COMPOSITIONS AND METHODS FOR DETECTION OF VIRAL PATHOGENS IN SAMPLES

CROSS-REFERENCE TO RELATED APPLICATIONS

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

SEQUENCE LISTING

[0002] The Sequence Listing writen in file DIA.0184.02_SeqList_ST26.xml is 296 kilobytes in size, was created August 31, 2023, and is hereby incorporated by reference.

BACKGROU D

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

[0004] Influenza is an acute respiratory' illness in humans caused by infection with the influenza (flu) vims, primarily types A and B. Influenza A viruses are further categorized into subtypes based on two major surface protein antigens, hemagglutinin (H), and neuraminidase (N). Influenza B viruses are not categorized into subtypes. The influenza vimses are RNA viruses in the family Orthomyxoviridae. Each of influenza types A and B (Flu A and Flu B, respectively) is a separate genus containing one species and many sub-species.

[0005] Influenza epidemics occur yearly around the world. Although both flu types A and B circulate in the population, type A is usually dominant. These yearly epidemics are partly due to antigenic variation in the H and N surface proteins of the virus. Transmission of influenza is primarily via airborne droplet (coughing or sneezing). Symptoms arise on average 1 to 2 days post-exposure and include fever, chills, headache, malaise, cough, and coryza. Gastrointestinal symptoms such as nausea, vomiting, and diarrhea can occur, primarily in young children. Complications due to influenza include pneumonia, which can cause increased morbidity and mortality in pediatric, elderly, and immune-compromised populations. In the United States, it is estimated that influenza results in more than 200,000 hospitalizations and up to 36,000 deaths annually. Large influenza outbreaks, or pandemics, occur rarely. In the 20 ^th century, three influenza pandemics occurred, in 1918, 1958, and 1968, each causing millions of deaths worldwide. Influenza may also affect other animals, including pigs, horses and birds. [0006] Coronaviruses are a family of RNA viruses that infect avians and mammals, including humans. Coronaviruses belong to the family Coronaviridae, which has four main sub-groupings, known as alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Human coronaviruses include alphacoronaviruses 229E and NL63 and betacoronaviruses OC43, HKU1, SARS-CoV and SARS-CoV-2 (the coronavirus that causes severe acute respiratory syndrome, or SARS), and MERS-CoV (the coronavirus that causes Middle East Respiratory Syndrome, or MERS). SARS-CoV-2 can cause severe lower respiratory tract infections (COVTD-19) and was 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. RSV is a member of the family Paramyxoviridae, in the genus Orthopneumo virus. There are 2 types of RSV, A and B, which are differentiated based on antigenic and surface protein variations. Most yearly epidemics contain a mix of RS V A and RSV B, but one subgroup can dominate during a season. RSV infection can cause severe respiratory illness among all ages but is more prevalent in pediatric, elderly, and immune-comprormsed populations. RSV can infect up to 80% of children less than 1 years of age. Bronchiolitis and pneumonia are the major clinical complications in infants and young children, resulting in an estimated 51,000-82,000 hospital admissions per year in the United States. RSV infection is also an important cause of severe respiratory disease and substantial number of deaths in the elderly, with an estimated annual cost of $150 million to $680 million for RSV pneumonia hospitalizations.

[0008] Given the morbidity, mortality, and economic costs associated with influenza, coronavirus, and RSV infections, there clearly exists a need for improved detection of these pathogens. This disclosure addresses this and other needs.

SUMMARY

[0009] This disclosure provides compositions, including kits and reagents, and methods for in vitro diagnostic analysis of influenza A virus (Flu A), influenza B virus (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. Preferably the in vitro diagnostic analysis utilizes polymerase chain reactions (PCR) or isothermal amplification reactions, though other in vitro assay methodologies are contemplated for use with the disclosed compositions. A particularly useful in vitro assay for use with the Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is a reverse transcription PCR (RT-PCR) assay, as these target nucleic acids are RNA viruses. A particularly useful and convenient in vitro assay for use with the Flu A, Flu B, S ARS- CoV-2, RSV A or RSV B target nucleic acids is a real-time, RT-PCR assay. A particularly useful and convenient in vitro assay for use with the Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is a transcription-mediated amplification (TMA) reaction. A particularly useful and convenient in vitro assay for use with the Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is a 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 nasopharyngeal (NP) swab specimens 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. Negative results do not preclude such infection. Conversely, positive results do not rule-out bacterial infections or co-infections wi th other viruses. The use of additional laboratory testing and clinical presentation may also be considered to obtain the final diagnosis of respiratory viral infection.

[0011] One aspect provides hybridization assay probes useful for detecting Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B target nucleic acid sequences. Preferably, such probe molecule species include a target hybridizing sequence that is substantially complementary to a probe target nucleic acid sequence in the viral genome, or an amplicon generated therefrom, being targeted for detection. In preferred embodiments, the probe target nucleic acid sequence consists of about 17 to about 100 contiguous bases contained within targeted viral genome (or 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 separately 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 to 57 and 59 to 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 separately 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 to 78, 80 to 87, 89 to 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 to 114.

[0017] Preferably, a probe molecule species is labeled, optionally distinguishably labeled such that any one probe molecule species can be distinguished from other probe molecule 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 disclosure concerns nucleic acid molecules that are amplification primers for use in 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 disclosure relates to pairs of such primers that can be used to amplify desired amplicons that contain a target nucleic acid sequence. These primers include one or more of the following primers 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 is different 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 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 generate corresponding amplicons for Flu A, Flu B, SARS-CoV-2, RSV A, and/or RSV B if the viral pathogen is present in the biological sample being tested.

[0020] In general, a primer pair includes a first primer that includes a pnming nucleotide sequence that is substantially complementary to a first target nucleic acid sequence of 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, and preferably by about 50-1,000 nucleotides, and each of them preferably consists of about 17 to about 100 contiguous bases of the viral genome to be detected. In some embodiments, one or more of the primers in one or more primer pairs further comprises a non-target hybridizing sequence, a primer upstream region having a nucleotide sequence that is not complementary to the primer’s target nucleotide sequence. [0021] Preferred first primers for generating a first Flu A amplicon have the priming nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:23. Preferred second primers for generating a first Flu A amplicon have the priming nucleotide sequence of SEQ ID NO:25 or SEQ ID NO:28.

[0022] Preferred first primers for generating a second Flu A amplicon have the 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. Preferred second primers for generating a second Flu A amplicon have the priming nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:27.

[0023] Preferred first primers for generating a Flu B amplicon have the priming nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:67. Preferred second primers for generating a Flu B amplicon have the priming nucleotide sequence of SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.

[0024] Preferred first primers for generating a SARS-CoV-2 amplicon have the 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. Preferred second primers for generating a SARS-CoV-2 amplicon have the 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] Preferred first primers for generating an RSV A amplicon have the priming nucleotide sequence of SEQ ID NO:79 or SEQ ID NO:88. Preferred second primers for generating an RSV A amplicon have the 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] Preferred first primers for generating an RSV B amplicon have the priming nucleotide sequence of SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 106. Preferred second primers for generating an RSV B amplicon have the priming nucleotide sequence of SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 115.

[0027] In some preferred embodiments, a probe and/or a primer contains one or more methylated cytosine bases. [0028] Another related aspect of the disclosure concerns compositions that contain such probes, primers, and primer pairs. Such compositions include dry or liquid compositions. Dried compositions include lyophilized reagents containing one or more of a primer and a probe. [0029] Another aspect of the disclosure relates to kits that include primers and/or probes. Such kits can also include 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 kit may be in a dried form or in an aqueous form.

[0030] Another aspect of the disclosure relates to a reagent that contains primers and/or probes. Such reagents can also include 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 a dried form or in an aqueous form.

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

[0032] Embodiment 1 : A composition or kit for analysis of one or more of a plurality of pathogen-associated target nucleic acid molecule species that may be present in a biological sample, comprising: (a) a first SARS-CoV-2 primers 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 primers 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 primer pair for generating a first Flu A amplicon from the biological sample, 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) an RSV A primer pair for generating an 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) an RSV B primer pair for generating an 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 the primer of (a)(i) comprises a target hybridizing sequence consisting of a nucleotide sequence that is 18 to 35 nucleotides in length contained within SEQ ID NO: 123 and containing SEQ ID NO: 122, and wherein the primer (a)(i) and the primer (a)(ii) generate an amplicon containing SEQ ID NO: 124.

