Compositions, methods, and kits for detecting adenovirus, metapneumovirus, and / or rhinovirus nucleic acids

Abstract

To provide methods, compositions, and kits for detecting Adenovirus, Metapneumovirus, and / or Rhinovirus nucleic acid.SOLUTION: The disclosed disclosure is related to methods, compositions, and kits for targeting Adenovirus, Metapneumovirus, and / or Rhinovirus nucleic acid. Compositions include amplification oligomers and / or detection probe oligomers. Kits and methods comprise at least one of these oligomers. Methods include uniplex and multiplex amplification and detection reactions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 476,753, filed March 25, 2017, which is incorporated herein by reference.

[0002] Field The present disclosure relates to the detection of infectious agents, more particularly to the detection of adenovirus, metapneumovirus, and / or rhinovirus. Compositions, methods, and kits for amplifying and / or detecting adenovirus, metapneumovirus, and / or rhinovirus using in vitro nucleic acid amplification techniques are described. [Background technology]

[0003] introduction Infectious diseases, whether bacterial, viral, or other in origin, pose acute and chronic challenges to human health. Many common infections affect the respiratory tract. Respiratory tract diseases are common in patients of all ages but are often more severe in the very young and very elderly. Viruses include DNA and RNA viruses.

[0004] Adenoviruses (Adeno or Adv) can cause infection in various organs, including the gastrointestinal tract, upper respiratory tract, and eyes. In individuals with a properly functioning immune system, adenovirus infections are typically not associated with life-threatening illness. However, adenoviruses can cause serious infections in immunocompromised patients, such as HIV-positive individuals, and in patients who have undergone bone marrow transplants. Over 50 different human adenovirus serotypes have been identified. Based on various characteristics of adenoviruses, they have been divided into six major subgroups (subgenera or species A-F), with recent literature indicating the existence of a seventh serotype.

[0005] Early approaches to detecting adenoviruses relied primarily on serological testing and cell culture. However, in immunosuppressed patients, the impaired immune response limits the use of serological testing, and evaluation of positive cultures is a relatively time-consuming process. The introduction of PCR-based assays has provided new methods for rapid, specific, and sensitive detection of adenoviruses. However, many of these diagnostic approaches do not effectively cover all adenovirus serotypes and use low-stringency conditions to enable detection of genetically highly diverse adenoviruses.

[0006] Adenovirus DNA sequence homology between different species is low. Even conserved regions within the adenovirus genome show limited homology between adenoviruses derived from different species. In many cases, considerable DNA sequence variation exists even between serotypes within the same species. These facts highlight the difficulty of developing molecular tests that facilitate reliable screening for adenovirus infection with the necessary broad specificity.

[0007] Human metapneumovirus (hMPV), first isolated in 2001, is now recognized as the second leading cause of acute respiratory tract illness in infants and adults. It is estimated that more than 50% of infants are infected by age 2 years, and nearly all children are infected by age 5 years. hMPV accounts for approximately 5-15% of respiratory tract illnesses in hospitalized young children (Alto, 2004, The Journal of the American Board of Family Practice / American Board of Family Practice 17:466-469; Williams et al., 2004, N Engl J Med 350:443-450). hMPV infection is a significant burden in at-risk infants, contributing to chronic lung disease, congestive heart disease, and immunodeficiency in premature infants (Martino et al., 2005, Biology of Blood and Marrow Transplantation: Journal of the American Society for Blood and Marrow Transplantation 11:781-796).

[0008] Two distinct genetic lineages of hMPV have been established, designated subtypes A and B. These lineages have been further divided into subgroups A1, A2, B1, and B2, as determined by phylogenetic analysis of sequence data, often utilizing the fusion protein and G glycoprotein genes. No significant differences in clinical symptoms have been observed between patients infected with different hMPV subgroups (Wei, H., Tsao, K., Huang, C., Huang, Y., Lin, T. J. Microbiol Immunol Infect. 2012 Sep 26. pii:S1684-1182(12)00151-X). While information regarding transmission and pathogenicity is inconclusive, hMPV likely spreads in a manner similar to common respiratory viruses such as influenza. hMPV has been shown to co-infect with other respiratory pathogens. hMPV appears to provide only partial immunity after infection, likely due to the various strains and subtypes circulating during a given season, and individuals can be reinfected and develop repeated illness. Infection occurs primarily in late winter and early spring, and the prevalence of each hMPV subtype appears to vary from year to year and by location. Similarly, the overall incidence of hMPV can vary from year to year, with its reported prevalence in symptomatic patients with respiratory tract infections ranging from 2 to 26%.

[0009] Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infections in humans, usually associated with the common cold. Colds caused by HRVs occur year-round, with peak incidence in the fall and spring, and are one of the leading reasons for work and school absences, with significant economic impact. Rhinoviruses can also cause lower respiratory tract infections, resulting in severe illness in children, the elderly, and immunosuppressed patients.

[0010] HRV, which includes over 100 different serotypes, is a small, non-enveloped, positive-strand RNA virus. HRV is one of six genera in the Picornaviridae family, which also includes enteroviruses (EVs). Reverse transcription-polymerase chain reaction (RT-PCR) has been developed in the past few years to detect HRV in clinical specimens (e.g., Billaud et al.). (Bromqvist et al. (1999) J. Clin. Microbiol. 37:2813-2816; Kares et al. (2003) J. Clin. Virol. 2004 February, 29(2):99-104; Loens et al. (2003) J. Clin. Microbiol. 41:1971-1976; Savolainen et al. (2002) J. General Virol. 83:333-340; Steininger et al. (2001) J. Clin. Microbiol. 39:129-133). Most of these RT-PCR methods utilize conserved sequences in the 5' non-coding region of picornavirus genomes.

[0011] The ability to specifically detect HRV, especially avoiding false positives due to the similarity between HRV and EV, is important for both diagnosis and the selection of appropriate available treatments. Specific assays for HRV are also important for the development of new drugs. For example, in clinical trial design, it is important to correctly identify participants as having HRV infection if the trial is designed to evaluate drugs for use in treating HRV infection. In addition, in other clinical trials, it may be important to exclude individuals infected with HRV. Furthermore, HRV detection assays need to be simple to perform, provide easily interpretable results, and be relatively inexpensive for practical use.

[0012] Traditional methods for distinguishing HRV from EV have been performed by either virus neutralization assays, selection with HRV-specific primer pairs, differentiation of the two viral amplification products based on size differences, sequencing of the amplification products and comparison with known HRV and EV sequences, or hybridization using HRV- or EV-specific probes. These approaches can be time-consuming, expensive, and / or require skilled technicians with experience to accurately interpret assay results. There is still a need in the art for a molecular-based assay that can rapidly, sensitively and specifically detect multiple adenovirus serotypes.There is also still a need in the art for a rapid, sensitive and specific detection of multiple subtypes and subgroups of hMPV.In addition, there is still a need in the art for a method that can rapidly, sensitively and specifically detect RV, especially with regard to the ability to distinguish RV from EV. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Alto,2004,The Journal of the American Board of Family Practice / American Board of Family Practice 17:466-469 [Non-patent document 2] Williams et al.,2004,N Engl J Med 350:443-450 [Non-patent document 3] Martino et al., 2005, Biology of Blood and Marrow Transplantation: Journal of the American Society for Blood and Marrow Transplantation 11:781-796 [Non-patent document 4] Wei, H., Tsao, K., Huang, C., Huang, Y., Lin, TJ Microbiol Immunol Infect.2012 Sep 26.pii:S1684-1182(12)00151-X [Non-Patent Document 5] Billaud et al. (2003) J. Virol. Methods 108:223-228 [Non-patent document 6] Blomqvist et al. (1999) J. Clin, Microbiol. 37:2813-2816 [Non-Patent Document 7] Kares et al.(2003)J Clin Virol.2004 February, 29(2):99-104 [Non-patent document 8] Loens et al.(2003)J.Clin.Microbiol.41:1971-1976 [Non-Patent Document 9] Savolainen et al. (2002) J. General Virol. 83:333-340 [Non-Patent Document 10] Steininger et al. (2001) J. Clin. Microbiol. 39:129-133 Summary of the Invention

[0014] overview It is an object of the present disclosure to provide methods, compositions, and kits that can be used to specifically amplify and / or sensitively detect one or more of adenovirus, hMPV, and HRV nucleic acids. Advantageously, the methods, compositions, and kits can be used to specifically detect many (e.g., 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more) or all known serotypes and subgroups of adenovirus, hMPV, and / or HRV with high sensitivity.

[0015] 1. A composition or kit comprising at least a first and a second amplification oligomer, 10. A composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47.

[0016] 2. A composition or kit comprising at least a first and a second amplification oligomer, 159、1601-1602、1603-1604、1605-1610、1606-1611、1607-1612、1608-1613、1609-1624、1614-1615、1615-1625、1616-1617、1618-1626、1619-1627、1620-1628、1621-1629、1622-1630、1623-1631、1624-1632、1625-1633、1626-1634、1627-1640、1627-1641、1628-1635、1629-1636、1629-1637、1638-1641、1639-1642、1643-1650、1651-1652、1652-1663、1653-1664、1655-1665、1666-1675、1667-1676、1668-1677、1669-1678、1679-1680、1681-1682、1682-1683、1683-1690、1684-1691、1685-1692、1685-1693、1686-1694、1687-1695、1696-1697

[0017] 3. A composition or kit comprising at least a first and a second amplification oligomer, 76; and / or 312-346 of SEQ ID NO: 120, and / or 279-314 of SEQ ID NO: 101, and / or 455-506 of SEQ ID NO: 76, and / or 480-533 of SEQ ID NO: 120, and / or 455-506 of SEQ ID NO: 101, and / or 338-397 of SEQ ID NO: 76.

[0018] 4. A composition or kit comprising at least first and second amplification oligomers configured for two or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO: 47; and (B) for the second target nucleic acid, (i) the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length, comprising at least one metapneumovirus position in the range of nucleotide positions selected from nucleotides 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159; or (ii) a composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, comprising at least one rhinovirus position in the range of nucleotide positions selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76.

[0019] 5. A composition or kit comprising at least first and second amplification oligomers configured for two or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length, the metapneumovirus amplicon comprising at least one metapneumovirus position in a range of nucleotide positions selected from nucleotides 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159; and (B) for the second target nucleic acid, (i) the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47; or (ii) a composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, comprising at least one rhinovirus position in the range of nucleotide positions selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76.

[0020] 6. A composition or kit comprising at least first and second amplification oligomers configured for two or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, comprising at least one rhinovirus position in a range of nucleotide positions selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76; and (B) for the second target nucleic acid, (i) the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47; or (ii) the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length, the metapneumovirus amplicon comprising at least one metapneumovirus position in the range of nucleotide positions selected from nucleotide positions 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159.

[0021] 7. A composition or kit comprising at least first and second amplification oligomers configured for three or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO: 47; and (B) for a second target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length that includes at least one metapneumovirus position in a range of nucleotide positions selected from nucleotides 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159; and (C) For a third target nucleic acid, the first amplification oligomer and the second amplification oligomer are selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or a composition or kit configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, the composition or kit comprising at least one rhinovirus position in the range of nucleotide positions selected from 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76.

[0022] 8. The composition or kit of any one of claims 1 to 3, wherein the first amplification oligomer comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof.

[0023] 9. The composition or kit of any of claims 1-3 or 8, wherein the second amplification oligomer comprises a nucleic acid sequence containing at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof.

[0024] 10. The composition or kit of claim 4 or claim 7, wherein the first amplification oligomer configured to amplify an adenoviral amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof, and / or the second amplification oligomer configured to amplify an adenoviral amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof.

[0025] 11. The composition or kit of claim 5 or claim 6, wherein the second target nucleic acid is an adenoviral target nucleic acid and the first amplification oligomer configured to amplify an adenoviral amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify an adenoviral amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both.

[0026] 12. The composition or kit of claim 5 or claim 7, wherein the first amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both.

[0027] 13. The composition or kit of claim 4 or claim 6, wherein the second target nucleic acid is a metapneumovirus target nucleic acid and the first amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both.

[0028] 14. The composition or kit of claim 6 or claim 7, wherein the first amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both.

[0029] 15. The composition or kit of claim 4 or claim 5, wherein the second target nucleic acid is a rhinovirus target nucleic acid and the first amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both.

[0030] 16. The composition or kit of any one of claims 1, 4, 7, 8, 10, or 11, wherein, for the adenovirus target nucleic acid, the first amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73, 74.

[0031] 17. The composition or kit of claim 5, 6, or 9, wherein the second target nucleic acid is an adenovirus and the first amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73, 74.

[0032] 18. The composition or kit of any one of claims 1, 4, 7, 8, 10, or 11, wherein, for the adenovirus target nucleic acid, the second amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42, 43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149.

[0033] 19. The composition or kit of claim 5, 6, or 9, wherein the second target nucleic acid is an adenovirus and the second amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42, 43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149.

