers, and a buffer containing bovine serum albumin. An RNase inhibitor (e.g., RNase Inhibitor II) can also be included to protect the RNA target nucleic acid from degradation. Various control nucleic acids can also be included. Such controls can be, for example, non-infectious in vitro transcribed RNA of a specific viral sequence and / or non-infectious plasmid DNA containing a control sequence. If desired, two different sets of amplification primers and probes targeting different genomic regions of the virus to be detected can be used for any given target nucleic acid. Detection probe species are often dual-labeled with distinguishable reporter dyes and quenchers (donor / acceptor) that function together to provide real-time results.

[0119] Reverse transcription of RNA to cDNA and subsequent amplification of the DNA can be performed, for example, with a Panther Fusion automated instrument (Hologic, Inc.) or a Cepheid SmartCycler II instrument (Cepheid, Sunnyvale, CA). In this process, for each target nucleic acid to be detected, the probe species specifically anneals to a target sequence in the target nucleic acid molecule (e.g., a specific region of the Flu A genome), followed by primer extension and amplification. The TaqMan reagent chemistry utilizes the 5'-3' exonuclease activity of Taq polymerase to cleave the probe, thus separating the reporter dye from its quencher. This results in an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing the fluorescent signal. The amount of fluorescence at any given cycle depends on the number of amplification products (amplicons) present at that time. Fluorescence intensity is monitored during each PCR cycle by a real-time instrument. [Example]

[0120] 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 as shown herein and are prepared and performed as follows.

[0121] Several primer and probe mixtures (PPR mixes) were prepared in microcentrifuge tubes to contain a forward primer, a reverse primer, and a dual-labeled detection probe. The primer and probe combinations in these PPR mixes were as follows: Set A: SEQ ID NOs: 116, 117, and 118; Set B: SEQ ID NOs: 117, 118, and 119; Set C: SEQ ID NOs: 126, 127, and 128; Set D: SEQ ID NOs: 129, 130, and 181; Set E: SEQ ID NOs: 131, 132, and 133; Set F: SEQ ID NOs: 134, 135, and 136; Set G: SEQ ID NOs: 137, 138, and 139; Set H: SEQ ID NOs: 140, 141, and 142; Set I: SEQ ID NOs: 143, 144, and 145; Set J: SEQ ID NOs: 146, 154, and 170; Set K: SEQ ID NOs: 176, 193, and 239; Set L: SEQ ID NOs: 240, 241, and 242; Set M: SEQ ID NOs: 246, 247, and 248; Set N: SEQ ID NOs: 254, 256, and 258; Set O: SEQ ID NOs: 257, 261, and 262; and Set P: SEQ ID NOs: 263, 264, and 265.

[0122] The internal control 1x PPR mix (SEQ ID NOS: 272-274) contained 0.6 μM of each primer and 0.4 μM of probe. Similarly, the coronavirus 1x PPR mix contained 0.6 μM of each primer and 0.4 μM of 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 detection probe was labeled with Quasar 705 and Black Hole Quencher 2, and each coronavirus ("SARS-CoV-2") detection probe was labeled with FAM and Black Hole Quencher 1 (all available from BioSearch Technologies, Inc., Novato, CA or Glen Research, Inc., Sterling, VA).

[0123] An equal volume of internal control PPR mix (275 μL) was added to each coronavirus mixture (275 μL) to provide a 1.25× PPR mix (550 μL total volume). Amplification and detection reactions were prepared by combining 20 μL of each PPR mix and 5 μL of target nucleic acid eluate with a 1.25× master mix containing dNTPs, dUTP, Taq polymerase, reverse transcriptase, and RNase inhibitor. Each amplification and detection mixture was then overlaid with oil. SARS-CoV-2 target nucleic acid was prepared from a stock virus concentrate (1E3 TCID50 / mL). The stock virus was serially diluted into sample transport medium (containing lithium lauryl sulfate (LLS), EDTA, and sodium phosphate) to provide concentrations of 1E0, 1E-1, 1E-2, and 1E-3 TCID50 / mL. Each serial dilution was combined with HeLa cells at a concentration of 1E4 cells / mL. Amplification and detection reactions were set up with 3 or 4 reactions per condition. Negative reactions contained sample transport medium without coronavirus target nucleic acid. Reactions were performed in real time using a Panther Fusion system (available from Hologic, Inc., Marlborough MA) with thermal cycling, and Ct and RFU data were recorded (see Table 1). Table 1 [Table 1]

[0124] Results. In all tests performed except for Sets L and M, the internal control was positive (100%) and the negative control wells were negative (0%). The internal control for Sets L and M was 0 / 4. This indicates a problem with the internal control in these PPR mixes. Unless otherwise noted in Table 1, the designated set was positive for all replicates tested. Overall, Set A had the fastest mean Ct for all dilutions. Set N was separately retested and showed similar results in the retest (mean Ct was 33.05 for 1E0 TCID50 / ml and 0 for the lowest concentration). Sets A, C, D, F, E, N, O, and P were further tested for performance in PPR mixes containing primers and probes to detect other respiratory pathogens.