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

[0034] Embodiment 12: The composition or kit of any 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 composition or kit further comprises a detection probe oligomer, wherein the primer of (a)(i) is 18 to 35 nucleotides in length contained within SEQ ID NO: 125 and containing SEQ ID NO: 122, and wherein the detection probe oligomer comprises a target hybridizing sequence consisting of a sequence that is 25 to 40 nucleotides in length and 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 composition or kit of any of embodiments 1 to 14, wherein 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 of embodiments 1 to 15, wherein the first primer of l(c)(i), the second primer of l(c)(ii), the third primer of l(d(i), and the fourth primer of l(d)(ii) are each independently selected from the group consisting of: SEQ ID NOs: l, 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 of embodiment 1 to 16, wherein the first primer of 1 (e)(i) and the second primer of l(e)(ii) are each independently selected from the group consisting of: SEQ ID NOs:29, 58, 67, 68, 69, 70, and 314. Embodiment 18: The composition or kit of embodiments 1 to 17, 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 19: The composition or kit of any of embodiments 1 to 18, wherein the first primer of 1 (g)(i) and the second primer of 1 (g)(ii) are each independently selected from the group consisting of: SEQ ID NOs:99, 100, 101, 104, 105, 106, and 115. Embodiment 20: The composition or kit of any of embodiments 13 to 19, wherein each primer individually contains from 0 to 20 nucleotide analogs. Embodiment 21 : The composition or kit of any of embodiments 13 to 20, wherein each primer individually contains from 0 to 20 5-Me- C nucleotide analogs.

[0036] Embodiment 22: The composition or kit of embodiment 15, 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 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, wherein the composition or kit further comprises 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 an 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 from 0% to 100% nucleotide analogs. 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 from 0% to 100% 5-Me-C nucleotide analogs. 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 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, wherein each of the target capture oligonucleotide is 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 a reagent for an amplification and/or a detection and/or a target capture reaction. Embodiment 43: The composition of any one of embodiments 1 to 40, wherein the composition is a dried formulation. Embodiment 44: The composition of embodiment 43, wherein the dried formulation further comprises a reagent for an amplification and/or a detection and/or a target capture reaction. Embodiment 45: A kit containing the composition of any one of embodiments 41 to 44.

[0044] Embodiment 46: A kit or composition for the detection of amplicon 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) for an amplicon generated with a SARS-CoV-2 primer pair, the probe molecule species comprises a nucleic acid sequence that is substantially identical to a sequence selected form 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) for an amplicon was generated with a Flu A primer pair, the probe molecule species comprises a nucleic acid sequence that is substantially identical to a sequence selected form the group consisting of: SEQ ID NOs:6, 7, 8, 9, 10, 11, 12, 13, 14, 015, 16, 17, 18, 19, 20, 21, 22, 269, 280, 281, 282, 301, and 302; and/or (c) for an amplicon generated with a Flu B primer pair, the probe molecule species comprises a nucleic acid sequence that is substantially identical to a sequence selected form 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 an amplicon generated with a RSV A primer pair, the probe molecule species comprises a nucleic acid sequence that is substantially identical to a sequence selected form 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 an amplicon generated with a RSV B primer pair, the probe molecule species comprises a nucleic acid sequence that is substantially identical to a sequence selected form 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 from 0% to 100% nucleotide analogs. Embodiment 48: The composition or kit of embodiment 46 or 47, wherein each detection probe oligomer comprises a nucleotide sequence that is from 0% to 100% 5- Me-C nucleotide analogs. 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 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 dried formulation. Embodiment 54: A kit containing the composition of embodiment 52 or 53.

[0046] Embodiment 55: A reaction mixture comprising the composition of embodiment 41 and 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 and 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 of 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 under stringent hybridization conditions target nucleic acid molecules from the biological sample with one or more of the detection probe oligonucleotides of embodiment 50 or 51; (b) detecting the presence or absence of a detectable label, the presence indicating that a probe:target nucleic acid duplex formed in step (a); (c) based on whether the presence of a detectable label was detected in step (b), determining that the biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and/or RSV 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 at step (c). Embodiment 60: The method of embodiment 58 or 59, wherein target nucleic acid forming the probe:target nucleic acid duplex at step (b) is an amplicon generated using a primer pair.

[0048] Embodiment 61 : A method of 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 target nucleic acid molecules from the biological sample with one or more primer pairs of one of embodiments 1 to 11 to form an amplification reaction mixture; (b) performing a nucleic acid amplification reaction to generate an amplicon from any target nucleic acid molecule in the amplification reaction mixture of step (a); and (c) determining whether an amplicon was generated at 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, before step (a), a target capture reaction is performed to extract the 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 performed using one or more detection probe oligonucleotides. Embodiment 67 : The method of embodiment 66, wherein the detection probe oligonucleotides are labeled with donor/acceptor label pairs. Embodiment 68: The method of embodiment 66 or embodiment 67, wherein the detection probe oligonucleotides are differentially labeled to allow for the determination of which of SARS-CoV-2, Flu A, Flu B, and RSV A and RSV B are contained 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 media contains one or more of a buffer, a chelator, an anionic detergent and a degrative enzyme.

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

[0052] Embodiment 78: A composition or kit or method for analysis of one or more of a plurality of pathogen-associated target nucleic acid molecule 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 the biological sample, the primer pair comprising (i) a first SARS-CoV- 2 primer and (ii) a second SARS-CoV-2 primer, wherein the primer of l(a)(i) comprises a target hybridizing sequence consisting of a nucleotide sequence that is 18 to 35 contiguous nucleotides in length contained within SEQ ID NO: 123 and containing SEQ ID NO: 122, and wherein the primer of 1 (a)(i) and the primer of 1 (a)(ii) generate an amplicon that contains SEQ ID NO: 124 and has 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 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 primer pair for generating a first Flu A amplicon from the biological sample, 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 (1) an RSV A primer pair for generating an 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) an RSV B primer pair for generating an 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 or kit or method of embodiment 78, wherein the primer of (a)(ii) comprises a target-hybridizing sequence consisting of a nucleotide sequence that is 22 to 25 nucleotides in length. Embodiment 80: The composition or kit or method of embodiment 78 wherein the target-hybridizmg sequence of the primer of (a)(i) is SEQ ID NO: 116 or 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 of (a)(i) contains at least one nucleotide analog; wherein the primer of (a)(i) contains at least one nucleotide analog that is a 5-Me-C; wherein the primer of (a)(ii) is 24 nucleotides in length and contains at least one nucleotide analog; wherein the primer of (a)(ii) contains at least one nucleotide analog that is a 5-Me-C; or combinations thereof. Embodiment 82: The composition or kit or method of embodiment 78 to 81 , wherein the targethybridizing sequence of the primer of (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 consisting of a nucleotide sequence that is 25 to 40 contiguous nucleotides in length and is contained within SEQ ID NO: 124. Embodiment 85: The composition or kit or method of embodiment 84, wherein the detection probe oligomer target-hybridizing sequence 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 detection probe oligomer target-hybridizing sequence is SEQ ID NO: 118.

[0054] Embodiment 87: The composition or kit or method of any of embodiments 78 to 86, further comprising 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: (a) for the second SARS-CoV-2 primer pair of 1 (b), the targethybridizing sequence of l(b)(i) and the target-hybridizing sequence of l(b)(ii) are selected from the groups consisting of: 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; and SEQ ID NOs:240 and 247; (b) for the first Flu A primer pair of 1(c), the target-hybridizing sequence of l(c)(i) and the targethybridizing sequence of 1 (c)(ii) are selected from the groups 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 of 1(d), the target-hybridizing sequence of l(d)(i) and the targethybridizing sequence of 1 (d)(ii) are selected from the groups consisting of: SEQ ID NOs:23 and 25; SEQ ID NOs:23 and 6; and SEQ ID NOs:23 and 28; (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 groups consisting of: SEQ ID NOs:67 and 58; SEQ ID NOs:67 and 68; and SEQ ID NOs:67 and 70; (e) for the RSV A primer pair of 1(1), the target-hybridizing sequence of l(Q(i) and the target-hybridizing sequence of l(f)(ii) are selected from the groups 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 (1) 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 groups 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 contains from 0 to 20 nucleotide analogs. Embodiment 89: The composition or kit or method of embodiment 88, wherein at least one of the nucleotide analogs is a 5-Me-C analog.