[0034] 20. The composition or kit of any one of claims 2, 5, 7, 8, 12, or 13, wherein, for the metapneumovirus target nucleic acid, the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52, 53, 151, 152, 153, 154, and 160.

[0035] 21. The composition or kit of claim 4, 6, or 9, wherein the second target nucleic acid is a metapneumovirus and the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52, 53, 151, 152, 153, 154, and 160.

[0036] 22. The composition or kit of any one of claims 2, 5, 7, 8, 12, or 13, wherein, for the metapneumovirus target nucleic acid, the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 56, 68, 158, 177, and 178.

[0037] 23. The composition or kit of claim 4, 6, or 9, wherein the second target nucleic acid is a metapneumovirus and the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 56, 68, 158, 177, and 178.

[0038] 24. The composition or kit of any one of claims 3, 6, 7, 8, 14, or 15, wherein, for the rhinovirus target nucleic acid, the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 50, 51, 59, 60, 65, 75, 77-86, 102-108, and 121-130.

[0039] 25. The composition or kit of claim 4, 5, or 9, wherein the second target nucleic acid is a rhinovirus and the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 50, 51, 59, 60, 65, 75, 77-86, 102-108, and 121-130.

[0040] 26. The composition or kit of any one of claims 3, 6, 7, 8, 14, or 15, wherein, for the rhinovirus target nucleic acid, the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 57, 95-100, 115-119, and 137.

[0041] 27. The composition or kit of claim 4, 5, or 9, wherein the second target nucleic acid is a rhinovirus and the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 57, 95-100, 115-119, and 137.

[0042] 28. The composition or kit of any one of claims 1 to 27, wherein the composition or kit further comprises at least one detection probe oligomer.

[0043] 29. The composition or kit of any one of claims 1, 4, 7, 8-11, and 16-19, wherein the composition or kit further comprises an adenovirus detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, and 140.

[0044] 30. The composition or kit of any one of claims 5 or 6, wherein the second target nucleic acid is an adenovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, and 140.

[0045] 31. The composition or kit of any one of claims 1, 4, 7, 8-11, and 16-19, wherein the composition or kit further comprises an adenovirus detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, the 18 to 36 nucleobases all selected from consecutive nucleobases within SEQ ID NO: 138.

[0046] 32. The composition or kit of any one of claims 15 or 6, wherein the second target nucleic acid is an adenovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, the 18 to 36 nucleobases all selected from consecutive nucleobases within SEQ ID NO: 138.

[0047] 33. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13, 20, 21, 22, and 23, wherein the composition or kit further comprises a metapneumovirus detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, and 176.

[0048] 34. The composition or kit of any one of claims 4 or 6, wherein the second target nucleic acid is a metapneumovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, and 176.

[0049] 35. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13, 20, 21, 22, and 23, wherein the composition or kit further comprises a metapneumovirus detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from consecutive nucleobases within SEQ ID NO: 161 or SEQ ID NO: 155.

[0050] 36. The composition or kit of any one of claims 4 or 6, wherein the second target nucleic acid is a metapneumovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from consecutive nucleobases within SEQ ID NO: 161 or SEQ ID NO: 155.

[0051] 37. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15, and 24-27, wherein the composition or kit further comprises a rhinovirus detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 49, 54, 87-94, 109-114, and 131-136.

[0052] 38. The composition or kit of any one of claims 4 or 5, wherein the second target nucleic acid is a rhinovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 49, 54, 87-94, 109-114, and 131-136.

[0053] 39. The composition or kit of any one of claims 28-38, wherein at least one of the detection probe oligomers comprises at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof.

[0054] 40. The composition or kit of any one of claims 28-39, wherein at least one of the detection probe oligomers comprises a detectable label.

[0055] 41. The composition or kit of claim 40, wherein the detectable label is a fluorophore.

[0056] 42. The composition or kit of claim 40 or claim 41, wherein the detection probe oligomer is a dual-labeled detection probe oligomer.

[0057] 43. The composition or kit of claim 42, wherein the detection probe oligomer comprises a fluorescently detectable label and a quencher moiety capable of quenching the fluorescence emission from the fluorescent label.

[0058] 44. The composition or kit of any one of claims 1, 4, 7-11, 16-19, 28-32, and 39-43, wherein the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify an adenoviral target nucleic acid.

[0059] 45. The composition or kit of claim 5 or claim 6, wherein the second target nucleic acid is an adenovirus, and the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify an adenovirus target nucleic acid.

[0060] 46. ​​The composition or kit of claim 44 or 45, wherein each of the one or more additional amplification oligomers comprises a target-hybridizing sequence independently selected from the group consisting of SEQ ID NOs: 1-9, 11-16, 25-28, 31-35, 38, 42-46, 61, 62, and 71-74.

[0061] 47. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13, 20-23, 28, 33-36, and 39-43, wherein the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a metapneumovirus target nucleic acid.

[0062] 48. The composition or kit of claim 4 or claim 6, wherein the second target nucleic acid is metapneumovirus, and the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a metapneumovirus target nucleic acid.

[0063] 49. The composition or kit of claim 47 or 48, wherein each of the one or more additional amplification oligomers comprises a target-hybridizing sequence independently selected from the group consisting of SEQ ID NOs: 52, 53, 56, 68, 151, 152, 153, 154, 158, 160, 177, and 178.

[0064] 50. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15, 24-28, and 37-43, wherein the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a rhinovirus target nucleic acid.

[0065] 51. The composition or kit of claim 4 or claim 5, wherein the second target nucleic acid is a rhinovirus, and the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a rhinovirus target nucleic acid.

[0066] 52. The composition or kit of claim 47 or 48, wherein each of the one or more additional amplification oligomers comprises a target-hybridizing sequence independently selected from the group consisting of SEQ ID NOs: 50, 51, 57, 59, 60, 65, 75, 77-86, 95-100, 102-108, 115-119, 121-130, and 137.

[0067] 53. The composition or kit of any one of claims 1, 4, 7-11, 16-19, 28-32, and 39-47, wherein the composition or kit comprises at least first and second amplification oligomers, and one or more additional amplification oligomers configured to amplify an adenovirus target nucleic acid, each of the amplification oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 61, 62, 71, 72, 73, and 74.

[0068] 54. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13, 20-23, 28, 33-36, 39-43, 46-49, and 53, wherein the composition or kit comprises at least first and second amplification oligomers, and one or more additional amplification oligomers configured to amplify a metapneumovirus target nucleic acid, each of the amplification oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 52, 53, 56, and 58.

[0069] 55. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15, 24-28, 37-43, and 50-54, wherein the composition or kit comprises at least first and second amplification oligomers, and one or more additional amplification oligomers configured to amplify a rhinovirus target nucleic acid, each of the amplification oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 50, 51, 57, 59, 60, and 65.

[0070] 56. The composition or kit of any one of claims 1, 4, 7-11, 16-19, 28-32, 39-47, and 53-55, wherein the composition or kit further comprises two adenovirus detection probe oligomers, each of the detection probe oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 63 and 64.

[0071] 57. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13, 20-23, 28, 33-36, 39-43, 46-49, and 53-56, wherein the composition or kit further comprises three metapneumovirus detection probe oligomers, each of the detection probe oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69, and 70.

[0072] 58. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15, 24-28, 37-43, and 50-57, wherein the composition or kit further comprises three rhinovirus detection probe oligomers, each of the detection probe oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 49, and 54.

[0073] 59. A composition or kit according to any one of the preceding claims, wherein the composition or kit further comprises a nucleic acid target capture probe comprising a target hybridizing sequence and an immobilized probe binding region.

[0074] 60. The composition or kit of claim 59, wherein the target hybridizing sequence is a poly K nucleotide sequence.

[0075] 61. The composition or kit of claim 60, wherein the poly K nucleotide sequence is a random poly GU sequence.

[0076] 62. The immobilized probe binding region is preferably T 0-4 A 10-36 62. The composition or kit of claim 59, 60, or 61, wherein the nucleotide sequence is a homopolymer nucleotide sequence comprising a nucleotide sequence selected from the group consisting of:

[0077] 63. The composition or kit of any one of claims 1-62, wherein the composition further comprises an enzyme, a buffer, a dNTP, or a combination thereof.

[0078] 64. Determine the presence or absence of adenovirus target nucleic acid, metapneumovirus target nucleic acid, rhinovirus target nucleic acid, or a combination thereof in a sample. A method for determining (A) contacting a sample with an amplification oligomer combination according to any one of claims 1 to 58; (B) performing an in vitro nucleic acid amplification reaction in which either an adenovirus target nucleic acid, a metapneumovirus target nucleic acid, or a rhinovirus target nucleic acid in the sample is used with a combination of amplification oligomers configured to amplify the target nucleic acid to generate an amplification product; (C) detecting the amplification product; Thereby determining the presence or absence of the target nucleic acid in the sample.

[0079] 65. The method of claim 64, wherein the sample is derived from a human.

[0080] 66. The method of claim 65, wherein the sample is a mucosal sample.

[0081] 67. The method of claim 65 or claim 66, wherein the sample is obtained using a nasopharyngeal swab.

[0082] 68. The method of any one of claims 64 to 67, wherein prior to step (A), a sample preparation step is carried out to separate any target nucleic acid in the sample from other sample components.

[0083] 69. The method of claim 68, wherein the sample preparation step comprises a target capture step.

[0084] 70. The method of claim 69, wherein the target capture step comprises contacting the sample with a nucleic acid target capture probe comprising a target hybridizing sequence and an immobilized probe binding region.

[0085] 71. The method of claim 70, wherein the target hybridizing sequence is a poly K nucleotide sequence.

[0086] 72. The method of claim 71, wherein the poly K nucleotide sequence is a random poly GU sequence.

[0087] 73. The immobilized probe binding region is preferably T 0-4 A 10-36 73. The method of claim 70, 71, or 72, wherein the nucleotide sequence is a homopolymer nucleotide sequence comprising a nucleotide sequence selected from the group consisting of:

[0088] 74. The method of any one of claims 64-73, wherein the detecting step (C) is carried out using one or more detection probe oligomers.

[0089] 75. The method of claim 74, wherein each of the one or more detection probe oligomers is individually selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140, 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 48, 49, 54, 87-94, 109-114, and 131-136.

[0090] 76. The method of claim 74 or 75, wherein at least one of the detection probe oligomers comprises at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof.

[0091] 77. The method of claim 74, 75, or 76, wherein at least one of the detection probe oligomers comprises a detectable label.

[0092] 78. The method of claim 77, wherein the detectable label is a fluorophore.

[0093] 79. The method of claim 77 or claim 78, wherein the detection probe oligomer is a dual-labeled detection probe oligomer.

[0094] 80. The method of claim 79, wherein the detection probe oligomer comprises a fluorescently detectable label and a quencher moiety capable of quenching the fluorescent emission from the fluorescent label.

[0095] 81. The method of any one of claims 64 to 80, wherein the in vitro nucleic acid amplification reaction comprises thermal cycling.

[0096] 82. The method of any one of claims 64 to 81, wherein the in vitro nucleic acid amplification reaction comprises PCR with a polymerase enzyme having 5' to 3' exonuclease activity.

[0097] 83. The method of any one of claims 75 to 80, wherein the in vitro nucleic acid amplification reaction is carried out using an enzyme having 5' to 3' exonuclease activity.

[0098] 84. The method of any one of claims 77 to 80, wherein the in vitro nucleic acid amplification reaction is carried out using an enzyme with 5' to 3' exonuclease activity and the amplification product is detected by determining a fluorescence value above a predetermined threshold.

[0099] 85. A system for performing one or more steps of the method of claim 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84.

[0100] 86. The system of claim 85, wherein the system is an automated system.

[0101] 87. The system of claim 85 or 86, wherein the system performs all of the steps of the method.

[0102] 88. A method for the in vitro detection of an adenovirus target nucleic acid, a metapneumovirus target nucleic acid, a rhinovirus target nucleic acid, or a combination thereof in a sample, the method comprising contacting the adenovirus target nucleic acid, the metapneumovirus target nucleic acid, and / or the rhinovirus target nucleic acid with a detection probe oligomer of any of claims 29-43, wherein hybridization of the detection probe oligomer to the target nucleic acid to which the detection probe oligomer is configured to hybridize indicates the presence of the target nucleic acid.

[0103] 89. The method of claim 88, wherein the method comprises contacting an amplification product from the adenovirus target nucleic acid, the metapneumovirus target nucleic acid, and / or the rhinovirus target nucleic acid with the detection probe oligomer, and wherein hybridization of the detection probe oligomer to the amplification product to which the detection probe oligomer is configured to hybridize indicates the presence of the target nucleic acid from which the amplification product was generated.

[0104] 90. The method of claim 88 or 89, wherein the in vitro detection reaction is carried out using an enzyme having 5' to 3' exonuclease activity.