[0125] Primer-probe mixes were prepared generally as described above, except that each PPR mix further contained primers and probes for amplification and detection of Flu A, Flu B, RSV A, and RSV B (final concentrations of 0.6 μM for each primer and 0.4 μM for each probe). The primers and probes used were SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:92, SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:115. The purpose of this experiment was to determine the performance of SARS-CoV-2 PPR mixes in a multiplex configuration. Samples were prepared as described above, and assays were performed on a Panther Fusion system. Ct data are shown in Table 2 below. Table 2 [Table 2]

[0126] Results: Set A showed the fastest Ct at 1E0 concentration and was the only set to show detection at 1E-1 concentration.

[0127] Example 2 Amplification and detection of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B in clinical samples Remaining nasopharyngeal (NP) swab specimens from SARS-CoV-2-positive individuals were analyzed in a multiplex real-time PCR assay containing primers and probes for amplification and detection of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B target nucleic acids. These NP swab samples were also tested in the Panther Fusion SARS-CoV-2 assay (catalog number PRD-06391) for comparative performance. Nucleic acid extraction, amplification, and detection reactions were performed on the Panther Fusion instrument according to the manufacturer's instructions.

[0128] For this example, 500 μL of each of the 22 remaining NP swab specimens was combined with 780 μL of sample transport medium. Nine of the 22 remaining specimens were also further diluted to an estimated 3× the reported LoD for the commercially available Panther Fusion SARS-CoV-2 assay (LoD=1E-2 TCID50 / mL for inactivated cultured SARS-CoV-2 isolate (US-WA1 / 2020, BEI Resources; NR52281)). The 3X LoD was determined using the separately obtained Cts for the nine samples and a desired Ct of 36.4. These values ​​were then substituted into the dilution factor formula to obtain the dilution factor required to reach the 3X LoD (2 36.4-Ct= (X)LoD / (3)LoD (where X is the dilution factor). Aliquots (500 μL) of the combined specimen and transport medium from each specimen were separately combined with lysis reagent (710 μL) in a Panther Fusion Lysis Tube (Hologic, Marlborough, MA). After incubation, 360 μL of the lysed specimen was combined with 450 μL of target nucleic acid isolation reagent containing capture oligonucleotides and a solid support. The lysis and target nucleic acid isolation reactions are generally described in U.S. Patent Nos. 6,110,678, WO 2021 / 097358, and 9,051,601. The target nucleic acids isolated from each clinical specimen were then eluted from the capture reaction to provide individual eluates corresponding to each of the NP swab specimens.

[0129] Nucleic acid amplification and detection reactions were performed as follows: 5 μL of eluate from each sample condition was added to a well of a multi-wall plate. 20 μL of rehydrated real-time PCR reaction mixture was also included in each well. The components of the real-time PCR reaction mixture are described above and include the experimental PPR mix (1× concentration of each primer: 0.6 μM and 1× concentration of each probe: 0.4 μM). The primers and probes in the PPR mix in this example were SEQ ID NOs: 5, 9, 14, 20, 23, 25, 26, 27, 59, 67, 68, 75, 79, 92, 101, 102, 115, 117, 118, 119, 176, 193, 239, 272, 273, and 274. The probes for detecting Flu A amplification products were labeled with FAM, the probes for detecting Flu B amplification products were labeled with Quasar 670, the probes for detecting RSV A and RSV B amplification products were labeled with Cal Orange 560, the probes for detecting SARS-CoV-2 were labeled with Cal Red 610, and the probes for detecting the internal control nucleic acid were labeled with Quasar 705. All probes contained BHQ-1 or BHQ-2 quenchers (labels available from LGC Biosearch Technologies, Petaluma, CA). Each eluate was then added to wells of a multiwell plate containing the amplification and detection reaction mixture from the Panther Fusion SARS-CoV-2 assay (set up according to the manufacturer's instructions). The amplification and detection reactions were then performed, and Ct and RFU data were collected. Data obtained with the PPR mix were compared to those obtained with the commercially available assay. The results are shown in Table 3 below. Table 3 [Table 3]

[0130] All internal control reactions were positive. All negative control wells were negative. This PPR mix demonstrated 100% sensitivity (22 / 22) and a 100% positive predictive value (compared to the commercial assay) (22 / 22), including with samples diluted to approximately 3X LoD. Comparable Ct values ​​for specimens tested undiluted using both the experimental and commercial systems indicate that detection of clinical SARS-CoV-2 titers was not delayed by the presence of Flu A, Flu B, RSV A, and RSV B primers and probes. The Ct values ​​for diluted specimens (3X LoD) were approximately 1 Ct earlier than expected (calculation not shown). This indicates that detection of diluted samples is not delayed in the multiplex system.