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

[0057] Embodiment 92: The composition or kit or method of embodiment 87, wherein 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 second SARS- CoV-2 primer pair, wherein the target-hybridizing sequence of l(b)(i), the target-hybridizing sequence of l(b)(ii), and the second SARS-CoV-2 region detection probe oligomer targethybridizing sequence are selected from the groups 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, and 128; 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 lD NOs: 131, 129, and 130; SEQ lD NOs: 131, 129, 133, and 130; SEQ ID N Os: 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, and 193; 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, and 242; 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, wherein 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 first Flu A primer pair, wherein the target-hybridizing sequence of l(c)(i), the target-hybridizing sequence l(c)(ii), and the detection probe oligomer target-hybridizing sequence are selected from the groups 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 of embodiment 87, 92, or 93, wherein 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 second Flu A primer pair, wherein the targethybridizing sequence of 1(d)(1), the target-hybridizing sequence of l(d)(ii), and the detection probe oligomer target-hybridizing sequence are selected from the groups 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; 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 generated by the Flu B primer pair, wherein the detection probe oligomer target-hybridizing sequence 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 or kit or method of embodiment 87, 92, 93, 94, or 95, wherein 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 A primer pair, wherein the detection probe oligomer target-hybridizing sequence 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 of embodiment 87, 92, 93, 94, 95, or 96, wherein 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 l(g)(i), the target-hybridizing sequence of l(g)(ii), and the detection probe oligomer target-hybridizing sequence are selected from the groups 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; 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 from 0% to 100% nucleotide analogs, each detection probe oligomer comprises a nucleotide sequence that is from 0% to 100% 5-Me-C nucleotide analogs, or each detection probe oligomer comprises a nucleotide sequence containing at least one nucleotide analog, and at least one nucleotide analog is a 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 of any one of embodiments 78 to 101, wherein the composition is an aqueous formulation. Embodiment 103: The composition of any one of embodiments 78 to 101, wherein the composition is a dried formulation. Embodiment 104: The composition of embodiment 102 or embodiment 103, further comprising: a reagent for an amplification reaction, a reagent for a detection reaction, a reagent for a target capture reaction, or a combination thereof. Embodiment 105: A kit containing the composition of any one of embodiments 102 to 104.

[0061] Embodiment 106: The method of any one of embodiments 92 to 101 for determining whether a biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and/or RSV B, comprising the step of: (a) contacting under stringent hybridization conditions target nucleic acid molecules from the biological sample with one or more of the detection probe oligonucleotides of 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, 154, 193, 242, 246, 258, and 265; (b) detecting the presence or absence of a detectable label, the presence indicating that a probe:target nucleic acid duplex formed in step (a); and (c) based on whether the presence of a detectable label was detected in step (b), determining that the biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and/or RSV 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 at step (c). Embodiment 108: The method of embodiment 106 or 107, wherein target nucleic acid forming the probe:target nucleic acid duplex at step (b) is an amplicon generated using a primer pair.

[0062] Embodiment 109: The method for determining whether a biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and/or RSV B, comprising: (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 an amplicon from any target nucleic acid molecule in the amplification reaction mixture of step (a); and (c) determining whether an amplicon was generated at 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 wherein the nucleic acid amplification reaction is 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 performed using one or more detection probe oligonucleotides. Embodiment 113: The method of embodiment 112, wherein the detection probe oligonucleotides are labeled with donor/ acceptor label pairs. Embodiment 114: The method of embodiment 113, wherein the detection probe oligonucleotides are differentially labeled to allow for the determination of which of SARS-CoV-2, Flu A, Flu B, RSV A, and/or RSV B are contained 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 media contains one or more of a buffer, a chelator, an anionic detergent and a degrative enzyme.

[0064] Embodiment 119: A SARS-CoV-2 primer that comprises a target hybridizing sequence that is from 18 to 35 nucleotides in length, wherein the target nucleic acid sequence contains the sequence of SEQ ID NO: 122 and is contained within SEQ ID NO: 123. Embodiment 120: A SARS-CoV-2 primer that comprises a target hybridizing sequence that is from 18 to 35 nucleotides in length and the target hybridizing sequence hybridizes to SEQ ID NO: 124. Embodiment 121: An amplicon comprising a nucleotide sequence that is from 89 to 100 contiguous nucleotides in length and 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 further comprising a reverse complement nucleic acid strand. Embodiment 123: A SARS CoV-2 detection probe oligomer comprising a target hybridizing sequence that is from 18 to 27 nucleotides in length and the target hybridizing sequence is contained in SEQ ID NOs: 124 and 125. Embodiment 124: An amplicon comprising a nucleotide sequence that is from 80 to 90 nucleotides in length, and the sequence is contained within SEQ ID NO: 150. Embodiment 125: The amplicon of embodiment 124, wherein the amplicon contains 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 the reverse complement thereof. Embodiment 128: A forward primer and probe combination, wherein (i) the forward primer comprises a target hybridizing sequence that is from 13 to 28 nucleotides in length, is contained within SEQ ID NO: 169, and contains SEQ ID NO: 153, and (ii) the detection probe comprises a target hybridizing sequence that is from 17 to 40 nucleotides in length, is contained within SEQ ID NO: 169 and contains SEQ ID NO: 157. Embodiment 129: A forward and reverse primer set, wherein the forward primer comprises a target hybridizing sequence that is from 13 to 28 nucleotides in length, is contained within SEQ ID NO: 151, and contains SEQ ID NO: 153, and wherein the reverse primer comprises a target hybridizing sequence that is from 15 to 35 nucleotides in length, is contained withing SEQ ID NO: 171, and contains SEQ ID NO: 172. Embodiment 130: An amplicon that is from 75 to 95 nucleotides in length and is generated using a SARS CoV-2 forward primer comprising a target hybridizing sequence that is from 13 to 28 nucleotides in length, is contained within SEQ ID NO: 169, and contains SEQ ID NO: 153, and a SARS-CoV-2 reverse primer comprising a target hybridizing sequence that is from 15 to 35 nucleotides in length, hybridizes to SEQ ID NO: 173, and contains SEQ ID NO:172. Embodiment 131: An amplicon that is from 68 to 103 nucleotides in length and comprises 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 from 15 to 35 nucleotides in length, the reverse primer is from 15 to 35 nucleotides in length, and the primer pair generates an amplicon of any one of embodiments 131 to 134. Embodiment 136: An amplicon that is from 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 for any one of embodiments 136 to 138 comprising a target hybridizing sequence that is from 15 to 40 nucleotides in length, contained within SEQ ID NO:249, is not contained in SEQ ID NO:251, and hybridizes to SEQ ID NO:250. Embodiment 240: A combination comprising a SARS- CoV-2 reverse primer and SARS-CoV-2 detection probe for amplifying and detecting an amplicon from any one for embodiments 136 to 138, wherein the reverse primer comprises a target hybridizing sequence that is from 15 to 21 nucleotides in length, contained within SEQ ID NO:250, is not contained within SEQ ID NO:252, and hybridizes to SEQ ID NO:249, and wherein the detection probe comprises a target hybridizing sequence that is from 15 to 40 nucleotides in length, contained within SEQ ID NO:249, is not contained in SEQ ID NO:251, and hybridizes to SEQ ID NO: 250. Embodiment 141 : A kit that contains any one of the primers of embodiment 119, 120, 128, 130, 135, or 140. Embodiment 142: A kit that contains any one of the detection probes of embodiment 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 amplicon that is at least 90% identical to the amplicon of embodiment 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 embodiment 121 to 127, 130 to 134, 136, 137, or 138.

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

DETAILED DESCRIPTION

[0066] It should be noted that, as used in this specification and the appended claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “an oligonucleotide” includes a plurality of oligonucleotides and the like. The conjunction “or” is to be interpreted in the inclusive sense, i.e., as equivalent to “and/or,” unless the inclusive sense would be unreasonable in the context. [0067] It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present disclosure, such that slight and insubstantial deviations are within the scope of the present teachings herein. In general, the term “about” indicates insubstantial variation in a quantity of a component of a composition not having any significant effect on the activity or stability of the composition. All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints”; thus, for example, “within 10-15” includes the values 10 and 15 and all whole and partial (when applicable) values there between.

[0068] Unless specifically noted, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). “Consisting essentially of’ means that additional component(s), composition(s), or method step(s) that do not materially change 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 specificity that distinguishes the target nucleic acid from other known respiratory pathogens. Any component(s), composition(s), or method step(s) that have a material effect on the basic and novel characteristics of the present disclosure would fall outside of this term.