[0105] 91. The method of any one of claims 88 to 90, wherein the in vitro detection reaction is carried out using an enzyme with 5' to 3' exonuclease activity, and the target nucleic acid or the amplification product produced therefrom is detected by determining a fluorescence value above a predetermined threshold.

[0106] 92. A system for carrying out an in vitro detection reaction according to any one of claims 88 to 91.

[0107] 93. The system of claim 92, wherein the system is an automated system.

[0108] 94. The system of claim 92 or 93, wherein the system performs all of the steps of the method.

[0109] 95. A dry composition comprising one or more of the amplification oligomers of any one of claims 1-27.

[0110] 96. A dry composition comprising one or more of the amplification oligomers of any one of claims 44-55.

[0111] 97. A dry composition comprising one or more of the detection probe oligomers of any of claims 29-43 or 56-58.

[0112] 98. A dry composition comprising a combination of amplification oligomers and / or detection probe oligomers according to any one of claims 1-58.

[0113] 99. The dry composition of any one of claims 95-98, wherein the dry composition further comprises an enzyme, a dNTP, or both.

[0114] 100. The dry composition of claim 99, wherein the enzyme has 5' to 3' exonuclease activity.

[0115] 101. The dry composition of claim 99 or claim 100, wherein the enzyme is a polymerase enzyme.

[0116] 102. The dry composition of any one of claims 95-101, wherein the dry composition has an inorganic salt concentration of 10 mM or less.

[0117] 103. The dry composition of any one of claims 95-102, wherein the dry composition has an inorganic salt concentration of 7 mM or less.

[0118] 104. The dry composition of any one of claims 95-103, wherein the dry composition has an inorganic salt concentration of 5 mM or less.

[0119] 105. The dry composition of any one of claims 95-101, wherein the dry composition has an inorganic salt concentration of about 0.5 mM to about 10 mM. In certain embodiments, for example, the following are provided: (Item 1) 1. A composition or kit comprising at least a first and a second amplification oligomer, 10. A composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47. (Item 2) 1. A composition or kit comprising at least a first and a second amplification oligomer, 159、1601-1602、1603-1604、1605-1610、1606-1611、1607-1612、1608-1613、1609-1624、1614-1615、1615-1625、1616-1617、1618-1626、1619-1627、1620-1628、1621-1629、1622-1630、1623-1631、1624-1632、1625-1633、1626-1634、1627-1640、1627-1641、1628-1635、1629-1636、1629-1637、1638-1641、1639-1642、1643-1650、1651-1652、1652-1663、1653-1664、1655-1665、1666-1675、1667-1676、1668-1677、1669-1678、1679-1680、1681-1682、1682-1683、1683-1690、1684-1691、1685-1692、1685-1693、1686-1694、1687-1695、1696-1697 (Item 3) 1. A composition or kit comprising at least a first and a second amplification oligomer, 76; and / or 312-346 of SEQ ID NO: 120, and / or 279-314 of SEQ ID NO: 101, and / or 455-506 of SEQ ID NO: 76, and / or 480-533 of SEQ ID NO: 120, and / or 455-506 of SEQ ID NO: 101, and / or 338-397 of SEQ ID NO: 76. (Item 4) 1. A composition or kit comprising at least first and second amplification oligomers configured for two or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO: 47; and (B) for the second target nucleic acid, (i) the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length, comprising at least one metapneumovirus position in a range of nucleotide positions selected from nucleotides 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159; or (ii) a composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, comprising at least one rhinovirus position in the range of nucleotide positions selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76. (Item 5) 1. A composition or kit comprising at least first and second amplification oligomers configured for two or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length, the metapneumovirus amplicon comprising at least one metapneumovirus position in a range of nucleotide positions selected from nucleotides 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159; and (B) for the second target nucleic acid, (i) the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47; or (ii) a composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, comprising at least one rhinovirus position in the range of nucleotide positions selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76. (Item 6) 1. A composition or kit comprising at least first and second amplification oligomers configured for two or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, comprising at least one rhinovirus position in a range of nucleotide positions selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76; and (B) for the second target nucleic acid, (i) the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47; or (ii) the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length, the metapneumovirus amplicon comprising at least one metapneumovirus position in the range of nucleotide positions selected from nucleotide positions 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159. (Item 7) 1. A composition or kit comprising at least first and second amplification oligomers configured for three or more target acids, (A) for a first target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify an adenoviral amplicon of at least about 50 nucleotides in length, comprising at least one adenoviral position in a range of nucleotide positions selected from 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO: 47; and (B) for a second target nucleic acid, the first amplification oligomer and the second amplification oligomer are configured to amplify a metapneumovirus amplicon of at least about 50 nucleotides in length that includes at least one metapneumovirus position in a range of nucleotide positions selected from nucleotides 966 to 1147 of SEQ ID NO: 150, and / or nucleotides 844 to 1027 of SEQ ID NO: 159, and / or 1000 to 1040 of SEQ ID NO: 150, and / or 880 to 915 of SEQ ID NO: 159, and / or 1027 to 1080 of SEQ ID NO: 150, and / or 913 to 958 of SEQ ID NO: 159, and / or 1073 to 1115 of SEQ ID NO: 150, and / or 953 to 995 of SEQ ID NO: 159; and (C) For a third target nucleic acid, the first amplification oligomer and the second amplification oligomer are selected from 230 to 556 of SEQ ID NO: 120, and / or 199 to 525 of SEQ ID NO: 101, and / or 80 to 410 of SEQ ID NO: 76, and / or 263 to 303 of SEQ ID NO: 120, and / or 231 to 264 of SEQ ID NO: 101, and / or 106 to 156 of SEQ ID NO: 76, and / or 312 to 346 of SEQ ID NO: 120, and / or a composition or kit configured to amplify a rhinovirus amplicon of at least about 50 nucleotides in length, the composition or kit comprising at least one rhinovirus position in the range of nucleotide positions selected from 279 to 314 of SEQ ID NO: 101, and / or 455 to 506 of SEQ ID NO: 76, and / or 480 to 533 of SEQ ID NO: 120, and / or 455 to 506 of SEQ ID NO: 101, and / or 338 to 397 of SEQ ID NO: 76. (Item 8) 4. The composition or kit of any one of items 1 to 3, wherein the first amplification oligomer comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof. (Item 9) 9. The composition or kit of any of items 1 to 3 or 8, wherein the second amplification oligomer comprises a nucleic acid sequence containing at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof. (Item 10) 8. The composition or kit of claim 4 or 7, wherein the first amplification oligomer configured to amplify an adenoviral amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof, and / or the second amplification oligomer configured to amplify an adenoviral amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof. (Item 11) 7. The composition or kit of claim 5 or 6, wherein the second target nucleic acid is an adenovirus target nucleic acid, and the first amplification oligomer configured to amplify an adenovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify an adenovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both. (Item 12) 8. The composition or kit of claim 5 or 7, wherein the first amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both. (Item 13) 7. The composition or kit of claim 4 or 6, wherein the second target nucleic acid is a metapneumovirus target nucleic acid and the first amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a metapneumovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both. (Item 14) 8. The composition or kit of claim 6 or 7, wherein the first amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof; or the second amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both. (Item 15) 6. The composition or kit of claim 4 or 5, wherein the second target nucleic acid is a rhinovirus target nucleic acid and the first amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or the second amplification oligomer configured to amplify a rhinovirus amplicon comprises a nucleic acid sequence containing at least one 5-Me-dC, at least one non-Watson-Crick base, at least one degenerate base, or a combination thereof, or both. (Item 16) 12. The composition or kit of any one of items 1, 4, 7, 8, 10, or 11, wherein, for the adenovirus target nucleic acid, the first amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73, 74. (Item 17) 10. The composition or kit of item 5, 6, or 9, wherein the second target nucleic acid is an adenovirus and the first amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73, 74. (Item 18) 12. The composition or kit of any one of items 1, 4, 7, 8, 10, or 11, wherein, for the adenovirus target nucleic acid, the second amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42, 43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149. (Item 19) 10. The composition or kit of item 5, 6, or 9, wherein the second target nucleic acid is an adenovirus and the second amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42, 43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149. (Item 20) 14. The composition or kit of any one of items 2, 5, 7, 8, 12, or 13, wherein, for the metapneumovirus target nucleic acid, the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52, 53, 151, 152, 153, 154, 160. (Item 21) 10. The composition or kit of item 4, 6, or 9, wherein the second target nucleic acid is a metapneumovirus and the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52, 53, 151, 152, 153, 154, 160. (Item 22) 14. The composition or kit of any one of items 2, 5, 7, 8, 12, or 13, wherein, for the metapneumovirus target nucleic acid, the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 56, 68, 158, 177, and 178. (Item 23) 10. The composition or kit of item 4, 6, or 9, wherein the second target nucleic acid is a metapneumovirus and the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 56, 68, 158, 177, 178. (Item 24) 25. The composition or kit of any one of claims 3, 6, 7, 8, 14, or 15, wherein, for the rhinovirus target nucleic acid, the first amplification oligomer comprises, consists of, or consists essentially of a target-hybridizing sequence selected from the group consisting of SEQ ID NOs: 50, 51, 59, 60, 65, 75, 77-86, 102-108, and 121-130. 10. The composition or kit of item 4, 5, or 9, wherein the second target nucleic acid is a rhinovirus and the first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 50, 51, 59, 60, 65, 75, 77-86, 102-108, 121-130. (Item 26) 16. The composition or kit of any one of items 3, 6, 7, 8, 14, or 15, wherein, for the rhinovirus target nucleic acid, the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 57, 95-100, 115-119, and 137. (Item 27) 10. The composition or kit of item 4, 5, or 9, wherein the second target nucleic acid is a rhinovirus and the second amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 57, 95-100, 115-119, and 137. (Item 28) 28. The composition or kit according to any one of items 1 to 27, wherein the composition or kit further comprises at least one detection probe oligomer. (Item 29) 20. The composition or kit of any one of items 1, 4, 7, 8-11, and 16-19, wherein the composition or kit further comprises an adenovirus detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, and 140. (Item 30) 7. The composition or kit of any one of items 5 or 6, wherein the second target nucleic acid is an adenovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140. (Item 31) Items 1, 4, 7, 8-11, and 16, wherein the composition or kit further comprises an adenovirus detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from consecutive nucleobases in SEQ ID NO: 138. A composition or kit described in any one of claims 1 to 19. (Item 32) 7. The composition or kit of any one of items 15 or 6, wherein the second target nucleic acid is an adenovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from contiguous nucleobases in SEQ ID NO: 138. (Item 33) 24. The composition or kit of any one of items 2, 5, 7, 8, 9, 12, 13, 20, 21, 22, and 23, wherein the composition or kit further comprises a metapneumovirus detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, and 176. (Item 34) 7. The composition or kit of any one of items 4 or 6, wherein the second target nucleic acid is a metapneumovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, and 176. (Item 35) 24. The composition or kit of any one of items 2, 5, 7, 8, 9, 12, 13, 20, 21, 22, and 23, wherein the composition or kit further comprises a metapneumovirus detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from consecutive nucleobases within SEQ ID NO: 161 or SEQ ID NO: 155. (Item 36) 7. The composition or kit of any one of items 4 or 6, wherein the second target nucleic acid is a metapneumovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence that is 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from contiguous nucleobases within SEQ ID NO: 161 or SEQ ID NO: 155. (Item 37) 28. The composition or kit of any one of items 3, 6, 7, 8, 9, 14, 15, and 24 to 27, wherein the composition or kit further comprises a rhinovirus detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 49, 54, 87 to 94, 109 to 114, and 131 to 136. (Item 38) 6. The composition or kit of any one of items 4 or 5, wherein the second target nucleic acid is a rhinovirus, and the composition or kit further comprises a detection probe oligomer comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 49, 54, 87-94, 109-114, and 131-136. (Item 39) 39. The composition or kit of any one of items 28 to 38, wherein at least one of the detection probe oligomers comprises at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof. (Item 40) 40. The composition or kit of any one of items 28 to 39, wherein at least one of the detection probe oligomers comprises a detectable label. (Item 41) 41. The composition or kit according to item 40, wherein the detectable label is a fluorophore. . (Item 42) 42. The composition or kit of claim 40 or 41, wherein the detection probe oligomer is a dual-labeled detection probe oligomer. (Item 43) 43. The composition or kit of claim 42, wherein the detection probe oligomer comprises a fluorescently detectable label and a quencher moiety capable of quenching fluorescence emission from the fluorescent label. (Item 44) 44. The composition or kit of any one of items 1, 4, 7-11, 16-19, 28-32, and 39-43, wherein the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify an adenovirus target nucleic acid. (Item 45) 7. The composition or kit of claim 5 or 6, wherein the second target nucleic acid is an adenovirus, and the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify an adenovirus target nucleic acid. (Item 46) 46. ​​The composition or kit of item 44 or 45, wherein each of the one or more additional amplification oligomers comprises a target-hybridizing sequence independently selected from the group consisting of SEQ ID NOs: 1-9, 11-16, 25-28, 31-35, 38, 42-46, 61, 62, and 71-74. (Item 47) 44. The composition or kit of any one of items 2, 5, 7, 8, 9, 12, 13, 20-23, 28, 33-36, and 39-43, wherein the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a metapneumovirus target nucleic acid. (Item 48) 7. The composition or kit of claim 4 or 6, wherein the second target nucleic acid is a metapneumovirus, and the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a metapneumovirus target nucleic acid. (Item 49) 49. The composition or kit of item 47 or 48, wherein each of the one or more additional amplification oligomers comprises a target-hybridizing sequence independently selected from the group consisting of SEQ ID NOs: 52, 53, 56, 68, 151, 152, 153, 154, 158, 160, 177, 178. (Item 50) 44. The composition or kit of any one of items 3, 6, 7, 8, 9, 14, 15, 24-28, and 37-43, wherein the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a rhinovirus target nucleic acid. (Item 51) 6. The composition or kit of claim 4 or 5, wherein the second target nucleic acid is a rhinovirus, and the composition or kit further comprises one or more additional amplification oligomers, each of the amplification oligomers configured to amplify a rhinovirus target nucleic acid. (Item 52) 49. The composition or kit of item 47 or 48, wherein each of the one or more additional amplification oligomers comprises a target-hybridizing sequence independently selected from the group consisting of SEQ ID NOs: 50, 51, 57, 59, 60, 65, 75, 77-86, 95-100, 102-108, 115-119, 121-130, and 137. (Item 53) 48. The composition or kit of any one of items 1, 4, 7-11, 16-19, 28-32, and 39-47, wherein the composition or kit comprises at least a first and a second amplification oligomer, and one or more additional amplification oligomers configured to amplify an adenovirus target nucleic acid, each of the amplification oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 61, 62, 71, 72, 73, and 74. (Item 54) 54. The composition or kit of any one of items 2, 5, 7, 8, 9, 12, 13, 20-23, 28, 33-36, 39-43, 46-49, and 53, wherein the composition or kit comprises at least first and second amplification oligomers and one or more additional amplification oligomers configured to amplify a metapneumovirus target nucleic acid, each of the amplification oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 52, 53, 56, and 58. (Item 55) 55. The composition or kit of any one of items 3, 6, 7, 8, 9, 14, 15, 24-28, 37-43, and 50-54, wherein the composition or kit comprises at least a first and a second amplification oligomer, and one or more additional amplification oligomers configured to amplify a rhinovirus target nucleic acid, each of the amplification oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 50, 51, 57, 59, 60, and 65. (Item 56) 56. The composition or kit of any one of items 1, 4, 7-11, 16-19, 28-32, 39-47, and 53-55, wherein the composition or kit further comprises two adenovirus detection probe oligomers, each of the detection probe oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 63 and 64. (Item 57) 57. The composition or kit of any one of items 2, 5, 7, 8, 9, 12, 13, 20-23, 28, 33-36, 39-43, 46-49, and 53-56, wherein the composition or kit further comprises three metapneumovirus detection probe oligomers, each of the detection probe oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69, and 70. (Item 58) 58. The composition or kit of any one of items 3, 6, 7, 8, 9, 14, 15, 24-28, 37-43, and 50-57, wherein the composition or kit further comprises three rhinovirus detection probe oligomers, each of the detection probe oligomers independently comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 49, and 54. (Item 59) The composition or kit of any one of the preceding items, wherein the composition or kit further comprises a nucleic acid target capture probe comprising a target hybridizing sequence and an immobilized probe binding region. (Item 60) 60. The composition or kit of item 59, wherein the target hybridizing sequence is a poly K nucleotide sequence. (Item 61) 61. The composition or kit of item 60, wherein the poly K nucleotide sequence is a random poly GU sequence. (Item 62) The immobilized probe binding region is preferably T 0-4 A 10-36 62. The composition or kit of items 59, 60, or 61, wherein the nucleotide sequence is a homopolymer nucleotide sequence comprising a nucleotide sequence selected from the group consisting of: (Item 63) 63. The composition or kit of any one of items 1 to 62, wherein the composition further comprises an enzyme, a buffer, a dNTP, or a combination thereof. (Item 64) Determining the presence of absence of adenovirus target nucleic acid, metapneumovirus target nucleic acid, rhinovirus target nucleic acid, or a combination thereof in the sample 1. A method for: (A) contacting a sample with the amplification oligomer combination according to any one of items 1 to 58; (B) performing an in vitro nucleic acid amplification reaction in which either an adenovirus target nucleic acid, a metapneumovirus target nucleic acid, or a rhinovirus target nucleic acid in the sample is used with a combination of amplification oligomers configured to amplify the target nucleic acid to generate an amplification product; (C) detecting the amplification product; Thereby determining the presence or absence of the target nucleic acid in the sample. (Item 65) 65. The method of item 64, wherein the sample is derived from a human. (Item 66) Item 66. The method of item 65, wherein the sample is a mucosal sample. (Item 67) 67. The method of claim 65 or 66, wherein the sample is obtained using a nasopharyngeal swab. (Item 68) 68. The method according to any one of items 64 to 67, wherein prior to step (A), a sample preparation step is carried out to separate any target nucleic acids in the sample from other sample components. (Item 69) 69. The method of claim 68, wherein the sample preparation step comprises a target capture step. (Item 70) 71. The method of claim 69, wherein the target capture step comprises contacting the sample with a nucleic acid target capture probe comprising a target hybridizing sequence and an immobilized probe binding region. 71. The method of claim 70, wherein the target hybridizing sequence is a poly K nucleotide sequence. (Item 72) 72. The method of claim 71, wherein the poly K nucleotide sequence is a random poly GU sequence. (Item 73) The immobilized probe binding region is preferably T 0-4 A 10-36 73. The method of claim 70, 71, or 72, wherein the nucleotide sequence is a homopolymer nucleotide sequence comprising a nucleotide sequence selected from the group consisting of: (Item 74) 74. The method according to any one of items 64 to 73, wherein the detecting step (C) is carried out using one or more detection probe oligomers. (Item 75) 75. The method of claim 74, wherein each of the one or more detection probe oligomers is individually selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140, 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 48, 49, 54, 87-94, 109-114, and 131-136. (Item 76) 76. The method of claim 74 or 75, wherein at least one of the detection probe oligomers comprises at least one 5-Me-dC, or at least one non-Watson-Crick base, or at least one degenerate base, or a combination thereof. (Item 77) 77. The method of claim 74, 75, or 76, wherein at least one of the detection probe oligomers comprises a detectable label. (Item 78) 78. The method of claim 77, wherein the detectable label is a fluorophore. (Item 79) 79. The method of claim 77 or claim 78, wherein the detection probe oligomer is a dual-labeled detection probe oligomer. (Item 80) 80. The method of claim 79, wherein the detection probe oligomer comprises a fluorescently detectable label and a quencher moiety capable of quenching fluorescence emission from the fluorescent label. (Item 81) 81. The method of any one of items 64 to 80, wherein the in vitro nucleic acid amplification reaction comprises thermal cycling. (Item 82) 82. The method according to any one of items 64 to 81, wherein the in vitro nucleic acid amplification reaction comprises PCR with a polymerase enzyme having 5' to 3' exonuclease activity. (Item 83) 81. The method according to any one of items 75 to 80, wherein the in vitro nucleic acid amplification reaction is carried out using an enzyme having 5' to 3' exonuclease activity. (Item 84) 81. The method of any one of items 77 to 80, wherein the in vitro nucleic acid amplification reaction is carried out using an enzyme having 5' to 3' exonuclease activity, and the amplification product is detected by determining a fluorescence value above a predetermined threshold. (Item 85) 8. A system for performing one or more steps of the method described in items 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84. (Item 86) Item 86. The system of item 85, wherein the system is an automated system. (Item 87) 87. The system of claim 85 or 86, wherein the system performs all of the steps of the method. (Item 88) 44. A method for the in vitro detection of an adenovirus target nucleic acid, a metapneumovirus target nucleic acid, a rhinovirus target nucleic acid, or a combination thereof in a sample, the method comprising contacting the adenovirus target nucleic acid, the metapneumovirus target nucleic acid, and / or the rhinovirus target nucleic acid with a detection probe oligomer according to any one of items 29 to 43, wherein hybridization of the detection probe oligomer with the target nucleic acid to which the detection probe oligomer is configured to hybridize indicates the presence of the target nucleic acid. (Item 89) 89. The method of claim 88, wherein the method comprises contacting an amplification product from the adenovirus target nucleic acid, the metapneumovirus target nucleic acid, and / or the rhinovirus target nucleic acid with the detection probe oligomer, wherein hybridization of the detection probe oligomer to the amplification product to which the detection probe oligomer is configured to hybridize indicates the presence of the target nucleic acid from which the amplification product was generated. (Item 90) 90. The method according to item 88 or 89, wherein the in vitro detection reaction is carried out using an enzyme having 5' to 3' exonuclease activity. (Item 91) 91. The method according to any one of items 88 to 90, wherein the in vitro detection reaction is carried out using an enzyme having 5' to 3' exonuclease activity, and the target nucleic acid or the amplification product produced therefrom is detected by determining a fluorescence value above a predetermined threshold. (Item 92) A system for carrying out an in vitro detection reaction according to any one of items 88 to 91. (Item 93) Item 93. The system of item 92, wherein the system is an automated system. (Item 94) Item 94. The system of item 92 or 93, wherein the system performs all of the steps of the method. (Item 95) 28. A dry composition comprising one or more of the amplification oligomers according to any one of items 1 to 27. (Item 96) 56. A dry composition comprising one or more of the amplification oligomers according to any one of items 44 to 55. (Item 97) A dry composition comprising one or more of the detection probe oligomers according to any of items 29 to 43 or 56 to 58. (Item 98) 59. A dry composition comprising a combination of amplification oligomers and / or detection probe oligomers according to any one of items 1 to 58. (Item 99) 99. The dry composition of any one of items 95 to 98, wherein the dry composition further comprises an enzyme, a dNTP, or both. (Item 100) 99. The dry composition of claim 98, wherein the enzyme has 5' to 3' exonuclease activity. (Item 101) The dry composition according to claim 99 or 100, wherein the enzyme is a polymerase enzyme. 102. The dry composition according to any one of items 95 to 101, wherein the dry composition has an inorganic salt concentration of 10 mM or less. (Item 103) 103. The dry composition according to any one of items 95 to 102, wherein the dry composition has an inorganic salt concentration of 7 mM or less. (Item 104) 104. The dry composition according to any one of items 95 to 103, wherein the dry composition has an inorganic salt concentration of 5 mM or less. (Item 105) 102. The dry composition according to any one of items 95 to 101, wherein the dry composition has an inorganic salt concentration of about 0.5 mM to about 10 mM. DETAILED DESCRIPTION OF THE INVENTION