[0131] NP swab specimens from Flu A, Flu B, RSV A, or RSV B positive individuals were analyzed using the PPR mix described above. These NP swab samples were also tested with the Panther Fusion Flu A / B / RSV Assay (Cat. No. PRD-04328) for performance comparison. Nucleic acid extraction, amplification, and detection reactions were set up and performed generally as described above. Ct and RFU data were collected and compared with data obtained with the PPR mix and the commercially available assay. The results are shown in Table 4 below. Table 4 [Table 4-1] [Table 4-2]

[0132] All internal control reactions were positive. All negative control wells were negative. Flu A was detected in 16 of 17 clinical samples (94.12%). Flu B was detected in 2 of 3 clinical samples (66.67%). RSV was detected in 21 of 22 clinical samples (95.45%). These same clinical specimens were also tested using a control assay (Ct data not shown). The control assay showed negative results for each of the same three clinical specimens, which showed negative results when tested with the PPR mix (100% agreement between the test assay and the commercial assay). These comparable results indicate a problem with the samples. Ct data between the test assay and control assay showed no significant differences for the Flu A and RSV systems (p>0.05 for each paired t-test). No statistical comparisons were made for the Flu B system (n=2).

[0133] Example 3 Amplification and detection of Flu A and RSV A using Flu A, Flu B, SARS-CoV-2, and RSV A / RSV B multiplex primer / probe mixtures The purpose of this experiment was to test a multiplex PPR mix containing modified Flu A primer and probe combinations. The primer and probe mixture (PPR mix) was prepared to contain SEQ ID NOs: 5, 9, 14, 23, 25, 67, 68, 75, 79, 92, 101, 102, 115, 117, 118, 119, 176, 193, 239, 266, 267, 269, 272, 273, and 274. Target nucleic acids were prepared from stock panels to provide 1E4, 1E3, and 1E2 copies per reaction for each target. Each serial dilution was combined with 1E4 copies / mL of HeLa cells (each condition was run in triplicate). Nucleic acid extraction and real-time amplification and detection reactions were set up and performed on a Panther Fusion device generally as described above. Ct and RFU data were collected and analyzed. Table 5 [Table 5-1] [Table 5-2]

[0134] Example 4 Single-plex CoV amplification and detection oligonucleotide combination The purpose of this experiment was to test the performance of several candidate Torch oligonucleotides and T7 promoter primers in the BiPhasic real-time TMA assay. Single-plex reactions were run for each condition. Reactions were typically prepared and TMA assays were performed as described herein.

[0135] A series of target capture reagents were prepared containing a target capture oligonucleotide (SEQ ID NO:299) and one of two T7 promoter primers (SEQ ID NOs:294 and 295). These target capture reagents were then split into three each to accommodate separate testing with three different promoter reagents (each containing a different torch oligonucleotide). Each of these three promoter reagents contained a T7 promoter primer (SEQ ID NO:193) and one of three different torch oligonucleotides (SEQ ID NOs:284, 285, and 286). An amplification reagent was also prepared containing a non-T7 primer (SEQ ID NO:283). These reagents were prepared and reactions were performed generally as described above.

[0136] Each different reaction condition was run in five replicates with wells containing either sample transport medium alone (negative control wells) or various concentrations of target nucleic acid. The target nucleic acid in this experiment was 30, 100, 300, or 15,000 copies of in vitro transcript (SEQ ID NO: 291) per mL. The results are shown in Table 6 below. Table 6 [Table 6-1] [Table 6-2]

[0137] These conditions showed positive signals in 1-5 replicates of each of the negative control wells. False-positive results are likely due to intra- and / or intermolecular interactions of the torch molecules. Despite these false-positive results, the condition containing molecular torch SEQ ID NO:286 showed low signal-to-background in positive wells (approximately 0-1500 RFU in negative wells compared with approximately 7,500-11,000 RFU in positive wells) and therefore outperformed the other two torch combinations. The T7 promoter primers in both target capture reagents (SEQ ID NOs:294 and 295) performed equally well.

[0138] A second experiment was performed to test additional Torch and T7 promoter primer combinations. Each condition was tested again as a single-plex reaction. In this second experiment, the target capture reagent contained SEQ ID NO:300 and one of SEQ ID NO:296 or SEQ ID NO:297; the amplification reagent contained SEQ ID NO:184; and the promoter reagent contained SEQ ID NO:298 and one of SEQ ID NOs:287-290.