[0069] The term “complement” refers to a nucleic acid molecule that comprises a contiguous nucleotide sequence that is complementary to a contiguous nucleic acid sequence of another nucleic acid molecule (for standard nucleotides A:T, A:U, C:G). Two nucleic acid sequences are “sufficiently complementary” when, their respective contiguous nucleic acid sequences are at least 70% complementary. See, e.g., Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2 ^nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0070] By “substantially homologous,” “substantially corresponding,” or “substantially corresponds” is meant 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 which may contain a target nucleic acid. Biological samples include peripheral blood, mucus, plasma, serum, saliva, cerebrospinal fluid, urine, bone marrow, or other body fluid, biopsy tissue, or other materials of biological origin, as well as solutions or compositions containing materials of biological origin, for example, a bronchial lavage fluid. Samples can be obtained from several sources, including a clinical source wherein the sample is collected in order to determine the presence or absence of a target nucleic acid in the sample and in turn provide a patient with a diagnosis. A sample may be chemically and/or mechanically treated to disrupt tissue or cell structure, thereby releasing intracellular components into a solution. 0072] A “nucleotide” includes both nucleotides and nucleosides, including deoxyribonucleotides (e.g., dATP, dCTP, dGTP, dTTP), ribonucleotides (e.g., rATP, rCTP, rGTP, rUTP), and analogs thereof. Nucleotides comprise a purine or pyrimidine base linked glycosidically to a ribose or a deoxyribose sugar and a phosphate group attached to the ribose or deoxyribose sugar. Nucleosides comprise a purine or pyrimidine base linked glycosidically to a ribose or a deoxyribose sugar but lack the phosphate residues that are present in a nucleotide. Nucleotides and nucleosides, as used herein, refer to a monomer of DNA or RNA, respectively. See e.g., Kornberg and Baker, DNA Replication, 2 ^nd Ed. (Freeman, San Francisco, 1992).

[0073] The term “analog,” in reference to a chemical compound, refers to compound having a structure like that of another one, but differing from it in respect of one or more different atoms, functional groups, or substructures that are removed or replaced with one or more other atoms, functional groups, or substructures. In the context of a nucleotide or nucleoside, an analog refers to a compound that, like the nucleotide/side of which it is an analog, can be incorporated into a nucleic acid molecule (e.g., a primer, a probe and/or an amplification product). Nucleotide/side analogs are commonly added to synthetic oligonucleotides (such as primers and probes) using phosphorami dite chemistry techniques and devices. Nucleotide/side analogs are commonly added to amplification products by including the analog in a reaction mixture wherein a suitable polymerase, for example, a DNA polymerase, will incorporate the analog into the amplification product. Nucleotide/side (hereinafter “nucleotide”) analogs include synthetic nucleotides having 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, and the like.

[0074] “DNA” refers to deoxyribonucleic acid, and a DNA oligonucleotide (e.g., a primer or a probe) refers polymer of deoxyribonucleotides linked by phosphodiester bonds. DNA oligonucleotides can be single-stranded (ssDNA) or double-stranded (dsDNA), and can include both single 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 doublestranded (dsRNA), and can include both single and double-stranded (or “duplex”) regions. Single-stranded DNA (or regions thereof) and ssRNA can, if sufficiently complementary, hybridize to form double-stranded complexes (or regions). By “RNA equivalent,” “DNA equivalent,” “RNA equivalent bases,” and “DNA equivalent bases” is meant RNA and DNA molecules having similar complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (ribose versus deoxyribose) and may differ, for example, by the presence of uracil in RNA and thymine in DNA. The differences between RNA and DNA equivalents do not contribute to differences in homology (or sequence identity) because the equivalents have the same degree of complementarity' to a particular sequence.

[0075] The term “oligonucleotide” (or “oligomer” or “oligo”) means a multimeric compound comprising two or more joined RNA nucleotides, DNA nucleotides, analogs of RNA nucleotides, analogs of DNA nucleotides, or combinations thereof. Oligonucleotides can include other molecules that may be present in a joined sequence of nucleotides and that do not prevent hybridization of the polynucleotide with a second molecule having a complementary sequence. For example, an oligonucleotide can include two or more joined nucleotides on a first side of a linker molecule and two or more joined nucleotides on a second side of the linker molecule, as is often the configuration of a molecular torch. Oligonucleotides are preferably a polymeric chain of from 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 sequence of letters, it will be understood that the nucleotides are in 5'-3' orientation from left to right and that “A” denotes adenosine (dATP/rATP) or an analog thereof, “C” denotes cytidine (dCTP/rCTP) or an analog thereof, “G” denotes guanosine (dGTP/rGTP) or an analog thereof, “U” denotes uracil (rUTP) or an analog thereof, and “T” denotes thymidine (dTTP) or an analog thereof, unless otherwise noted. Oligonucleotides of the disclosure comprise the four natural nucleotides and often comprise non-natural 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 made up of a variety' of linkages known in the art, including one or more sugar-phosphodiester linkages, peptide-nucleic acid bonds (PNAs), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the oligonucleotide may be either ribose or deoxyribose, or similar compounds having known substitutions, such as, for example, 2'-(9-methyl ribose and 2' halide substitutions (e.g., 2'-O-Me or 2'-F). The 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 e.g., The Biochemistry of the Nucleic Acids, pages 5-36, Adams, et al., ed., 11 ^th ed., 1992; and PCT pub. No. WO 93/13121.

[0076] A “probe” is an oligonucleotide that hybridizes specifically to a target nucleic acid sequence in a nucleic acid, preferably in an amplified nucleic acid, under conditions that promote hybridization, to form a detectable hybrid. Probe oligonucleotides comprise one or more of a contiguous nucleotide sequence, a target hybridizing sequence, a non-target hybridizing sequence, detectable labels, linkers, and nucleotide analogs. Probes preferably have oligonucleotide lengths from about 10 contiguous nucleotides up to 100 contiguous nucleotides and comprise RNA, DNA, analogs, modified backbones, and combinations thereof. A probe may comprise target-specific sequences and optionally other sequences that are non-target hybridizing sequences (e.g., a sequence that does not hybridize the nucleic acid to be detected). Such non-target hybridizing sequences can include, for example, sequences that contnbute to three-dimensional conformation of the probe as are common with molecular torches and molecular beacons. See, e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, and 6,361,945. A probe may contain a detectable label that is either attached to the end of the probe or attached internally on the probe. 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 is attached. Labels include dyes, particles, chromophores (e.g., an atom or molecule that imparts a detectable color), combinatorial fluorescence 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 radiolabels. Labels and related detections methods are well known. See, e.g., Styer and Haugland, (1967), Proc. Natl. Acad. Sci. U.S.A. 98:719; U.S. Pat. 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” means a fluorescent chemical compound that can re-emit light upon light excitation Fluorophores include, for example, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, CalFluor Red™, CalFluor Orange™, stilbene, Quasar dyes (e.g., Quasar 570, Quasar 670, Quasar 705), Lucifer Yellow, Cascade Blue™, Texas Red, Alexa dyes, phycoerythin, Bodipy, and others known in the art. See, e.g., Haugland, Molecular Probes Handbook (Eugene, OR), 6 ^th Edition; The Synthegen catalog (Elouston, TX); Lakowicz, Principles of Fluorescence Spectroscopy, 2 ^nd 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 a light emitting label such as a fluorophore to absorb emitted light when in close proximity to the fluorophore. Quenchers are well-known in the art and include, e.g., Black Hole Quencher™ (or BHQ™, BHQ-1™, or BHQ-2™), Blackberry Quencher, Dabcyl, QSY, and Tamra™ compounds, to name a few.

[0079] Linear probes, molecular torches, and beacons are preferably labeled with an interactive pair of detectable labels. Examples of detectable labels that are preferred as members of an interactive pair of detectable labels interact with each other by FRET or non- FRET energy transfer mechanisms, often referred to as “donor/acceptor pairs” wherein the donor initially absorbs and then transfers the energy, and the acceptor is the moiety to which the energy is subsequently transferred. When the two labels of a donor/acceptor pair are held sufficiently close that energy emitted by one label can be received or absorbed by the second label, the two labels are said to be in “energy transfer relationship” with each other. This is the case, for example, when a molecular beacon or molecular torch is maintained in the “closed” state by formation of a stem duplex, and fluorescent emission from a fluorophore attached to one arm of the probe is quenched by a quencher moiety on the opposite arm. This is also the case when, for example, a linear probe is labeled with a fluorophore and a quencher at a distance along the linear probe that fluorescent emission from the attached fluorophore is quenched by the attached quencher. In these instances, the spatial separation of the fluorophore and quencher molecules (e.g., by “opening” the molecular torch or beacon or by hydrolyzing the linear probe molecule). Examples of donor/acceptor pairs, include pairing a fluorophore such as fluorescein, IAEDANS, EDANS, coumarin, BODIPY FL, BODIPY, lucifer yellow, eosin, erythrosine, tetramethylrhodamine, CalOrange, CalRed, Quasar, Texas Red, CY5, or CY3 with a quencher such as tetramethylrhodamine, fluororescein, 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] Synthetic techniques and methods of bonding labels to nucleic acids and detecting labels are well known in the art. See, e.g., Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2 ^nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333; and published European Pat. App. No. 0 747 706).