[0120] Detailed Description Nucleic acid oligomer sequences are disclosed that can serve as primers for amplifying and detecting adenovirus, metapneumovirus, and / or rhinovirus nucleic acids. These target nucleic acids can be detected in samples using in vitro nucleic acid amplification methods such as PCR (e.g., Taqman™ PCR) or transcription-mediated amplification methods such as TMA or NASBA. Probes for detecting amplified nucleic acid sequences are also described. The detection probes specifically hybridize to at least a portion of the amplified sequence after or during the amplification process. The methods disclosed herein can be used to amplify and detect adenovirus, metapneumovirus, and / or rhinovirus nucleic acids present in samples from or derived from animals and humans.

[0121] The disclosed nucleic acid sequences and methods are useful for amplifying and detecting adenovirus, metapneumovirus, and / or rhinovirus nucleic acids from or derived from virus particles present in a sample in a relatively short time, resulting in a rapid diagnosis that allows for the initiation of effective treatment and limits the spread of the virus. The methods are useful for screening individuals with adenovirus, metapneumovirus, and / or rhinovirus infection, particularly for screening patients at high risk of death or serious complications from adenovirus, metapneumovirus, and / or rhinovirus infection, such as young, elderly, or immunocompromised individuals. The methods are also useful for rapid screening of large numbers of samples. The methods are useful because they minimize the risk of laboratory personnel being exposed to infectious pathogens, thereby limiting the risk of viral infection and spread. Thus, the methods and compositions disclosed herein address the need for rapid, sensitive, and specific testing of clinical samples that may contain adenovirus, metapneumovirus, and / or rhinovirus.

[0122] The disclosed probe sequences can be used as primers, and the disclosed primers can be used as probes.The same applies to the disclosed probe domains and primer domains.Therefore, the probe domains disclosed herein can be used as primer domains.Similarly, the primer domains disclosed herein can be used as probe domains.