[0139] Each different reaction condition was run in five replicates with wells containing either sample transport medium alone (negative control wells) or various concentrations of target nucleic acid. The target nucleic acid in this experiment was 30 copies, 100 copies, 300 copies, or 15,000 copies of in vitro transcript (SEQ ID NO: 292) per mL. The results are shown in Table 7 below. Table 7 [Table 7] *Numbers in the promoter reagent lines are sequence numbers.

[0140] These conditions did not exhibit false-positive signals. Conditions containing either promoter primer and molecular Torch SEQ ID NO:289 in the target capture reagent performed well, as did conditions containing promoter primer SEQ ID NO:296 and Torch SEQ ID NO:288 in the target capture reagent. However, conditions containing Torch SEQ ID NO:289 exhibited fast TTimes (13-21 minutes) and high RFU values ​​(1,000-4,000) against low background. Furthermore, conditions containing Torch SEQ ID NO:289 exhibited low spread in these TTime and RFU values ​​across replicates and target concentrations.

[0141] Example 5 A multiplex assay to determine the performance of primers and probes for the detection of SARS-CoV-2 in the presence of target capture oligomers, primers, and probes for the detection of influenza A and influenza B, respectively. A multiplex reaction was prepared to test the ability of primers and probes to amplify and detect two regions of SARS-CoV-2 target nucleic acid in the presence of primers and probes for detecting influenza target nucleic acid. The oligonucleotides used in this experiment were as follows: target capture reagent: SEQ ID NOS: 310, 311, 299, and 300 (target capture oligonucleotides) and SEQ ID NOS: 307, 308, 295, and 309 (T7 promoter primers); amplification reagent: SEQ ID NOS: 305, 306, 283, and 184 (non-T7 primers); and promoter reagent: SEQ ID NOS: 307, 308, 193, and 309 (T7 promoter primers) and SEQ ID NOS: 301, 302, 303, 289, and 304 (torch oligomers). SEQ ID NOs: 291 and 292 were used as target nucleic acids (5 copies / mL, 10 copies / mL, 20 copies / mL, 30 copies / mL, and 100 copies / mL). Negative reactions were with sample transport medium alone. Each reaction condition was run in 20 replicates. Table 8 [Table 8]

[0142] In this example, the detection limits for the multiplex reaction were 13.9 copies / mL for SEQ ID NO:291 and 17.2 copies / mL for SEQ ID NO:292 as determined by probit analysis at 95% probability.

[0143] Example 6 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 the IVT (SEQ ID NOS: 291 and 292) were tested with individual primer and probe sets (see Table 9) and the combined oligo set 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 set were tested at seven concentration levels of the IVT (SEQ ID NOS: 291 and 292) ranging from 1 x 10^7 (1E+7) copies / reaction to 10 copies / reaction.

[0144] Two SARS-CoV-2 primer and probe (PPR) mixes were prepared as generally described in Example 1 above. These SARS-CoV-2 PPR mixes contained the indicated primers and probes, containing analog nucleotides and varying arrangements of fluorophores / quenchers (see Table 9). Quasar 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 included only IC PPR mix. Positive and negative reactions were each run in replicates of six reactions for stage 1 and 12 reactions for stage 2. Table 9 [Table 9]

[0145] Table 10 shows a summary of the results for the individual and combined primer / probe mixes. IC reactions showed 100% detection in all tested conditions. No problems were observed with the combined oligo sets. Linearity and sensitivity data for the combined PPR mixes listed in (iii) above are summarized in Table 11. These results show that 100% detection of both IVTs was observed down to 100 cp / rxn. PCR efficiency was high with R^2 values ​​>0.99 and a slope of -3.33 (y = -3.3266x + 39.484). IC showed 100% detection at all dilutions. Table 10: Summary of results for individual and combined oligo sets (Stage 1) [Table 10] Table 11: IVT sensitivity results for combination oligo sets (Tier 2) [Table 11]

[0146] Example 7 Exemplary Oligonucleotide Sequences Table 12 illustrates several primer, probe, and target capture oligonucleotide sequences that are useful as compositions, in kits, as diagnostic reagents, and / or in methods for the amplification or detection of one or more of Flu A, Flu B, RSV-A, and RSV-B. Table 12 below illustrates only the nucleotide sequences. It is understood that these sequences may further include detectable labels, sugar modifications (e.g., 2'-methoxy), base modifications (e.g., methylated bases), and other chemical entities not represented in the illustrated sequential arrangement of symbols. Table 12 [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Table 12-9] pU = propyne dU mC = 5-methylcytosine [F*] = fluorophore [Q*] = Quencher mN (mA, mC, mG, mT, mK, or mR) = 2'O-methyl nucleotide (rxl) = rxl internucleotidyl linker attached to an acridinium ester compound (e.g., U.S. Pat. No. 5,585,481) (C9) = 9 carbon spacer * All sequences are written in the 5' to 3' orientation unless otherwise indicated. Sequence symbols follow Table 1 of the World Intellectual Property Organization (WIPO) Handbook on Industrial Property Information and Documentation, Standard ST.25 (1998) ("WIPO ST.25 (1998)").