[0081] An “amplification primer” or “primer” is an optionally modified oligonucleotide that hybridizes to a target nucleic acid sequence, or its complement, and can participate in a nucleic acid amplification reaction. Primer oligonucleotides comprise one or more of a contiguous nucleotide sequence, a target hybridizing sequence, a non-target hybridizing sequence, linkers, and nucleotide analogs. Primers preferably have oligonucleotide lengths from about 10 contiguous nucleotides up 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 an organism to be detected and a sequence (or its complement) contained within an amplified nucleic acid molecule that hybridizes specifically to at least a portion of the primer oligonucleotide using standard hydrogen bonding. Primers hybridize to a target nucleic acid sequence and typically have a 3' end that can be extended by a DNA polymerase (e.g., promoter providers are a type of primer that do not have an extendable 3' end; see, e.g., U.S. Pat. No. 7,939,260) that incorporates nucleotides complementary to the target nucleic acid sequence to generate a double stranded portion thereof. Primers include, but are not limited to, non-T7 primers, promoter primers, promoter providers, T7 promoter primers. [0082] By “capture oligonucleotide” is meant at least one nucleic acid oligonucleotide that allows for joining 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 includes two binding regions: a target nucleic acid-binding region and an immobilized probe-binding region, usually contiguous on the same oligonucleotide, although the capture oligonucleotide may include a target nucleic acid-binding region and an immobilized probe-binding region that are present on two different oligonucleotides joined together by one or more linkers. The target hybndizing region of a capture probe can be specific for the target nucleic acid (e.g., sufficiently complementary to the target nucleic acid sequence) or non-specific for the target nucleic acid. Target capture systems that include a capture oligonucleotide are described in U.S. Pat. Nos. 6,110,678, WO 2021/097358, and 9,051,601.

[0083] By “immobilized probe” or “immobilized nucleic acid” is meant a nucleic acid that joins, directly or indirectly, a capture oligonucleotide to an immobilized support. An immobilized probe is an oligonucleotide joined to a solid support that facilitates separation of bound target nucleic acids from unbound material in a sample. [0084] The term “solid support” means any suitable medium present in the solid phase to which an immobilized probe or other agent can be covalently or non-covalently affixed or immobilized.

[0085] By “separating” or “purifying” or “isolating” is meant that one or more components of the biological sample are removed from one or more other components of the sample. Sample components include nucleic acids in a generally aqueous solution phase that can also include other materials, for example, 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. It can be formed using various 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 different amplification products, or amplicons, are generated by means of using two or more pairs of amplification primers in the same amplification reaction.

[0088] By “target nucleic acid” or “target” is meant a nucleic acid containing a target nucleic acid sequence. 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. By “target nucleic acid sequence” (also referred to as “target nucleotide sequence,” “target sequence,” “target region,” “target nucleic acid molecule”) is meant a specific deoxyribonucleotide or ribonucleotide molecule or nucleotide sequence comprising all or part of the nucleotide sequence of a single-stranded nucleic acid molecule, and the deoxyribonucleotide or ribonucleotide sequence complementary thereto.

[0089] By “transcription-associated amplification” is meant any type of nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. One example of a transcription-associated amplification method, called “Transcription-Mediated Amplification” (TMA), generally employs an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-template complementary oligonucleotide, and optionally may include one or more analogous oligonucleotides. Variations of TMA are well known in the art and are described, for example, in U.S. Pat. Nos. 5,437,990; 5,399,491; 5,554,516; 5,130,238; 4,868,105; and 5,124,246; published PCT application nos. 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) that includes chelators (EDTA, EGTA and/or trisodium salt of methyl glycine diacetic acid (Trilon M, Na3MGDA, BASF)), and lithium lauryl sulfate (LLS). Sample transport media formulations can further contain one or more of the following: a Good’s buffer; a stabilizing agent such as propylene glycol, poly ol, sugar, sugar alcohol, lactic acid, boric acid, or a boric acid derivative; sodium xylene sulfonate; a surfactant; trisodium citrate; and one or more degrative enzymes such as an amylase, a lipase, and a protease.

[0091] "Target Capture Reagent” or "TCR” is a HEPES-buffered solution (pH 6.4) that includes lithium chloride and EDTA, together with magnetic solid support particles (1 micron SERA-MAGTM MG-CM particles, Seradyn, Inc. Indianapolis, IN) having (dT)14 immobilized probe molecules covalently bound thereto. TCR contains multiple oligos that may include one or more target capture oligos (TCO) and, optionally, one or more promoter primers. For multiplex BiPhasic TMA amplification reactions, a 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 Al. For multiplex PCR amplification reactions, a TCR can contain a TCO configured to hybridize to each target nucleic acid to be amplified and/or detected. Alternatively for multiplex amplification reactions, a TCR contains a “wobble probe TCO” (see, e.g., U.S. Pat. No. 9,051,601), which non-specifically hybridizes to the 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] "Amplification Reagent," "AMP Reagent,” or "AR” is a Tris-buffered solution (pH 7-8) that includes 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, an AMP Reagent contains one or more primers configured to hybridize to each target nucleic acid 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 Flu A, Flu B, SARS-CoV-2, RSV A, RSV B, and combinations thereof. In some embodiments, AMP Reagent further comprises reverse transcriptase, RNA polymerase, salts, and cofactors. The reverse transcriptase can be, but it not limited to MMLV reverse transcriptase. The RNA polymerase can be, but is not limited to, T7 RNA polymerase.

[0093] "Promoter Reagent” or "PR" is a Tris buffered solution that includes 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 primer in the Promoter Reagent targets the same target nucleic acid as the promoter primer in the TCR. The promoter primer may have the same sequence as the promoter primer in the TCR or it may have a sequence that is different from the promoter primer in the TCR. For multiplex amplification, a Promoter Reagent contains 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 contains 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, S ARS-CoV-2, RSV A, RSV B, and combinations thereof. In some embodiments, the Promoter Reagent further comprises reverse transcriptase, RNA polymerase, salts and cofactors. The reverse transcriptase can be, but it not limited to MMLV reverse transcriptase. The RNA polymerase can be, but is not limited to, T7 RNA polymerase. "Enzyme Reagents" used in amplification or pre-amplification reaction mixtures are HEPES- buffered solutions (pH 6.5-8) that include MMLV reverse transcriptase, T7 RNA polymerase, salts, and cofactors.

[0094] Described herein are compositions, including kits and reagents, and methods for selectively detecting nucleic acids of various viral pathogens, specifically, influenza A (Flu A), influenza B (Flu B), S ARS-CoV-2, respiratory syncytial virus A (RSV A), and respiratory syncytial virus B (RSV B), in a sample. These compositions and methods can be used, for example, in diagnostic applications for screening clinical samples, nasopharyngeal samples, bronchoalveolar samples, donated blood 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 target may be used as primers or probes in any suitable primer/probe-based in vitro nucleic acid amplification method adapted for amplification of an intended target nucleic acid.

[0096] The amplification primers are useful as components of singleplex or multiplex amplification reactions wherein amplicon species can be produced from target-specific primers in the reaction mixture. A multiplex amplification reaction includes pnmer pairs for amplifying two or more of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B, or additionally includes primers for one or more of 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 connection with 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 replication enzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying out these various amplification techniques respectively can be found in U.S. Pat. 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 particularly preferred embodiments, 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 strains, and to accommodate for mismatches/mutations between a primer or a probe and its corresponding target nucleic acid sequences in viral target nucleic acid, degenerate bases and non-Watson Crick (NWC) base pairing can, in some preferred embodiments, be included in a primer or probe oligonucleotide. A NWC position in an oligonucleotide refers to a position where the oligonucleotide is configured to hybridize to at least one target nucleic acid sequence with a non-Watson Crick pairing, such as G-U, G-T, or G-A (either the G or the U/T/A can be the base in the oligonucleotide) or using a nucleotide analog, such as G-inosine or G-5-MethylC, or . In some embodiments, the NWC position is configured to hybridize via a wobble (G-U or G-T) or purine-purine (G-A) pair. In some embodiments, when one or more degenerate bases have been identified in the target nucleic acid sequence for a single primer or probe, multiple primer species or probe species may be synthesized to include all base combinations.