[0123] The amplification oligomers disclosed herein are intended to allow several different amplicon species to be generated using an assortment of target-specific primers (e.g., two or more, three or more, four or more, The amplification systems disclosed herein are further contemplated as components of multiplex amplification reactions that can be generated from multiplexed amplification reactions (e.g., from five or more, six or more, or even ten or more). For example, it is contemplated that two or more of the amplification systems disclosed herein can be combined to provide a multiplexed assay with robust and broad target detection capabilities, e.g., the ability to amplify and detect nucleic acids from at least two, at least three, at least four or more microorganisms. For example, the amplification systems disclosed herein can be combined to provide a multiplexed assay for the detection of the following targets: adenovirus target nucleic acid and at least one additional target nucleic acid, metapneumovirus target nucleic acid and at least one additional target nucleic acid, rhinovirus target nucleic acid and at least one additional target nucleic acid, adenovirus target nucleic acid and metapneumovirus target nucleic acid and at least one additional target nucleic acid, adenovirus target nucleic acid and rhinovirus target nucleic acid and at least one additional target nucleic acid, rhinovirus target nucleic acid and metapneumovirus target nucleic acid and at least one additional target nucleic acid, adenovirus target nucleic acid and metapneumovirus target nucleic acid and rhinovirus target nucleic acid and at least one additional target nucleic acid. The multiplex assays described herein involve providing two or more amplification systems that each amplify and detect different subtypes or subgroups of a species, different species of microorganisms, or combinations thereof.

[0124] To aid in the understanding of aspects of the present disclosure, some terms used herein will be explained in more detail. All other scientific and technical terms used herein are defined in accordance with the principles of the Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), The Harper Collins The terms "term" and "term" have the same meaning as commonly understood by one of ordinary skill in the relevant art, such as those provided in Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY), and in the references cited herein. Unless otherwise stated, the techniques used or contemplated herein are standard methods well known to those skilled in the art of molecular biology.

[0125] definition It should be noted that an entity preceded by "a," "an," or "the" refers to one or more of that entity. For example, "a nucleic acid" is understood to refer to one or more nucleic acids. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0126] Sample. A "sample" or "specimen," including a "biological" or "clinical" sample, may contain or be suspected of containing adeno, hMPV, and / or HRV microorganisms, or components thereof, such as nucleic acids or fragments of nucleic acids. A sample may be a complex mixture of components. Samples include "biological samples," including any tissue or material derived from a living or dead mammal or microorganism, including, for example, blood, plasma, serum, blood cells, saliva, mucus, and cerebrospinal fluid. Samples may also include samples of in vitro cell culture components, including, for example, conditioned media resulting from the growth of cells and tissues in culture media. Samples may be treated to physically or mechanically disrupt tissue or cellular structures and release intracellular nucleic acids into solutions that may contain enzymes, buffers, salts, detergents, and the like, to prepare a sample for analysis. In one step of the methods described herein, a sample suspected of containing at least adeno, hMPV, and / or HRV target nucleic acids is provided. This step therefore excludes the physical step of obtaining the sample from a subject.

[0127] Nucleic acid. Nucleic acid refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs, where the nucleosides are linked together by phosphodiester or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and their analogs. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages (in "peptide nucleic acids" or PNA, see PCT Publication No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of a nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy and 2'-halide substitutions (e.g., 2'-F). The nitrogenous bases may be conventional bases (A, G, C, T, U), their analogs (e.g., inosine, 5-methyl-2'-deoxycytosine (5-Me-dC), isoguanine; The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43:617-24), or derivatives of purine or pyrimidine bases (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5 or 6 positions, purine bases with modified or replaced substituents at the 2, 6, and / or 8 positions, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4These include -alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines; U.S. Patent Nos. 5,378,825, 6,949,367, and PCT Publication No. WO93 / 13121. Nucleic acids can contain "abasic" residues, in which the backbone does not contain a nitrogenous base for one or more residues (U.S. Patent No. 5,585,481). Nucleic acids can contain only conventional sugars, bases, and linkages as found in RNA and DNA, or can contain conventional components and substitutions (e.g., nucleic acids containing conventional bases linked by a 2'-methoxy backbone, or a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked to RNA that mimics the sugar conformation, enhancing hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., 2004, Biochemistry 43(42):13233-41). Nucleic acids may contain modified bases to alter the function or behavior of the nucleic acid, such as the addition of a 3'-terminal dideoxynucleotide to prevent additional nucleotides from being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art, but nucleic acids may also be purified from natural sources using routine techniques.

[0128] Polynucleotides and oligonucleotides. These terms refer to nucleic acid strands. Throughout this application, nucleic acids are designated from the 5' end to the 3' end. Standard nucleic acids, such as DNA and RNA, are typically synthesized "3' to 5'," i.e., by adding nucleotides to the 5' end of a growing nucleic acid. Oligonucleotides, used interchangeably with "oligomer" and "oligo," generally refer to nucleic acids having fewer than 1,000 nucleotide (nt) residues, ranging from about 5 nt residues to about 900 nt residues, from about 10 nt residues to about 800 nt residues, including polymers with lower limits of about 12-15 nt and upper limits of about 40-600 nt; other embodiments range from lower limits of about 15-20 nt and upper limits of about 22-100 nt. These ranges are merely exemplary, and it is understood that oligonucleotides can include each integer within the range. Oligonucleotides may be purified from naturally occurring sources or synthesized using any of a variety of well-known enzymatic or chemical methods. The term oligonucleotide does not indicate any specific function for the reagent, but is used generally to cover all such reagents described herein.Oligonucleotide can perform a variety of different functions.For example, if oligonucleotide is specific to complementary strand, can hybridize with it, and can be further extended in the presence of nucleic acid polymerase, it can function as primer; if oligonucleotide contains the sequence recognized by RNA polymerase and allows transcription, it can provide promoter (for example, T7 promoter); if oligonucleotide is appropriately positioned and / or modified, it can function to prevent hybridization or prevent primer extension.

[0129] Nucleotide. A nucleotide is a subunit of nucleic acid consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of the ribose (2'-O-Me or 2' methoxy). As used herein, a methoxy oligonucleotide containing a "T" residue has a methoxy group at the 2' position of the ribose moiety and uracil at the base position of the nucleotide. A "C residue" present in an oligonucleotide (e.g., a primer or probe) includes methylated cytosine (e.g., 5-Me-dC) and unmethylated cytosine, unless the context indicates otherwise.

[0130] Non-nucleotide units. A non-nucleotide unit is a unit that does not significantly participate in hybridization of a polymer. Such a unit may, for example, not participate in any significant hydrogen bonding with a nucleotide, and may exclude units that have one of the five nucleotide bases or analogs thereof as components.

[0131] Target nucleic acid. The target nucleic acid is a nucleic acid that contains the "target sequence" to be amplified. The target nucleic acid may be DNA or RNA, and may be either single-stranded or double-stranded. The target nucleic acid may contain other sequences other than the target sequence that can be amplified. Typical target nucleic acids are or are derived from Adv, hMPV, and HRV genomes.

[0132] Target sequence. The term target sequence refers to the specific nucleotide sequence of a target nucleic acid to be amplified. If the target nucleic acid is originally single-stranded, the term "target sequence" also refers to the sequence complementary to the target sequence present in the target nucleic acid. If the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands. As used herein with respect to a region of an adeno, hMPV, or HRV nucleic acid, the term "target sequence" or "target nucleic acid" refers to the process by which an oligonucleotide stably hybridizes to a target sequence in a manner that allows for amplification and / or detection as described herein. In one embodiment, the oligonucleotide is complementary to the target sequence and does not contain mismatches. In another embodiment, the oligonucleotide is complementary but contains one, two, three, four, five, or more mismatches with the target sequence. In another embodiment, the oligonucleotide is complementary to the target sequence but contains one or a combination of degenerate nucleotide residues, non-Watson-Crick residues, or nucleoside analogs. Preferably, the oligonucleotide that stably hybridizes to the target sequence contains at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 consecutive nucleotides complementary to the target sequence. At least 10 and up to 50 are inclusive ranges, and are therefore understood to include 10, 50, and each integer therebetween. As used herein, the term "configured to target a sequence" means that the target hybridization region of the amplification oligonucleotide is designed to have a polynucleotide sequence that can target the sequence of a reference adeno, hMPV, or HRV region. Such amplification oligonucleotides are not limited to targeting only that sequence, but rather are useful as compositions, in kits, or in methods for targeting adeno, hMPV, or HRV target nucleic acids, as described herein. The term "configured to" refers to the actual arrangement of the polynucleotide sequence configuration of the amplification oligonucleotide target hybridizing sequence.

[0133] Isolation, separation, and purification refer to removing nucleic acids from their natural environment, but these terms do not necessarily imply any degree of purification. These terms mean that one or more components of a sample are removed or separated from other sample components. Sample components typically include the target nucleic acid in a generally aqueous solution phase that may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids. Separating or purifying removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components. Percent purity ranges include all integers and rational numbers within the range.

[0134] Region. This term refers to a portion of a nucleic acid, said portion being smaller than the entire nucleic acid. For example, if the nucleic acid being referred to is an oligonucleotide promoter provider, the term "region" may be used to refer to a smaller promoter portion of the entire oligonucleotide. Similarly, and by way of example only, if the nucleic acid is a target nucleic acid, the term "region" may be used to refer to a smaller region of the nucleic acid.

[0135] "RNA and DNA equivalents" refer to RNA and DNA molecules that have essentially the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. Because equivalents have the same degree of complementarity to a particular sequence, differences between RNA and DNA equivalents do not contribute to differences in homology. Unless otherwise indicated, a reference to an adeno-, hMPV-, or HRV nucleic acid includes the corresponding Adv-, hMPV-, or HRV-RNA and its DNA equivalent.

[0136] As used herein, an oligonucleotide having a nucleic acid sequence "comprising," "consisting," or "consisting essentially of" a sequence selected from a specified group of sequences means that the oligonucleotide has, as its basic and novel feature, the ability to stably hybridize under stringent hybridization conditions to a nucleic acid having the exact complement of one of the recited nucleic acid sequences of the group. The exact complement includes the corresponding DNA or RNA sequence.

[0137] Corresponding: As used herein, a nucleic acid "corresponds" to a specific nucleic acid if the nucleic acid is 100% identical to or complementary to that specific nucleic acid.

[0138] Substantially corresponding. As used herein, a nucleic acid that "substantially corresponds" to a designated nucleic acid sequence means that the oligonucleotide referred to is sufficiently similar to the reference nucleic acid sequence, so that the oligonucleotide has the same hybridization properties as the reference nucleic acid sequence, in that it hybridizes to the same target nucleic acid sequence under stringent hybridization conditions. A substantially corresponding nucleic acid differs from the designated nucleic acid by at least one nucleotide. This variation can be expressed in terms of the percentage of identity or complementarity between the nucleic acid and the designated nucleic acid. Thus, a nucleic acid substantially corresponds to a reference nucleic acid sequence when these percentages of base identity or complementarity are less than 100% to about 80% (including all integers and rational numbers therein).

[0139] Blocking Moiety. As used herein, a "blocking moiety" is a substance used to "block" the 3' end of an oligonucleotide or other nucleic acid so that it cannot be efficiently extended by a nucleic acid polymerase. Oligomers not intended for primer-based extension by a nucleic acid polymerase may contain a blocker group that replaces the 3' OH to prevent enzyme-mediated extension of the oligomer in an amplification reaction. For example, blocked amplification oligomers and / or detection probes present during amplification may not have a functional 3' OH but may instead contain one or more blocking groups located at or near the 3' end. In some embodiments, the blocking group near the 3' end may be within five residues of the 3' end and is sufficiently large to restrict polymerase binding to the oligomer. In other embodiments, the blocking group is covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, such as alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin.

[0140] Amplification Oligomers. An "amplification oligomer," which may also be referred to as an "amplification oligonucleotide" or "primer," is an oligomer that has at least its 3' end complementary to a target nucleic acid (a "target hybridizing sequence"), hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase in the amplification process. Another example of an amplification oligomer is a "promoter-based amplification oligomer," which contains a target hybridizing sequence and a promoter sequence for initiating transcription by an appropriate polymerase. Amplification oligonucleotides range in size from about 10 to about 70 nt in length (including all integers and rational numbers therein). In one embodiment, an amplification oligomer may optionally contain one or a combination of degenerate nucleotide residues, non-Watson-Crick residues, or nucleoside analogs. Thus, an amplification oligomer designated as comprising at least one degenerate nucleobase is a collection of amplification oligomer species that each independently have one of the nucleic acid residues represented by the degenerate nucleotide.

[0141] Amplification. Amplification refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, or its complement, or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Amplification of a "fragment" refers to the production of amplified nucleic acids containing less than the entire target nucleic acid or its complement, for example, by using amplification oligonucleotides that hybridize to and initiate polymerization at an internal position of the target nucleic acid. Known amplification methods include both thermal cycling amplification and isothermal amplification. Isothermal amplification is preferred in some embodiments. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods (see, e.g., U.S. Pat. No. 4,786,600; U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; U.S. Pat. Nos. 5,427,930 and 5,516,663; and U.S. Pat. No. 5,422,252; U.S. Pat. No. 5,547,861; and U.S. Pat. No. 5,648,211).