[0147] Table 13 illustrates, for example, nucleic acid sequences representing all or part of one strand of an amplification product, sequences that primers or probes comprise, sequences that primers or probes contain, and target nucleic acid sequences. Table 13 [Table 13-1] [Table 13-2] [Table 13-3]

[0148] All of the articles, devices, systems, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the devices, systems, and methods of the present disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the above articles and methods without departing from the spirit and scope of the present disclosure. All such variations and equivalents apparent to those skilled in the art, whether currently existing or later developed, are deemed to be within the spirit and scope of the present disclosure. It will also be recognized that computer-based embodiments of the present disclosure can be implemented using any suitable hardware and software.

[0149] All patents, patent applications, and publications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes.

Claims

1. A composition or kit for determining the presence or absence of SARS-CoV-2 in a sample, wherein the composition or kit comprises a first SARS-CoV-2 primer set comprising first and second SARS-CoV-2 primers capable of amplifying a first target region of SARS-CoV-2 nucleic acid, wherein the first SARS-CoV-2 primer comprises a target hybridize region of 18 to 35 nucleic acid bases in length, comprising SEQ ID NO: 123 and comprising SEQ ID NO: 122, wherein the first SARS-CoV-2 primer and the second SARS-CoV-2 primer generate a first SARS-CoV-2 amplicon of 57 to 89 nucleotides in length, comprising SEQ ID NO:

124.

2. (i) The first SARS-CoV-2 primer comprises the nucleotide sequence of SEQ ID NO: 116, SEQ ID NO: 119, or SEQ ID NO: 121; (ii) The second SARS-CoV-2 primer comprises the nucleotide sequence of SEQ ID NO: 117; or (iii) The first SARS-CoV-2 primer comprises the nucleotide sequence of SEQ ID NO: 116, SEQ ID NO: 119, or SEQ ID NO: 121, and the second SARS-CoV-2 primer comprises the nucleotide sequence of SEQ ID NO:

117. The composition or kit according to claim 1.

3. The composition or kit according to claim 1, further comprising a first SARS-CoV-2 detection probe oligomer.

4. The composition or kit according to claim 3, wherein the first SARS-CoV-2 detection probe oligomer comprises a target hybridize sequence of 25 to 40 consecutive nucleotides, contained in SEQ ID NO: 124 or SEQ ID NO:

125.

5. The composition or kit according to claim 4, wherein the first SARS-CoV-2 detection probe oligomer comprises SEQ ID NO:

118.

6. The aforementioned composition or kit is (a) A second SARS-CoV-2 primer set comprising third and fourth SARS-CoV-2 primers capable of amplifying a second target region of SARS-CoV-2 nucleic acid; (b) A first Flu A primer set comprising first and second influenza A primers capable of amplifying a first target region of influenza A nucleic acid; (c) A second Flu A primer set comprising third and fourth influenza A primers capable of amplifying a second target region of influenza A nucleic acid; (d) A Flu B primer set comprising first and second influenza B primers capable of amplifying the target region of influenza B nucleic acid; (e) an RSV A primer set comprising first and second RSV A primers capable of amplifying the target region of an RSV A nucleic acid; and (f) An RSV B primer set comprising first and second RSV B primers capable of amplifying the target region of RSV B nucleic acid, The composition or kit according to claim 1, further comprising one or more of the following.