[0099] Primers useful for conducting amplification reactions can have different lengths to accommodate the presence of extraneous sequences that do not participate in target binding and that may not substantially affect amplification or detection procedures. For example, promoter primers useful for performing amplification reactions in accordance with the disclosure have at least a minimal sequence that hybridizes to the desired target nucleic acid sequence and a promoter sequence positioned upstream of that minimal sequence. However, insertion of sequences between the target binding sequence and the promoter sequence could change the length of the primer without compromising its utility in the amplification reaction. Additionally, the lengths of the amplification primers and detection probes are matters of choice as long as the sequences of these oligonucleotides conform to the minimal 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 include a sequence of bases substantially complementary to the selected target nucleic acid sequence in the Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B genome (or amplicon representing the corresponding region and its flanking or surrounding regions). Such probes may 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 a designed amplification and detection reaction. For example, in PCR the extension and detection reactions are carried out such that an oligonucleotide would hybridize to its target nucleic acid sequence at a reaction temperature of about 60°C. Probes in accordance with the disclosure have sequences complementary to, or corresponding to, a pre-selected target region of a particular viral target nucleic acid targeted by the probe. Preferred probes have a probe sequence, which includes the target-hybridizing sequence of bases together with any base sequences that are not complementary to the nucleic acid that is to be detected, in the length range of from 10-100 nucleotides.

[0102] Amplification of nucleic acids by transcription-mediated amplification (TMA) is a common technique in molecular biology, typically requiring sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. In an illustrative BiPhasic TMA reaction, a T7 (promoter) primer and TCO are hybridized to the target sequence during target capture, followed by removal of excess T7 primer during a wash step prior to a 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 a first amplification reaction. During the first amplification phase, AMP reagent, and optionally Enz me Reagent, is introduced. In the presence of reverse transcriptase, the T7 primer hybridized to the captured target is extended, creating a cDNA copy. The 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 produces multiple RNA transcripts from the template. The NT7 primer subsequently hybridized to the RNA transcripts and is extended, producing promoterless cDNA copies of the target RNA template. The RNA strands are degraded by RNase activity of the reverse transcriptase. Because no free T7 primer is available in the phase 1 amplification mixture, the reaction does not proceed further. The second phase is started with the addition of a Promoter Reagent, thus initiating exponential amplification and detection of the cDNA pool produced in phase 1.

[0103] Plate Setup: In some embodiments, four different plates are set up for use on two automated Kingfisher devices. The reactions can also be carried out in tubes or other containers. Plate 1 (TCR plate) contains the sample. Target Capture Reagent (e.g., 100 pL) is added to this plate. The TCO and T7 primer hybridize to target nucleic acid (e g., 400 pL sample). The TCO:target nucleic acid:T7 primer (pre-amplification hybrid) are captured using magnetic beads (immobilized probe on a solid support) and a magnet. For capture, plate 1 is placed into a heat block and heated to 60-65°C (e.g., 62°C) for 20-30 minutes followed by incubation at lower temperatures (e.g., 23°C) for 20 minutes to2 hours. For single phase TMA, T7 primer may be absent from the TCR mixture. In some embodiments, sample is combined with TCR containing TCO and, for BiPhasic amplification, T7 primer. The mixture of sample and TCR is then removed from the plate or tube and beads are washed twice with wash buffer. Plate 2 is a deep-well plate and holds 200-500 pL/well wash buffer. The wash buffer contains detergent and alcohol used to wash the captured pre-amplification hybrid. Captured hybrid is then moved to plate 3, which contains 200-500 pL/well wash buffer and is used to provide a second wash of the captured pre-amplification hybrid. Captured hybrid is moved to plate 4, which contains 50 pL/well AMP reagent. Plate 4 is processed for real-time isothermal amplification and detection.

[0104] BiPhasic Transcription-Mediated Amplification and Real-Time detection. First Phase Amplification: Plate 4 is first incubated at about 42-44°C for 5-15 minutes (e.g., 43°C for 5 minutes). Following this first incubation, 25 pL of Enzyme Reagent containing reverse transcriptase and T7 RNA polymerase, are added to each well and plate 4 is incubated about 5 minutes at about 42-44°C to generate a first amplification product. Second Phase Amplification: 25 pL of Promoter Reagent containing T7 primer and probe oligonucleotides is added to the first amplification product, and the reaction is incubated at 42-43°C for 30-60 minutes to allow formation of a second amplification product. Amplification product is detected in real time by recording 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, typically requiring sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, 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 concurrently with amplification in the same tube within the same instrument. Such a combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis. The concept of combining amplification with product analysis has become known as “real-time” PCR. See, e.g., U.S. Pat. Nos. 6,174,670 and 8,137,616. In real-time PCR, the formation of PCR products is monitored in each cycle of the PCR. The amplification is usually measured in thermocyclers that have additional devices for signal generation and detection from labels attached to probe oligonucleotide species during the amplification reaction. A number of such devices are known in the art for performing multiplex diagnostic assays with three, four, or more distinguishably labeled hybridization probes within one reaction vessel.

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

[0107] In TaqMan probe formats, a single-stranded hybridization probe for a given target is labeled with a donor/ acceptor pair of detectable labels. When the donor (e g., a fluorophore moiety) is excited with light of a suitable 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 a PCR reaction cycle, the hybridization probe binds to the target DNA and is degraded by the 5'-3' exonuclease activity of the Taq polymerase during the subsequent elongation phase. As a result, the excited donor moiety and the acceptor moiety become spatially separated, thus allowing for unquenched signal from the donor (e.g., a fluorescent emission) that is detected by the device. See, e.g., U.S. Pat. No. 5,538,848.

[0108] Molecular beacon and molecular torch formats typically also include hybridization probes labeled with a donor/acceptor pair, with each of the donor moiety and the acceptor moiety being 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 moiety and the acceptor moiety (e g., the fluorescent moiety and the quencher moiety) are in spatial vicinity in solution. After hybridization of the probe’s target hybridizing region to the desired target nucleic acid sequence, the donor moiety and the acceptor moiety are separated from one another such that after excitation of the donor moiety with light of a suitable wavelength, its emission can be measured. See, e.g., U.S. Pat. No. 5,118,801. [0109] In some preferred embodiments, real-time PCR methods for amplifying and detecting multiple target DNA sequences in a multiplex assay are used. Such methods involve providing a composition or reaction mixture containing nucleic acids from biological sample, probes, primers, and a suitable polymerase activity to catalyze amplification, subjecting the reaction mixture to a thermocyling protocol such that amplification of the multiple target sequences occurs, and monitoring hybridization of each of the probe molecule species (e.g., pairs of FRET hybridization probes) at least once after a plurality of amplification cycles. In embodiments where the viral target nucleic acid(s) to be detected is/are comprised of one or more RNA molecules, such methods typically involve first converting RNA to DNA (e.g., a “complementary” DNA or “cDNA”) using a reverse polymerase activity.

[0110] In such multiplex embodiments, the composition or reaction mixture typically comprises at least 2, preferably 3-8 detection probes. Preferably each detection probe comprises a donor/acceptor label pair. For multiplex assays configured to differentiate each of the target nucleic acids from the others, the detection probes each comprise a distinguishable donor/acceptor label pair. In addition, such a composition or reaction mixture also comprises several reagents, including one or more of the following: buffers 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 complete, the reaction is monitored to detect stable hybridization between one or more of the distinguishably labeled probe species present in the reaction and its corresponding target nucleic acid sequence (carried in an amplicon generated using the corresponding primer pair for the particular viral (or other) pathogen to be detected). Based on whether the donor moieties from each of the different donor/acceptor pairs are detected, it can then be determined if the biological sample contains Flu A, Flu B, SARS-CoV-2, RSV A, and/or RSV B and/or such other pathogens as are targeted in the particular assay.

[0112] Certain preferred kits will comprise one or more of a probe, a primer, a capture oligonucleotide, internal control oligonucleotides, other ancillary oligonucleotides, a buffer, dNTPs, DNA polymerase, reverse transcriptase, and instructions for using components of the kit (or a link to a website providing such instructions).