[0142] "Assay conditions" means conditions that allow stable hybridization of an oligonucleotide to a target nucleic acid. Assay conditions do not require preferential hybridization of an oligonucleotide to a target nucleic acid.

[0143] In the cyclic amplification method of detecting amplicons in real time, the term "threshold cycle" (Ct) is a measure of the time of the appearance of a signal associated with the amplification of a target, and is generally 10 times the standard deviation of the normalized reporter signal. When amplification reaches the "threshold cycle", it is generally considered that there is a positive amplification product of the sequence to which the probe binds. The identity of the amplification product can then be determined by methods known to those skilled in the art, such as gel electrophoresis, nucleic acid sequencing, and other such well-known methods.

[0144] Real-Time Amplification: As used herein, the term "real-time amplification" refers to the amplification of a target nucleic acid that is monitored by a real-time detection means.

[0145] Amplicon. This term, used interchangeably with "amplification product," refers to a nucleic acid molecule produced during an amplification procedure that is complementary to or homologous to a sequence contained within a target sequence. These terms can be used to refer to a single-stranded amplification product, a double-stranded amplification product, or one of the strands of a double-stranded amplification product.

[0146] Probe. A probe, also known as a "detection probe" or "detection oligonucleotide," is a term that refers to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization and allow detection of the target sequence or amplified nucleic acid. Detection can be either direct (e.g., a probe directly hybridized to its target sequence) or indirect (e.g., a probe bound to its target via an intermediate molecular structure). Probes can be DNA, RNA, analogs thereof, or combinations thereof, and can be labeled or unlabeled. The "target sequence" of a probe generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through standard base pairing. The probe may contain target-specific sequences and other sequences that contribute to the three-dimensional structure of the probe (e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication No. 2006 / 0068417). In a preferred embodiment, the detection probe contains a 2' methoxy backbone, which can lead to higher signal acquisition. In another preferred embodiment, the probe contains a fluorophore covalently attached to the 5' end of the probe and a quencher at the 3' end. Such probes are known as Taqman™ probes. In another embodiment, the probe may optionally contain one or a combination of degenerate nucleotide residues, non-Watson-Crick residues, or nucleoside analogs. Thus, a probe designated as containing at least one degenerate nucleobase is a collection of probe species that each independently have one of the nucleic acid residues represented by the degenerate nucleotide.

[0147] Stable. By "stable" or "stable for detection" is meant that the temperature of the reaction mixture is at least 2° C. below the melting temperature of the nucleic acid duplex.

[0148] Label. As used herein, "label" refers to a moiety or compound directly or indirectly attached to a probe that is detected or provides a detectable signal. Direct labeling can occur through a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonding, hydrophobic and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can occur using a bridging moiety or "linker," such as a binding pair member, antibody, or additional oligomer, that is directly or indirectly labeled and can amplify the detectable signal. Labels include radionuclides, ligands (e.g., biotin, avidin), enzymes or enzyme substrates, reactive groups, or chromophores (e.g., dyes, particles, or beads that impart a detectable color), light-emitting compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent labels (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604)), or fluorophores. Labels can be detectable in homogeneous assays in which bound labeled probes in a mixture exhibit a detectable change, e.g., instability or specific degradation characteristics, that differ from unbound labeled probes (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Fluorophore embodiments include those that absorb light in the range of about 495-650 nm and emit light in the range of about 520-670 nm, including those known as FAM™, TET™, CAL FLUOR™ (Orange or Red), and QUASAR™ compounds. Fluorophores may also be used in conjunction with quencher molecules that absorb light when in close proximity to the fluorophore, reducing background fluorescence. Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHER™ (or BHQ™, BHQ-1™, or BHQ-2™) or TAMRA™ compounds.The synthesis and the method of binding labels to nucleic acids and the method of detecting labels are well known (for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10, and U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174 and 4,581,333).Two or more labels and two or more types of labels can be present on a specific probe, or detection can use a mixture of probes, each of which is labeled with a compound that produces a different detectable signal (for example, U.S. Patent Nos. 6,180,340 and 6,350,579).

[0149] Capture Oligonucleotide. As used herein, "capture oligonucleotide," "target capture oligonucleotide," or "capture probe" refers to a nucleic acid oligomer that hybridizes to a target sequence in a target nucleic acid via a binding partner on an immobilized probe, binds to the binding partner, and captures the target nucleic acid to a support. One example of a capture oligomer includes an oligonucleotide containing two binding regions: a target hybridizing sequence and an immobilized probe binding region. In a variation of this example, the two regions may be present on two different oligomers linked to each other by one or more linkers. In another embodiment of a capture oligomer, the target hybridizing sequence is a sequence containing a random or nonrandom poly-K, poly-GU, poly-GT, or poly-U sequence for nonspecifically binding to the target nucleic acid and linking it to the immobilized probe on the support. (See, e.g., PCT Publication No. WO2008 / 016988 and U.S. Patent No. 9,051,601.) The immobilized probe binding region may be a nucleic acid sequence called a tail. The tail is approximately 10-40 nucleotides long (e.g., A 10 ~A 40 ) or about 14 to 33 nt (e.g., T3A 14 ~T3A30 ) in some embodiments. 0-4 A 10-36 It may contain sequences.

[0150] Immobilized Oligonucleotide. As used herein, "immobilized oligonucleotide," "immobilized probe," or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly binds a capture oligomer to a support, facilitating separation of the target nucleic acid bound to the capture probe from unbound material in a sample. One embodiment of an immobilized probe is an oligomer bound to a support, such as nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other composition, one embodiment of which is a magnetically attractable particle.

[0151] Complementarity. "Complementary" means that the nucleotide sequences of similar regions of two single-stranded nucleic acids, or two different regions of the same single-stranded nucleic acid, have a nucleotide base composition that allows the single-stranded regions to hybridize together under stringent hybridization or amplification conditions in a stable double-stranded hydrogen-bonded region. The sequences that hybridize to each other can be fully complementary or partially complementary to the intended target sequence through standard nucleic acid base pairing (e.g., G:C, A:T, or A:U pairing). "Fully complementary" means a contiguous sequence that can hybridize to another sequence through hydrogen bonding between a series of complementary bases, which may be complementary at every position in the sequence through standard base pairing, or may not be complementary through standard A:T / U and G:C pairing, or may contain one or more residues that are modified nucleotides, modified nucleotides, or nucleotide analogs, such as abasic residues. A sufficiently complementary contiguous sequence is typically at least 80% or at least 90% complementary to the sequence to which the oligomer is intended to specifically hybridize (the range of percent complementarity includes all integers and rational numbers within that range). A "sufficiently complementary" sequence allows stable hybridization of a nucleic acid oligomer with its target sequence under appropriate hybridization conditions, even if the sequences are not perfectly complementary. A nucleotide sequence is "perfectly" complementary if a contiguous sequence of nucleotides in one single-stranded region can form a series of "canonical" hydrogen-bonded base pairs with a similar sequence of nucleotides in another single-stranded region, such that A pairs with U or T and C pairs with G.

[0152] Preferentially hybridize. "Preferentially hybridize" means that under stringent hybridization assay conditions, oligonucleotides hybridize to their target sequence or a copy thereof to form stable oligonucleotide:target sequence hybrids while minimizing the formation of stable oligonucleotide:non-target sequence hybrids. For example, probe oligonucleotides preferentially hybridize to a target sequence or a copy thereof to a sufficiently greater extent than non-target sequences, allowing one skilled in the art to accurately detect RNA copies or complementary DNA (cDNA) of the target sequence formed during amplification. Suitable hybridization conditions for probe, amplification, target capture, blocker, and other oligonucleotides are well known in the art and can be predicted based on sequence composition or determined using routine testing (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2002). nd ed.(Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY, 1989) §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, especially §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0153] Nucleic Acid Hybrid. "Nucleic Acid Hybrid," or "Hybrid," or "Duplex" refers to a nucleic acid structure containing two strands of hydrogen-bonded regions, each strand complementary to the other, which regions are sufficiently stable under stringent hybridization conditions to be detected by means including, but not limited to, chemiluminescent or fluorescent photodetection, autoradiography, or gel electrophoresis. Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA double-stranded molecules.

[0154] Sample preparation. Sample preparation refers to any step or method of processing a sample for subsequent amplification and / or detection of one or more Adv, hMPV, or HRV nucleic acids present in the sample. The target nucleic acid may be a minor component in the sample. Sample preparation may include any known method for isolating or concentrating components, such as viruses or nucleic acids, using standard microbiological methods. Sample preparation may include physical disruption and / or chemical lysis of cellular components to release the intracellular components into a substantially aqueous or organic phase, and removal of debris, such as by using filtration, centrifugation, or adsorption. Sample preparation may include the use of nucleic acid oligonucleotides to selectively or nonspecifically capture the target nucleic acid and separate it from other sample components (e.g., as described in U.S. Pat. Nos. 6,110,678, 9,051,601, and PCT Publication WO2008 / 016988).

[0155] Specificity. The term "specificity" in the context of an amplification system is used herein to refer to the characteristic of an amplification system that describes its ability to distinguish between target and non-target sequences, depending on the sequence and assay conditions. In the context of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of by-products (e.g., signal-to-noise ratio).

[0156] Sensitivity. The term "sensitivity" is used herein to refer to the precision with which a nucleic acid amplification reaction can be detected or quantified. The sensitivity of an amplification reaction is generally a measure of the minimum copy number of the target nucleic acid that can be reliably detected in an amplification system, and depends, for example, on the detection assay used and the specificity of the amplification, for example, the ratio of specific amplicon to by-products.

[0157] Relative Fluorescence Units. As used herein, the term "relative fluorescence units" ("RFU") is an arbitrary unit of measurement of fluorescence intensity. RFUs vary depending on the characteristics of the detection means used for the measurement.

[0158] In particular, references in the claims to "the sequence of SEQ ID NO: X" refer to the base sequence set forth in the corresponding sequence listing unless otherwise indicated, and do not require identity of the backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues).

[0159] A "degenerate" position in an oligomer refers to a position where two or more base pairs exist in a population of oligomers. For example, in SEQ ID NO: 81, the eighth nucleotide is R, which represents G or A. An oligomer having a degenerate position can be synthesized by providing a mixture of nucleotide precursors corresponding to the desired degenerate combination in the synthesis step where the introduction of a degenerate position is desired. The resulting oligomer is a mixture of species each containing one of the nucleotides represented by the degenerate designation.

[0160] A "non-Watson-Crick" (NWC) position in an oligomer refers to a position at which the oligomer is configured to hybridize to at least one nucleic acid sequence by non-Watson-Crick pairing, such as GU, GT, or GA (where either G or U / T / A can be the base in the oligomer). In some embodiments, the NWC position is configured to hybridize via a wobble (GU or GT) or purine-purine (GA) pair.

[0161] Oligonucleotides for Amplification of Adenovirus, Metapneumovirus, and / or Rhinovirus Oligonucleotides for amplifying each of the target nucleic acids of adenovirus, metapneumovirus, and / or rhinovirus typically contain at least two amplification oligomers per target. Some embodiments of the present disclosure may utilize two, three, four, five, six, or more amplification oligomers per target, for example, in multiplex amplification assays. Thus, by way of example, oligonucleotides for amplifying each target microorganism may contain one, two, three, four, five, or more forward amplification primers and one, two, three, four, five, or more reverse amplification primers. For example, oligonucleotides for amplifying adenovirus may contain one, two, three, four, five, or more forward amplification primers and one, two, three, four, five, or more reverse amplification primers. Oligonucleotides for amplifying hMPV may include one, two, three, four, five, or more forward amplification primers and one, two, three, four, five, or more reverse amplification primers. Oligonucleotides for amplifying HRV may include one, two, three, four, five, or more forward amplification primers and one, two, three, four, five, or more reverse amplification primers. Furthermore, a subtype or subgroup of a target microorganism may require at least two amplification oligomers, each containing a nucleotide sequence specific for one or more different member(s) of the subtype / subgroup of the microorganism.

[0162] Oligonucleotides for detecting each of the adenovirus, metapneumovirus, and / or rhinovirus target nucleic acids typically contain at least one detection oligomer per target. Some embodiments of the present disclosure may utilize two, three, four, five, six, or more detection probe oligomers per target, for example, in multiplex detection assays. For example, oligonucleotides for detecting adenovirus may contain one, two, three, four, or more detection probe oligomers. Oligonucleotides for detecting hMPV may contain one, two, three, four, or more detection probe oligomers. Oligonucleotides for detecting HRV may contain one, two, three, four, or more detection probe oligomers. Furthermore, a subtype or subgroup of a target microorganism may require at least two detection probe oligomers, each containing a nucleotide sequence specific for one or more different member(s) of the microorganism subtype / subgroup. Oligomer combinations for multiplex amplification and detection of one or more adenovirus, metapneumovirus, and rhinovirus target nucleic acids typically include at least two forward amplification oligomers, at least two reverse amplification oligomers, and at least two detection probe oligomers. Some embodiments of the present disclosure may utilize two, three, four, five, or even six or more amplification oligomers and two, three, four, five, or even six or more probes for each intended target nucleic acid. Thus, by way of example, oligonucleotides for multiplex amplification and detection of several targets may include 6 to 40 amplification oligomers and 3 to 15 detection probe oligomers.