7. The aforementioned composition or kit is (1) Two or more of the following: the second SARS-CoV-2 primer set, the first Flu A primer set, the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (2) Three or more of the following: the second SARS-CoV-2 primer set, the first Flu A primer set, the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (3) Four or more of the following: the second SARS-CoV-2 primer set, the first Flu A primer set, the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (4) Five or more of the following: the second SARS-CoV-2 primer set, the first Flu A primer set, the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (5) The second SARS-CoV-2 primer set, the first Flu A primer set, the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (6) The second SARS-CoV-2 primer set, (7) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set; (8) The Flu B primer set; (9) At least one primer set selected from the group consisting of the RSV A primer set and the RSV B primer set; (10) The RSV A primer; (11) The RSV B primer set; (12) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set and the first Flu A primer set and the second Flu A primer set; (13) The second SARS-CoV-2 primer set and the Flu B primer set; (14) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set and the RSV A primer set and the RSV B primer set; (15) The second SARS-CoV-2 primer set and the RSV A primer; (16) The second SARS-CoV-2 primer set and the RSV B primer; (17) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, and the Flu B primer set; (18) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, and at least one primer set selected from the group consisting of the RSV A primer set and the RSV B primer set; (19) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, and the RSV A primer; (20) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, and the RSV B primer; (21) At least one primer set selected from the group consisting of the Flu B primer set and the RSV A primer set and the RSV B primer set; (22) The Flu B primer set and the RSV A primer; (23) The Flu B primer set and the RSV B primer; (24) The RSV A primer set and the RSV B primer set; (25) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, and the Flu B primer set; (26) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, and at least one primer set selected from the group consisting of the RSV A primer set and the RSV B primer set; (27) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, and the RSV A primer set; (28) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, and the RSV B primer set; (29) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the Flu B primer set, and the RSV A primer set and the RSV B primer set; (30) The second SARS-CoV-2 primer set, the Flu B primer set, and the RSV A primer set; (31) The second SARS-CoV-2 primer set, the Flu B primer set, and the RSV B primer set; (32) The second SARS-CoV-2 primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (33) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, the Flu B primer set, and at least one primer set selected from the group consisting of the RSV A primer set and the RSV B primer set; (34) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, the Flu B primer set, and the RSV A primer set; (35) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, the Flu B primer set, and the RSV B primer set; (36) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, the RSV A primer set, and the RSV B primer set; (37) The Flu B primer set, the RSV A primer set, and the RSV B primer set; (38) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, the Flu B primer set, and the RSV A primer set; (39) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, the Flu B primer set, and the RSV B primer set; (40) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set, and the second Flu A primer set, the RSV A primer set, and the RSV B primer set; (41) The second SARS-CoV-2 primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; (42) At least one primer set selected from the group consisting of the first Flu A primer set and the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set; or (43) At least one primer set selected from the group consisting of the second SARS-CoV-2 primer set, the first Flu A primer set and the second Flu A primer set, the Flu B primer set, the RSV A primer set, and the RSV B primer set, The composition or kit according to claim 6, comprising:

8. (i) The third and fourth SARS-CoV-2 primers, if present, include SEQ ID NOs: 146 and 170, SEQ ID NOs: 126 and 127, SEQ ID NOs: 146 and 127, SEQ ID NOs: 263 and 264, SEQ ID NOs: 131 and 132, SEQ ID NOs: 181 and 129, SEQ ID NOs: 131 and 129, SEQ ID NOs: 262 and 257, SEQ ID NOs: 140 and 141, SEQ ID NOs: 134 and 135, SEQ ID NOs: 140 and 135, SEQ ID NOs: 143 and 144, SEQ ID NOs: 176 and 239, SEQ ID NOs: 143 and 239, SEQ ID NOs: 137 and 138, SEQ ID NOs: 176 and 138, SEQ ID NOs: 256 and 254, SEQ ID NOs: 137 and 254, SEQ ID NOs: 240 and 241, SEQ ID NOs: 256 and 241, SEQ ID NOs: 248 and 247, or SEQ ID NOs: 240 and 247; (ii) The first and second influenza A primers, if present, include SEQ ID NOs: 5 and 26, SEQ ID NOs: 5 and 27, SEQ ID NOs: 5 and 266, or SEQ ID NOs: 5 and 267; (iii) The third and fourth influenza A primers, if present, include SEQ ID NOs: 23 and 25, SEQ ID NOs: 23 and 6, or SEQ ID NOs: 23 and 28; (iv) The first and second influenza B primers, if present, include SEQ ID NOs: 67 and 58, SEQ ID NOs: 67 and 68, or SEQ ID NOs: 67 and 70; (v) The first and second RSV A primers, if present, include SEQ ID NOs: 79 and 72, SEQ ID NOs: 79 and 73, SEQ ID NOs: 79 and 74, SEQ ID NOs: 79 and 92, SEQ ID NOs: 79 and 93, SEQ ID NOs: 79 and 94, SEQ ID NOs: 79 and 95, or SEQ ID NOs: 79 and 96; (vi) The first and second RSV B primers, if present, include SEQ ID NOs: 99 and 104, SEQ ID NOs: 99 and 105, SEQ ID NOs: 100 and 115, SEQ ID NOs: 101 and 115, or SEQ ID NOs: 106 and 115, The composition or kit according to claim 6.

9. The composition or kit according to claim 6, further comprising one or more of a second SARS-CoV-2 detection probe oligomer, a first Flu A detection probe oligomer, a second Flu A detection probe oligomer, a Flu B detection probe oligomer, an RSV A detection probe oligomer, and an RSV B detection probe oligomer.