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

[0114] General Reagents and Methods. Unless otherwise indicated, amplifications were performed using a Panther Fusion instrument (Hologic, Inc.). Viral isolates used as amplification targets or controls were diluted in suitable media, e.g., Specimen Transport Media (Hologic, Inc., Cat. No. PRD-04423 or PRD-04339); Micro Test M4 Media (Remel, Inc. Cat. No. R12500), Micro Test M5 Viral Transport Medium (Remel, Inc. Cat. No. R12515), Micro Test M6 Viral Transport Medium (Remel, Inc. Cat. No. R12530), Micro Test M4RT Viral Transport Medium (Remel, Inc. Cat. No. R12505), or Copan Universal Transport Medium (Copan Diagnostics, Inc., Cat. No. 330C). Nucleic acid was extracted from viral isolates using a non-specific target capture procedure as described in U.S. Pat. No. 9,051,601. [0115] PCR reaction mixtures were typically assembled as follows: 19.05 pL Supermix (Promega GoTaq® Supermix); 0.35 pL MMLV Reverse Transcriptase (35 U); 0.6 pL GoTaq MDX Hotstart Taq (3U); 5 pL of nucleic acids (primers, probe, and target in suitable diluent); =25 pL total reaction volume. Promega, Madison, WI; New England Biolabs, Ipswich, MA; Sigma-Aldrich, St. Louis MO; Thermo Fisher, Waltham, MA; and others.

[0116] A representative RT-PCR assay based on TaqMan reagent chemistry to 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 is described below. The following description is provided solely for the sake of illustration.

[0117] A typical process begins by collecting a sample from a subject, for example, a nasopharyngeal swab specimen. Unless the sample is to be immediately assayed, the sample is typically placed in 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] Nucleic acids in the sample are isolated on the automated Panther Fusion system (Hologic, Inc.), or other system such as, for example, a KingFisher Flex Magnetic Particle Processor (ThermoFisher), a MagNA Pure LC System (Roche) and a MagNA Pure Total Nucleic Acid Isolation Kit (Roche; Cat. No. 03038505001), or aNuchSENS easy MAG System (bioMerieux) and Automated Magnetic Extraction Reagents (bioMerieux). Purified nucleic acids are then added to a PCR reaction mix along with a thermostable DNA polymerase and a reverse transcriptase. The reaction mix contains oligonucleotide primer pairs and targetspecific oligonucleotide probes for each of each of the target nucleic acids to be assayed, as well as a buffer containing dNTPs (dATP, dCTP, dGTP, dTTP (or dUTP)), MgCh, stabilizers, and bovine serum albumin. To protect RNA target nucleic acids from degradation, an RNase inhibitor (e.g., RNase Inhibitor II) can also be included. Various control nucleic acids may also be included. Such controls may be, for example, non-infectious in vitro transcribed RNA of specific viral sequences and/or non-infectious plasmid DNA containing control sequences. If desired, two different sets of amplification primers and probes targeting different genomic regions of the viruses to be detected can be used for any given target nucleic acid. Detection probe species are often dual-labeled with a distinguishable reporter dye and a quencher (donor/acceptor) which work in conjunction to provide real-time results.

[0119] Reverse transcription of RNA into cDNA and subsequent amplification of DNA may be performed, for example, on 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 anneals specifically to the target sequence of 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 the Taq polymerase to cleave the probe, thus separating the reporter dye from its quencher. This generates 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 is dependent on the number of amplification products (amplicons) present at that time. Fluorescence intensity is monitored during each PCR cycle by the real-time instrument.

EXAMPLE 1.

Real-Time PCR Amplification and Detection of Coronavirus Using Different Combinations of Primers and Probes

[0120] This example describes screening experiments testing primer and probe combinations for the real-time PCR amplification and detection of coronavirus. Reactions are generally prepared and performed as presented herein and as follows.

[0121] Several primer and probe mixtures (PPR mixes) were prepared in a microfuge tube to include a forward primer, a reverse primer, and a dual labeled detection probe. 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 1 x PPR mix (SEQ ID NOs:272-274) comprised 0.6 pM of each primer and 0.4 pM of the probe. Similarly, the coronavirus l x PPR mixes comprised 0.6 pM of each primer and 0.4 pM of the probe. These PPR mixes also contain 150 mM of KC1, 10 rnM MgC'b. and were brought to final volume using 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 an internal control PPR mix (275 pL) was added to each of a coronavirus mixture (275 pL) to provide a 1.25x PPR mixes (550 pL total volume). Amplification and detection reactions were prepared by combining 20 pL of each PPR mix and 5 L of a target nucleic acid eluate with a 1.25 x master mix containing dNTPs, dUTP, Taq polymerase, reverse transcriptase, and RNase inhibitor. Each of the amplification and detection mixtures was then overlaid with oil. The SARS-CoV-2 target nucleic acid was prepared from a stock virus concentration (1E3 TCID50/mL). The stock vims was serially diluted into a sample transport media (containing lithium lauryl sulfate (LLS), EDTA, and sodium phosphate) to provide 1E0, IE-1, IE-2, and IE-3 TClD50/mL concentrations. Each serial dilution was combined with HeLa cells at a 1E4 cell/mL concentration. Amplification and detection reactions were set up at 3 or 4 reactions per condition. Negative reactions include sample transport media without the coronavirus target nucleic acid. The reactions were performed in real-time using a Panther Fusion system (available from Hologic, Inc., Marlborough MA) by thermal cycling, and Ct and RFU data was recorded (see Table 1).

Table 1 [0124] Results. In all tests performed except for Sets L and M, the internal controls were positive (100%) and the negative control wells were negative (0%). Internal controls for Sets L and M were 0/4, indicating a problem with the internal control in these PPR mixes. Unless indicated otherwise in Table 1, the designated sets were positive for all replicates tested. Overall, Set A had the earliest average Ct for all dilutions. Set N was separately retested and showed similar results in the retest (avg Ct was 33.05 for 1E0 TCID50/ml, and 0 for the lower concentrations). 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 as generally described above except that each PPR mix further comprised primers and probes for the amplification and detection of Flu A, Flu B, RSV A, and RSV B (final concentration of 0.6 pM for each primer and 0.4 pM for each probe). Primers and probe 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 the SARS-CoV-2 PPR mixes in a multiplex configuration. Samples were prepared as described above, and the assay performed on a Panther Fusion system. Ct data is presented in Table 2, below.

Table 2

[0126] Results: Set A showed the fastest Ct at the 1E0 concentration and was the only set to show detection at the IE-1 concentration. EXAMPLE 2

Amplification and Detection of Flu A, Flu B, SARS-CoV-2, RSV A. and RSV B in Clinical Samples

[0127] Remnant nasopharyngeal (NP) swab specimens from SARS-CoV-2 positive individuals were analyzed in a multiplex real-time PCR assay containing primers and probes for the 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 with the Panther Fusion SARS-CoV-2 assay (Cat. No. PRD-06391) for performance comparisons. Nucleic acid extraction, amplification, and detection reactions were performed on a Panther Fusion instrument according to manufacturer’s instructions.

[0128] For this example, 500 pL of each of 22 remnant NP swab specimen were combined with 780 yL of sample transport media. Nine of the 22 remnant specimens were also further diluted to an estimated 3* the reported LoD for the Panther Fusion SARS-CoV-2 assay commercial assay (LoD = IE-2 TCID50/mL of the inactivated, cultured SARS-CoV-2 isolate (US-WA1/2020, BEI Resources; NR52281)). 3X LoD was determined using a separately obtained Ct for the 9 samples and a desired Ct of 36.4. These values were then plugged into a dilution factor equation to obtain the dilution factor needed to reach 3X LoD (2.sup.36.4-Ct = (X)LoD/(3)LoD, wherein X is the dilution factor). An aliquot of the combined specimen and transport media (500 pL) from each specimen was separately combined with a lysis reagent (710 pL) in a Panther Fusion Lysis Tube (Hologic, Marlborough, MA). Following an incubation, 360 pL of lysed specimen was combined with 450 pL of a target nucleic acid isolation reagent containing a capture oligonucleotide and a solid support. The lysis and target nucleic acid isolation reactions are generally described in U.S. Pat. Nos. 6,110,678, WO 2021/097358, and 9,051,601. 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 pL of eluate from each sample condition was added to a well of a multiwall plate. Also contained within each well was 20 pL of a rehydrated real-time PCR reaction mixture. Components of the real-time PCR reaction mixture are described above and comprised an experimental PPR mix (0.6 pM of each primer at 1 x concentration and 0.4 pM of each probe at 1 x concentration). Primers and probes in the PPR mix for 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. Probes for detecting Flu A amplification products were labeled with FAM, probes for detecting Flu B amplification products were labeled with Quasar 670, probes for detecting RSV A and RSV B amplification products were labeled with Cal Orange 560, probes for detecting SARS- CoV-2 were labeled with Cal Red 610, probes for detecting internal control nucleic acids were labeled with Quasar 705, and all probes contained a BHQ-1 or a BHQ-2 quencher (labels available from LGC Biosearch Technologies, Petaluma, CA). Each eluate was further added to a well of a multi well plate containing an amplification and detection reaction mixture from the Panther Fusion SARS-CoV-2 assay (set up according to manufacturer’s instructions). Amplification and detection reactions were then performed, and Ct and RFU data was collected. The data obtained with the PPR mix was compared to the data obtained with the commercial assay. Results are presented in Table 3, below.