[0163] Methods for detecting adenovirus, metapneumovirus, and / or rhinovirus target nucleic acids (including amplicons) optionally include a detection step using at least one probe that specifically binds to the amplification product (RNA or DNA amplicon, preferably DNA amplicon). Preferably, the probe is labeled and generates a signal that is detected homogeneously, i.e., without separating bound probe from unbound probe. Other examples of probes may be labeled with a fluorescent compound that emits a detectable signal only when the probe binds to its target, such as the Taqman™ detection probes described herein.

[0164] In one embodiment, at least one of the amplification oligomers is configured to specifically hybridize to a region within the adenoviral target sequence corresponding to nucleotides 1-99 of SEQ ID NO: 47 or corresponding to nucleotides 83-175 of SEQ ID NO: 47. In one embodiment, at least two amplification oligomers are used, each of which is 10 to about 50 nucleotides in length, and which are configured to specifically hybridize to a region within the adenoviral target sequence selected from the group consisting of nucleotides 1-99 of SEQ ID NO: 47 and nucleotides 83-175 of SEQ ID NO: 47, respectively, to generate an amplicon that can then be detected. In one embodiment, at least one of the amplification oligomers is configured to specifically hybridize to a region within the adenoviral target sequence corresponding to nucleotides 52-99 and / or 40-87 and / or 1-23 and / or 7-23 and / or 7-45 and / or 139-155 and / or 103-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47 to generate an amplicon that can then be detected. In one embodiment, at least one of the amplification oligomers is configured to specifically hybridize to a region within the adenoviral target sequence corresponding to nucleotides 52-74 and / or 76-99 and / or 40-56 and / or 65-87 and / or 1-18 and / or 7-23 and / or 28-45 and / or 27-45 and / or 26-45 and / or 139-155 and / or 103-123 and / or 159-175 and / or 83-99 and / or 83-98 of SEQ ID NO:47 to generate an amplicon that can then be detected.

[0165] Oligonucleotides for amplifying and / or detecting adenoviral target nucleic acids include oligonucleotide sequences selected from the group consisting of SEQ ID NOS: 1-46, 62-64, 71-75, and 138-149. Embodiments of amplification oligomers specific for adenoviral nucleic acids include amplification oligomers comprising, consisting of, or consisting essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOS: 1-9, 11-16, 25-28, 31-35, 38, 42-46, 61, 62, and 71-74, or a combination of two or more thereof. According to one embodiment, at least one first amplification oligomer comprises, consists of, or consists essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOS: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73, 74, or a combination of two or more thereof. According to one embodiment, at least one second amplification oligomer comprises, consists of, or consists essentially of a target-hybridizing sequence selected from the group consisting of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42, 43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or a combination of two or more thereof. In one embodiment, one or more of the amplification oligomers for amplifying an adenoviral target nucleic acid comprises at least one of 5-Me-dC, a non-Watson-Crick base, a degenerate base, or a combination thereof.

[0166] In one embodiment, one or more detector probes are configured to detect a sequence within a region corresponding to nucleotides 74-139 of SEQ ID NO:47, and / or nucleotides 56-103 of SEQ ID NO:47, and / or nucleotides 18-83 of SEQ ID NO:47, and / or nucleotides 23-83 of SEQ ID NO:47, and / or nucleotides 23-83 of SEQ ID NO:47, and / or nucleotides 23-83 of SEQ ID NO:47, and / or nucleotides 52-99 of SEQ ID NO:47. In one embodiment, detector probe oligonucleotides are provided that are 18-36 nucleobases in length, wherein the 18-36 nucleobases are all selected from contiguous nucleobases within SEQ ID NO:138.

[0167] Probes for specific detection of adenoviral sequences include oligomers selected from the group consisting of SEQ ID NOs: 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140, or combinations of two or more thereof. In one embodiment, one or more of the detection probe oligomers for detecting adenoviral target nucleic acids (including adenoviral amplicons) comprises at least one of 5-Me-dC, a non-Watson-Crick base, a degenerate base, or a combination thereof.

[0168] Oligonucleotides for amplifying and / or detecting hMPV target nucleic acids include oligonucleotide sequences configured to hybridize to a region of hMPV within the region corresponding to nucleotides 966-1147 of SEQ ID NO: 150, and / or nucleotides 844-1027 of SEQ ID NO: 159, and / or 1000-1040 of SEQ ID NO: 150, and / or 880-915 of SEQ ID NO: 159, and / or 1027-1080 of SEQ ID NO: 150, and / or 913-958 of SEQ ID NO: 159, and / or 1073-1115 of SEQ ID NO: 150, and / or 953-995 of SEQ ID NO: 159. Oligonucleotides for amplifying and / or detecting hMPV target nucleic acids include oligonucleotide sequences selected from the group consisting of SEQ ID NOs: 52, 53, 56, 67-70, 151-158, and 161-178. Amplification oligomer embodiments include amplification oligomers comprising, consisting of, or consisting essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52, 53, 56, 68, 151, 152, 153, 154, 158, 160, 177, 178, or a combination of two or more thereof. First amplification oligomer embodiments include amplification oligomers comprising, consisting of, or consisting essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52, 53, 151, 152, 153, 154, 160, or a combination of two or more thereof. Second amplification oligomer embodiments include amplification oligomers comprising, consisting of, or consisting essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 56, 68, 158, 177, 178, or a combination of two or more thereof. In one embodiment, one or more of the amplification oligomers for amplifying an hMPV target nucleic acid comprises at least one of 5-Me-dC, a non-Watson-Crick base, a degenerate base, or a combination thereof.

[0169] Probes for specific detection of hMPV sequences include oligomers selected from the group consisting of SEQ ID NOs: 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, or a combination of two or more thereof. In one embodiment, detection probe oligonucleotides are provided that are 18 to 36 nucleobases in length, wherein the 18 to 36 nucleobases are all selected from contiguous nucleobases within SEQ ID NO: 161 or SEQ ID NO: 155. In one embodiment, one or more of the detection probe oligomers for detecting hMPV target nucleic acids (including hMPV amplicons) comprise at least one of 5-Me-dC, a non-Watson-Crick base, a degenerate base, or a combination thereof.

[0170] Oligonucleotides for amplifying and / or detecting HRV target nucleic acids include oligonucleotide sequences configured to hybridize to a region of HRV within a region corresponding to nucleotides 230-556 of SEQ ID NO:120, and / or 199-525 of SEQ ID NO:101, and / or 80-410 of SEQ ID NO:76, and / or 263-303 of SEQ ID NO:120, and / or 231-264 of SEQ ID NO:101, and / or 106-156 of SEQ ID NO:76, and / or 312-346 of SEQ ID NO:120, and / or 279-314 of SEQ ID NO:101, and / or 455-506 of SEQ ID NO:76, and / or 480-533 of SEQ ID NO:120, and / or 455-506 of SEQ ID NO:101, and / or 338-397 of SEQ ID NO:76. Oligonucleotides for amplifying and / or detecting HRV target nucleic acids include oligonucleotide sequences selected from the group consisting of SEQ ID NOs: 48, 49, 50, 51, 54, 57, 59, 60, 65, 75, 77-100, 102-119, 121-137, or combinations of two or more thereof. Embodiments of amplification oligomers include amplification oligomers that comprise, consist of, or consist essentially of target-hybridizing sequences selected from the group consisting of SEQ ID NOs: 50, 51, 57, 59, 60, 65, 75, 77-86, 95-100, 102-108, 115-119, 121-130, 137, or combinations of two or more thereof. First amplification oligomer embodiments include amplification oligomers comprising, consisting of, or consisting essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 50, 51, 59, 60, 65, 75, 77-86, 102-108, 121-130, or a combination of two or more thereof. Second amplification oligomer embodiments include amplification oligomers comprising, consisting of, or consisting essentially of a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 57, 95-100, 115-119, 137, or a combination of two or more thereof. In one embodiment, an amplification oligonucleotide is provided that is 18-29 nucleobases in length, wherein the 18-29 nucleobases are all selected from contiguous nucleobases within SEQ ID NO: 77.In one embodiment, amplification oligonucleotides are provided that are 18 to 27 nucleobases in length, wherein the 18 to 27 nucleobases are all selected from the contiguous nucleobases in SEQ ID NO: 95. In one embodiment, amplification oligonucleotides are provided that are 18 to 35 nucleobases in length, wherein the 18 to 35 nucleobases are all selected from the contiguous nucleobases in SEQ ID NO: 96. In one embodiment, amplification oligonucleotides are provided that are 18 to 27 nucleobases in length, wherein the 18 to 27 nucleobases are all selected from the contiguous nucleobases in SEQ ID NO: 115. In one embodiment, amplification oligonucleotides are provided that are 18 to 27 nucleobases in length, wherein the 18 to 27 nucleobases are all selected from the contiguous nucleobases in SEQ ID NO: 137. In one embodiment, one or more of the amplification oligomers for amplifying an HRV target nucleic acid comprises at least one of 5-Me-dC, a non-Watson-Crick base, a degenerate base, or a combination thereof.

[0171] Probes for specific detection of HRV sequences include oligomers selected from the group consisting of SEQ ID NOs: 48, 49, 54, 87-94, 109-114, 131-136, or combinations of two or more thereof. In one embodiment, one or more of the detection probe oligomers for detecting HRV target nucleic acids (including HRV amplicons) comprise at least one of 5-Me-dC, a non-Watson-Crick base, a degenerate base, or a combination thereof.

[0172] Assays for detecting adeno-, hMPV-, and / or HRV-nucleic acids may include an internal control (IC) nucleic acid that is amplified and detected by using IC-specific primers and probes in the same reaction mixture used to amplify and detect the target nucleic acid. Amplification and detection of the IC-specific sequence indicates that the assay reagents and conditions were properly used, even if no target-specific signal is detected for a test sample (i.e., a negative sample). The IC may be used as an internal calibration for the assay to provide quantitative results. The IC may also be a randomized sequence derived from a natural source that is not the target nucleic acid.

[0173] Sample preparation Sample preparation for amplification and detection of target nucleic acid sequences may include methods for separating and / or concentrating microorganisms contained in the sample from other sample components. Sample preparation may include routine methods for disrupting or lysing the sample to release intracellular contents containing the target nucleic acid or a gene sequence containing the target nucleic acid. Sample preparation prior to amplification may include an optional target capture step to specifically or nonspecifically separate the target nucleic acid from other sample components. Nonspecific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, attachment of nucleic acids to a support that is washed to remove other sample components, or other methods for physically separating nucleic acids from a mixture containing adenoviral nucleic acids and other sample components.

[0174] Amplification of the adenovirus target region Amplification of target nucleic acid target region using two or more primers can be achieved by using various known nucleic acid amplification reactions.For example, amplification can be achieved by using PCR amplification (Mullis et al., U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159), by using DNA polymerase to generate additional dsDNA molecules, by using a thermal cycling reaction to separate dsDNA and a primer specific to a part of the separated strand, thereby generating multiple DNA strands.Also, well-known variations of the basic PCR method, such as PCR combined with real-time detection, such as Taqman PCR, can be used.

[0175] Nucleic Acid Detection Nucleic acid detection can be achieved by various methods. Detection methods may use a nucleic acid probe that contains a target hybridizing sequence complementary to a portion of the amplification product and detects the presence of a probe:product complex, or by using a complex of probes that can amplify a detectable signal associated with the amplification product (e.g., U.S. Patent Nos. 5,424,413, 5,451,503, 5,849,481, 5,639,604, and 5,283,174). A directly or indirectly labeled probe that specifically associates with the amplification product provides a detectable signal indicating the presence of the target nucleic acid in the sample. For example, if the target nucleic acid is adenovirus DNA, the amplification product will contain a sequence within or complementary to the adenovirus target sequence. The probe is configured to directly or indirectly bind to a portion of the amplification product to indicate the presence of adenovirus in the tested sample.

[0176] In embodiments where amplification products are detected near or at the end of the amplification step, a linear detection probe may be used to provide a signal indicating hybridization of the probe to the amplification product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is performed by chemiluminescence using a luminometer (see, e.g., International Patent Application Publication No. WO 89 / 002476). In other embodiments using real-time detection, the detection probe may be, for example, a hairpin probe such as a molecular beacon, a molecular torch, or a hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to the amplification product. Such a probe may include a target-hybridizing sequence and a non-target-hybridizing sequence. Various forms of such probes are described, for example, in U.S. Patent Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication Nos. 2006 / 0068417(A1) and 2006 / 0194240(A1).