10. (i) If the second SARS-CoV-2 primer set is present, the second SARS-CoV-2 detection probe oligomer includes SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136, SEQ ID NO: 139, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 154, SEQ ID NO: 193, SEQ ID NO: 242, SEQ ID NO: 246, SEQ ID NO: 258, SEQ ID NO: 261, or SEQ ID NO: 265; (ii) If the first Flu A primer set is present, the first Flu A detection probe oligomer is present and includes Sequence ID No. 20; (iii) If the second Flu A primer set is present, the second Flu A detection probe oligomer is present and includes SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 14; (iv) If the Flu B primer set is present, the Flu B detection probe oligomer is present and includes SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66; (v) If the RSV A primer set is present, the RSV A detection probe oligomer is present and includes SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 97, or SEQ ID NO: 98; and (vi) If the RSV B primer set is present, the RSV B detection probe oligomer is present and includes SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, or SEQ ID NO: 114 The composition or kit according to claim 9.

11. (i) If the second SARS-CoV-2 primer set is present, the third and fourth SARS-CoV-2 primers and the second SARS-CoV-2 detection probe oligomer are sequence numbers 146, 170, and 154; sequence numbers 126, 127, and 128; sequence numbers 146, 127, and 154; sequence numbers 146, 127, and 128; sequence number 2 63, 264, and 265; SEQ ID NOs: 131, 132, and 133; SEQ ID NOs: 181, 129, and 130; SEQ ID NOs: 131, 129, and 133; SEQ ID NOs: 131, 129, and 130; SEQ ID NOs: 262, 257, and 261; SEQ ID NOs: 140, 141, and 142; SEQ ID NOs: 134, 135, and 136; SEQ ID NOs: 140, 135, and 142; SEQ ID NOs: 140 , 135, and 136; SEQ ID NOs: 143, 144, and 145; SEQ ID NOs: 176, 239, and 193; SEQ ID NOs: 143, 239, and 145; SEQ ID NOs: 143, 239, and 193; SEQ ID NOs: 137, 138, and 139; SEQ ID NOs: 176, 138, and 193; SEQ ID NOs: 176, 138, and 139; SEQ ID NOs: 256, 254, and 258; SEQ ID NOs: 137, 254, and 139; Sequence IDs 137, 254, and 258; Sequence IDs 137, 254, 139, and 258; Sequence IDs 240, 241, and 242; Sequence IDs 256, 241, and 258; Sequence IDs 256, 241, and 242; Sequence IDs 248, 247, and 246; Sequence IDs 240, 247, and 242; or including Sequence IDs 240, 247, and 246; (ii) If the first Flu A primer set is present, the first and second influenza A primers and the first Flu A detection probe oligomer include SEQ ID NOs. 5, 26, and 20; SEQ ID NOs. 5, 27, and 20; SEQ ID NOs. 5, 266, and 20; or SEQ ID NOs. 5, 267, and 20; (iii) If the second Flu A primer set is present, the third and fourth influenza A type primers and the second Flu A detection probe oligomer include SEQ ID NOs. 23, 25, and 9; SEQ ID NOs. 23, 25, and 14; SEQ ID NOs. 23, 25, 9, and 14; SEQ ID NOs. 23, 6, and 9; SEQ ID NOs. 23, 6, and 14; SEQ ID NOs. 23, 6, 9, and 14; SEQ ID NOs. 23, 28, and 9; SEQ ID NOs. 23, 28, and 14; SEQ ID NOs. 23, 28, 9, and 14; SEQ ID NOs. 23, 25, and 8; SEQ ID NOs. 23, 25, and 13; SEQ ID NOs. 23, 25, 8, and 13; SEQ ID NOs. 23, 6, and 8; SEQ ID NOs. 23, 6, and 13; SEQ ID NOs. 23, 28, and 8; SEQ ID NOs. 23, 28, and 13; or SEQ ID NOs. 23, 28, 8, and 13; (iv) If the Flu B primer set is present, the first and second influenza B primers and the Flu The B detection probe oligomer includes SEQ ID NOs. 67, 70, and 59; SEQ ID NOs. 67, 70, and 63; SEQ ID NOs. 67, 70, and 64; SEQ ID NOs. 67, 70, and 65; SEQ ID NOs. 67, 70, and 66; SEQ ID NOs. 67, 70, 59, and 63; SEQ ID NOs. 67, 68, and 69; SEQ ID NOs. 67, 68, and 63; SEQ ID NOs. 67, 68, and 64; SEQ ID NOs. 67, 68, and 65; SEQ ID NOs. 67, 68, and 66; SEQ ID NOs. 67, 58, and 634; SEQ ID NOs. 67, 58, and 65; SEQ ID NOs. 67, 58, and 66; or SEQ ID NOs. 67, 58, 59, and 63; (v) If the RSV A primer set is present, the first and second RSV A primers and the RSV A detection probe oligomers include SEQ ID NOs. 79, 72, and 75; SEQ ID NOs. 79, 72, and 71; SEQ ID NOs. 79, 72, and 77; SEQ ID NOs. 79, 72, and 90; SEQ ID NOs. 79, 72, and 97; SEQ ID NOs. 79, 74, and 75; SEQ ID NOs. 79, 74, and 71; SEQ ID NOs. 79, 74, and 77; SEQ ID NOs. 79, 74, and 90; SEQ ID NOs. 79, 74, and 97; SEQ ID NOs. 79, 92, and 75; SEQ ID NOs. 79, 92, and 71; SEQ ID NOs. 79, 92, and 77; SEQ ID NOs. 79, 92, and 90; SEQ ID NOs. 79, 92, and 97; SEQ ID NOs. 79, 95, and 75; SEQ ID NOs. 79, 95, and 71; SEQ ID NOs. 79, 95, and 77; SEQ ID NOs. 79, 95, and 90; or SEQ ID NOs. 79, 95, and 97; and (vi) If the RSV B primer set is present, the first and second RSV B primers and the RSV The B detection probe oligomers include SEQ ID NOs: 100, 115, and 102; SEQ ID NOs: 101, 115, and 102; SEQ ID NOs: 106, 115, and 102; SEQ ID NOs: 100, 115, and 108; SEQ ID NOs: 101, 115, and 108; SEQ ID NOs: 106, 115, and 108; SEQ ID NOs: 100, 115, and 110; SEQ ID NOs: 101, 115, and 110; SEQ ID NOs: 106, 115, and 110; SEQ ID NOs: 100, 115, and 112; SEQ ID NOs: 101, 115, and 112; SEQ ID NOs: 106, 115, and 112; SEQ ID NOs: 100, 115, and 114; SEQ ID NOs: 101, 115, and 114; or SEQ ID NOs: 106, 115, and 114. The composition or kit according to claim 10.