Table 3

[0130] Internal control reactions were all positive. Negative control wells were all negative.

This PPR mix showed a sensitivity of 100% (22/22) and a positive predictive value (compared with commercial assay) of 100% (22/22), including with the samples diluted to about 3X LoD. Comparable Cts for the specimen tested neat using both the experimental system and the commercial system shows that detection of clinical titers of SARS-CoV-2 was not delayed by the presence of the Flu A, Flu B, RSV A, and RSV B primers and probes. Cts for the dilute specimen (3X LoD) were about 1 Ct earlier than estimated (calculation not shown), indicating that detection of dilute 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 above described PPR mix. These NP swab samples were also tested with the Panther Fusion Flu A/B/RSV assay (Cat. No. PRD-04328), for performance comparisons. Nucleic acid extraction, amplification, and detection reactions were set up and performed as is generally described above. Ct and RFU data was collected, and the data obtained with the PPR mix was compared to the data obtained with the commercial assay. Results are presented in Table 4, below.

Table 4

[0132] Internal control reactions were all positive. Negative control wells were all 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 the comparator assay (Ct data not shown here). The comparator assay showed a negative result for each of the same three clinical specimens showing negative when tested with the PPR mix (100% agreement between the test assay and the commercial assay). These corresponding results indicate a problem with the sample. The Ct data between the test assay and the comparator assay showed no significant differences for the Flu A system and for the RSV system (for each, p > 0.05 in paired t-test). No statistical comparison was done for the Flu B system (n = 2).

EXAMPLE 3

Amplification and Detection of Flu A and RSV A using a Flu A, Flu B, SARS-CoV-2, and RSV A/RSV B Multiplex Primer/Probe Mixture

[0133] The purpose of this experiment was to test a multiplex PPR mix containing a modified Flu A primer and probe combination. 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. The target nucleic acids were prepared from a stock panel to provide 1E4, 1E3, and 1E2 copies per reaction of 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, as is generally described above. Ct and RFU data were collected and analyzed.

Table 5

EXAMPLE 4

Singleplex CoV Amplification and Detection Oligonucleotide Combinations

[0134] The purpose of this experiment was to test the performance of several candidate torch oligonucleotides and T7 promoter primers in a BiPhasic, real-time, TMA assay. Testing was done as singleplex reactions for each condition. Reactions were generally prepared and TMA assay s performed as presented herein.

[0135] A series of target capture reagents were prepared to contain 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 each then split into three to accommodate separately testing along with 3 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 to contain a non-T7 primer (SEQ ID NO:283). These reagents were prepared, and the reactions were performed as is generally described above.

[0136] Each of the different reaction conditions were run in replicates of five (5) for wells containing sample transport media alone (negative control wells) or containing a target nucleic acid at various concentrations. The target nucleic acid for this experiment was an in vitro transcript (SEQ ID NO:291) at 30 copies, 100 copies, 300 copies or 15,000 copies per mL. Results are presented in Table 6, below.

Table 6

[0137] These conditions showed positive signals in from 1 to 5 replicates of each of the negative control wells. False positive results are possibly due to the torch molecules exhibiting intramolecular and/or intermol ecul ar interactions. Despite these false positive results, the conditions containing molecular torch SEQ ID NO: 286 showed low background to signal in the positive wells (negative reaction well RFU from about 0 to 1500, compared to positive reaction well RFU from about 7,500 to 11,000), thus out-performing combinations containing the other two torches. 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. Testing was again done as singlepl ex reactions for each condition. 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 of the different reaction conditions were run in replicates of five (5) for wells containing sample transport media alone (negative control wells) or containing a target nucleic acid at various concentrations. The target nucleic acid for this experiment was an in vitro transcript (SEQ ID NO:292) at 30 copies, 100 copies, or 300 copies or 15,000 copies per mL. Results are presented in Table 7, below.

Table 7

* Numbers in the Promoter Reagent row are SEQ ID NOs. [0140] These conditions showed no false positive signals. Conditions containing either promoter primer in the target capture reagent and the molecular torch SEQ ID NO:289 performed well, as did the condition containing promoter primer SEQ ID NO:296 in the target capture reagent and torch SEQ ID NO: 288. The conditions containing torch SEQ ID NO: 289, though, showed fast TTimes (13 minutes to 21 minutes) and high RFU values (1,000 to 4,000) over a low background. Furthermore, conditions containing torch SEQ ID NO: 289 showed a low spread of these TTime and RFU values between the replicates and across the target concentrations.

EXAMPLE S

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 Each of Influenza A and Influenza B.

[0141] A multiplex reaction was prepared to test the performance of primers and probes for amplifying and detecting two regions of a SARS-CoV-2 target nucleic acid in the presence of primers and probes for detecting influenza target nucleic acids. The oligonucleotides used in this experiment were as follows: in the target capture reagent were SEQ ID NOs: 310. 311, 299, and 300 (target capture oligonucleotides) and SEQ ID NOs: 307, 308, 295, and 309 (T7 promoter primers); in the amplification reagent were SEQ ID NOs: 305, 306, 283, and 184 (non-T7 primers); and in the promoter reagent were 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 sample transport media alone. Each reaction condition was run in replicates of twenty (20).

Table 8

[0142] In this example, the limit of detection for the multiplex reaction is 13.9 copies/mL of SEQ ID NO: 291 and 17.2 copies/mL of SEQ ID NO: 292, as determined by Probit analysis with a 95% probability. EXAMPLE 6

Linearity and Sensitivity Testing of SARS-CoV-2 Primer and Probe Mixes

[0143] The individual and combined linearity and sensitivity tests were conducted with in vitro transcripts (IVTs suspended in sample transport media (STM) and supplemented with 1,000 HeLa cells/mL). These experiments were carried out in two stages. In the first stage, IVTs (SEQ ID NOs:291 and 292) at 1,000 copies/reaction were tested with the individual primer and probe sets (see Table 9), as well as the combined oligo sets to gather information about their individual and combined performance characteristics of these primer and probe combinations. In the second stage, the linearity and the sensitivity of the combined oligo sets were tested with 7 concentration levels of the IVTs (SEQ ID NOs:291 and 292) ranging from 1 X 10 ^A 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 included the indicated primers and probes comprising analog nucleotides and varied placement 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. The negative reaction contained only the IC PPR Mix. Each of the positive reactions and the negative reaction were run in multiples of 6 reactions for stage 1 and multiples of 12 reactions for stage 2.

Table 9 [0145] Table 10 shows the results summary for the individual and combined primer/probe mixtures. The IC reaction showed 100% detection with all tested conditions. No issues with the combined oligo sets were observed. The linearity and the 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 seen down to 100 cp/rxn. PCR efficiency was high with an R 2 value >0.99 and slope at -3.33 (y = -3.3266x + 39.484). The IC exhibited 100% detection in all dilutions.

Table 10: Results Summary for Individual and Combined Oligo Sets (Stage 1).

Table 11: IVT Sensitivity Results for the Combined Oligo Sets (Stage 2).

EXAMPLE 7

Exemplary Oligonucleotide Sequences

[0146] 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. The following Table 12 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., a methylated base), and other chemical components that are not represented in the illustrated contiguous arrangements of symbols.

Table 12

SEQ ID NO Sequence (5' to 3') Oligo Type

pU = propyne dU mC = 5 -Methyl cytosine

[F*] = fluorophore

[Q*] = quencher mN (mA, mC, mG, mT, mK, or mR) = 2'0-Methyl nucleotide

(rxl) = rxl intemucleotidyl linker attached to acridinium ester compound (e.g., US Pat. No. 5,585,481) (C9) = 9 carbon spacer

* All sequences are written in the 5' to 3' orientation unless indicated otherwise. Sequence symbols are per Table 1 of World Intellectual Property Organization (WIPO) Handbook on Industrial Property Information and Documentation, Standard ST.25 (1998) (“WIPO ST.25 (1998)”).

[0147] Table 13 illustrates nucleic acid sequences that represent, for example, all or part of one strand of an amplification product, sequences in which primers or probes are contained, sequences which primers or probes contain, and target nucleic acid sequences.

Table 13 [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 this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the disclosure. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the disclosure. It will also be appreciated that computer-based embodiments of the instant disclosure can be implemented using any suitable hardware and software.

[0149] All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the 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.