12. The composition or kit according to any one of claims 1 to 11, wherein at least one of the primers further comprises an RNA polymerase promoter sequence.

13. The composition or kit according to any one of claims 3 to 11, wherein at least one of the primers or detection probe oligomers comprises one or more nucleotide analogs.

14. The composition or kit according to claim 13, wherein one or more nucleotide analogs are independently selected from the group consisting of 5-methylcytosine, propinedeoxyuridine, and 2'O-methylnucleotide.

15. The composition or kit according to any one of claims 3 to 11, wherein at least one detection probe oligomer comprises a detectable label, or a detectable label and a quencher, or a donor / acceptor label pair.

16. The composition or kit according to claim 15, wherein the composition or kit comprises two or more detection probe oligomers, each different detection probe oligomer comprising a detectable label that is distinguishable from the detectable label on the other detection probe oligomers.

17. The composition or kit according to any one of claims 1 to 11, further comprising one or more of the following: salt, polymerase enzyme, DNA polymerase, reverse transcriptase, RNA polymerase, dNTP, rNTP, buffer, one or more target capture oligonucleotides, sample transport medium, target capture reagent, amplification reagent, and promoter reagent.

18. The composition or kit according to any one of claims 1 to 11, wherein the primer and / or detection probe oligomer is present in an aqueous formulation or a dry formulation.

19. A method for amplifying and / or detecting SAR-CoV-2 virus in a sample, and optionally one or more of influenza virus type A, influenza virus type B, RSV virus type A, and RSV virus type B, wherein the method is (i) A step of contacting the sample with the composition according to any one of claims 1 to 11 to form an amplified reaction reaction; (ii) A step of carrying out a nucleic acid amplification reaction, wherein any SAR-CoV-2 virus target nucleic acid, and optionally influenza virus type A target nucleic acid, influenza virus type B target nucleic acid, RSV virus type A target nucleic acid, or RSV virus type B target nucleic acid, if present in the sample, is amplified to form an amplicon; and (iii) A step of detecting the presence or absence of the amplicon, wherein the detection of the presence of the SARS-CoV-2 amplicon indicates the presence of the SARS-CoV-2 virus in the sample, and optionally, the detection of the presence of influenza virus type A amplicon, influenza virus type B amplicon, RSV virus type A amplicon, and / or RSV virus type B amplicon indicates the presence of influenza virus type A, influenza virus type B, RSV virus type A, and / or RSV virus type B in the sample. A method that includes this.

20. The method according to claim 19, wherein the detection step is performed in real time.