[0177] Uniformly detectable labels, such as fluorescent labels and electronically detectable labels, are intended for use in the implementation of the present disclosure.Preferable examples of detectable labels as members of an interactive pair of labels interact with each other through FRET or non-FRET energy transfer mechanisms.Fluorescence resonance energy transfer (FRET) involves the non-radiative transfer of energy quantum from the absorption site to its utilization site in the molecule or molecular system through the resonance interaction between chromophores over a distance significantly longer than the interatomic distance, without conversion to thermal energy and without the dynamic collision of the donor and the acceptor.The "donor" is the moiety that first absorbs energy, and the "acceptor" is the moiety to which energy is subsequently transferred.In addition to FRET, there are at least three other "non-FRET" energy transfer processes that can transfer excitation energy from the donor to the acceptor molecule.

[0178] When two labels are held close enough together that energy emitted by one label can be accepted or absorbed by the second label, whether by a FRET or non-FRET mechanism, the two labels are said to be in an "energy transfer relationship" with one another, where fluorescence emission from a fluorophore attached to one part of the probe is quenched by a quencher moiety on another part of the probe (e.g., "Taqman" detection probe chemistry). Label moieties for Taqman probes include a fluorophore and a second moiety (i.e., a "quencher") with fluorescence-quenching properties. In this embodiment, the characteristic signal is likely to be fluorescence of a specific wavelength, although it may alternatively be a visible light signal. When fluorescence is involved, the change in emission is preferably due to FRET or to radiative energy transfer or non-FRET modes. When a detection probe having a pair of interacting labels in a "quenched" state is stimulated with light of the appropriate frequency, a fluorescent signal is generated at a first level, which may be very low. When this same probe is in an "unquenched" state and stimulated by light of the appropriate frequency, the fluorophore and quencher moieties are sufficiently separated from each other that energy transfer between them is substantially eliminated. Under these conditions, the quencher moiety cannot quench the fluorescence from the fluorophore moiety. When the fluorophore is stimulated by light energy of the appropriate wavelength, a second level of fluorescent signal is generated, higher than the first level. The difference between the two fluorescence levels is detectable and measurable.Examples of fluorophore / quencher label pairs that can be used in connection with the present disclosure include fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / D-ABCYL, coumarin / D-ABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / D-ABCYL, Lucifer Yellow / D-ABCYL, BODIPY / D-ABCYL, eosin / D-ABCYL, erythrosine / D-ABCYL, tetramethylrhodamine / D-ABCYL, CalOrange / BHQ1, CalRed / BHQ2, Texas Red / DABCYL, CY5 / BH1, CY5 / BH2, CY3 / BH1, CY3 / BH2, fluorescein / QSY7, FAM / BHQ1, and Quasar / BHQ2. Those skilled in the art will understand that when the donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or quenching of donor fluorescence. When the donor and acceptor species are the same, energy can be detected by the resulting fluorescence depolarization. Non-fluorescent acceptors, such as DABCYL and QSY7 dyes, advantageously eliminate the potential problem of background fluorescence resulting from direct (i.e., non-sensitized) acceptor excitation.

[0179] Synthetic techniques and methods for attaching labels to nucleic acids and detecting labels are well known in the art (e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual. 2nd ed. (Cold Spring, Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10, U.S. Patent No. 5,658,737 to Nelson et al., U.S. Patent No. 5,656,207 to Woodhead et al., U.S. Patent No. 5,547,842 to Hogan et al., U.S. Patent No. 5,283,174 to Arnold et al., U.S. Patent No. 4,581,333 to Kourilsky et al., and European Patent Application No. 0 747 706 to Becker et al.

[0180] kit The oligomers for use in the methods described herein are suitable for preparation as kits. Such kits may include containers, each containing one or more of the various oligomers, optionally together with one or more reagents (e.g., enzymes) necessary to carry out the methods described herein. The components of the kit may be supplied in a concentrated form. Typically, a set of instructions for using the components of the kit will also be included. When the kit includes a combination of oligomers, the individual oligomers may be provided individually with appropriate instructions for mixing them, or in a ready-to-mix combination thereof.

[0181] In one aspect, a kit is provided that includes the composition of the present disclosure and, optionally, a set of instructions for practicing the same.

[0182] Detection of target sequences and correlation with diagnosis Detection of an amplified target sequence characteristic of adenovirus, metapneumovirus, and / or rhinovirus in a biological sample from an individual is indicative of infection by adenovirus, metapneumovirus, and / or rhinovirus, respectively. [Example]

[0183] Example 1: Analysis of certain adenovirus amplification primers and probes material and method The first amplification reaction used: 1x to 2x Fast Start Master Buffer (Roche), 2 units of Fast Start Taq DNA polymerase (Roche), 100 nM of forward amplification primer (SEQ ID NO: 5), and 100 nM of reverse amplification primer (SEQ ID NO: 6 or SEQ ID NO: 8), and 100 nM of probe (SEQ ID NO: 10).

[0184] The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from adenovirus was added per reaction. Control reactions were performed by setting up the reactions as described above, except that no template nucleic acid was added. The amplification cycles used for both sets of amplification reactions were as follows: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0185] result [Table 1] The result is C T / Presented as RFU (threshold cycle / relative fluorescence units) values, these represent the average of 12 experiments using various adenovirus serotypes. No amplification was observed in any of the control reactions.

[0186] conclusion The primers and probes used appeared to be highly sensitive and specific for adenoviral nucleic acid.

[0187] Example 2: Analysis of certain additional adenovirus amplification primers and probes material and method The following reagents were used: 1x to 2x concentration of Fast Start Master Buffer (Roche), 2 units of Fast Start Taq DNA polymerase (Roche), 200 nM of forward amplification primer (SEQ ID NO: 11 or SEQ ID NO: 12), and 200 nM of reverse amplification primer (SEQ ID NO: 13 or SEQ ID NO: 15), and 200 nM of probe (SEQ ID NO: 17 or SEQ ID NO: 19).

[0188] The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from adenovirus was added per reaction. Control reactions were performed by setting up the reaction as described above, except that no template nucleic acid was added. The amplification cycles used were as follows: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0189] result [Table 2] The result is C T / Presented as RFU values, these represent the average of eight experiments using various adenovirus serotypes. No amplification was observed in any of the control reactions.

[0190] conclusion Combinations of SEQ ID NOs: 11, 13, and 19, SEQ ID NOs: 11, 15, and 19, SEQ ID NOs: 12, 15, and 19, or SEQ ID NOs: 12, 13, and 19 were highly sensitive and specific for adenoviral nucleic acids. The combination containing the forward primer of SEQ ID NO: 12 appears to have better sensitivity than the combination containing the forward primer of SEQ ID NO: 11. The combination containing SEQ ID NOs: 12, 15, and 19 appeared to be the most sensitive in these experiments.

[0191] Example 3: Adenovirus serotyping using SEQ ID NOs: 12, 15, and 19 material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 2 units of Fast Start Taq DNA Polymerase (Roche), 400 nM forward amplification primer (SEQ ID NO: 12) and 400 nM reverse amplification primer (SEQ ID NO: 15) with 400 nM probe (SEQ ID NO: 19). The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from adenovirus was added per reaction. A control reaction was set up but no template nucleic acid was added. The amplification cycles used were as follows: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0192] result [Table 3-1] [Table 3-2] [Table 3-3] "Serotype number - 1 x 10 x TCID 50 Set up the serotype column to reflect " / mL". T All values ​​are truncated. T Presented as / RFU value.

[0193] conclusion The combination of SEQ ID NOs: 12, 15, and 19 was able to detect all serotypes of adenovirus tested.

[0194] Example 4: Analysis of additional adenovirus probe combinations with primers of SEQ ID NOs: 12 and 15 material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 2 units of Fast Start Taq DNA Polymerase (Roche), 100 nM forward amplification primer (SEQ ID NO: 12) and 100 nM reverse amplification primer (SEQ ID NO: 15), with either 150 nM probe (SEQ ID NO: 21) and 50 nM probe (SEQ ID NO: 24), 100 nM probe (SEQ ID NO: 21) and 100 nM probe (SEQ ID NO: 24), or 50 nM probe (SEQ ID NO: 21) and 150 nM probe (SEQ ID NO: 24). The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from adenovirus was added per reaction. Control reactions were set up without the addition of template nucleic acid. The amplification cycles used were: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0195] result [Table 4] The result is C T / Presented as RFU values, these represent the average of six experiments using various adenovirus serotypes.

[0196] conclusion The probes of SEQ ID NO: 21 and SEQ ID NO: 24 in combination with SEQ ID NOs: 12 and 15 were able to sensitively and specifically detect adenovirus at the various concentrations tested.

[0197] Example 5: Analysis of additional probe and primer combinations for the detection of adenovirus material and method The following reagents were used: 1× Fast Start Master Buffer (Roche), 2 units of Fast Start Taq DNA polymerase (Roche), and either: (i) 50 mM forward amplification primer (SEQ ID NO: 25), 50 mM forward amplification primer (SEQ ID NO: 26), 50 mM reverse amplification primer (SEQ ID NO: 27), 50 mM reverse amplification primer (SEQ ID NO: 28), and 100 nM probe (SEQ ID NO: 21 and SEQ ID NO: 23); (ii) 50 mM forward amplification primer (SEQ ID NO: 26), 50 mM reverse amplification primer (SEQ ID NO: 27), and 100 nM probe (SEQ ID NO: 28). ), 50 mM of reverse amplification primer (SEQ ID NO:28), and 100 nM of probe (SEQ ID NO:21 and SEQ ID NO:23); (iii) 50 mM of forward amplification primer (SEQ ID NO:25), 50 mM of reverse amplification primer (SEQ ID NO:27), 50 mM of reverse amplification primer (SEQ ID NO:28), and 100 nM of probe (SEQ ID NO:21 and SEQ ID NO:23); (iv) 50 mM of forward amplification primer (SEQ ID NO:25), 50 mM of forward amplification primer ( (SEQ ID NO:26), 50 mM of reverse amplification primer (SEQ ID NO:28), and 100 nM of probe (SEQ ID NO:21 and SEQ ID NO:23); (v) 50 mM of forward amplification primer (SEQ ID NO:25), 50 mM of forward amplification primer (SEQ ID NO:26), 50 mM of reverse amplification primer (SEQ ID NO:28), and 100 nM of probe (SEQ ID NO:21 and SEQ ID NO:23); (vi) 50 mM of forward amplification primer (SEQ ID NO:25), 50 mM of forward amplification primer (SEQ ID NO:26), 50 mM of reverse amplification primer (SEQ ID NO:28), and 100 nM of probe (SEQ ID NO:21 and SEQ ID NO:23). primer (SEQ ID NO:26), 50 mM reverse amplification primer (SEQ ID NO:27), 50 mM reverse amplification primer (SEQ ID NO:28), and 100 nM probe (SEQ ID NO:23); or (vii) 50 mM forward amplification primer (SEQ ID NO:25), 50 mM forward amplification primer (SEQ ID NO:26), 50 mM reverse amplification primer (SEQ ID NO:27), 50 mM reverse amplification primer (SEQ ID NO:28), and 100 nM probe (SEQ ID NO:21).

[0198] The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from adenovirus was added per reaction. Two different concentrations were tested.

[0199] The amplification cycles used were: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0200] result Table 5a-5d. Amplification and detection using different concentrations and primer-probe combinations. [Table 5a] [Table 5b] [Table 5c] [Table 5d] Results are presented as RFU values ​​and represent the average of six experiments for each concentration.

[0201] conclusion Omitting one of the primers or probes from the assay made little difference in most cases, however omission of probe SEQ ID NO:23 resulted in reduced detection in this particular experiment.

[0202] Example 6: Analysis of primer and probe combinations for detecting Adenovirus 18 material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 3 units of Fast Start Taq DNA Polymerase (Roche), 150 nM forward amplification primers (SEQ ID NO:25 and SEQ ID NO:26) and 150 nM reverse amplification primers (SEQ ID NO:27 and SEQ ID NO:28) with 300 nM probe (SEQ ID NO:29). The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from adenovirus 18 was added per reaction. The amplification cycles used were: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0203] result [Table 6] "Serotype number - 1 x 10 x TCID 50 Set up the serotype column to reflect " / mL". T All values ​​are truncated. T Presented as / RFU value.

[0204] conclusion This combination of primers and probe successfully detects adenovirus 18.

[0205] Example 7: Analysis of additional primer and probe combinations for detecting adenovirus material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 3 units of Fast Start Taq DNA Polymerase (Roche), 150 nM forward amplification primers (SEQ ID NO:31 and SEQ ID NO:26) and 150 nM reverse amplification primers (SEQ ID NO:27 and SEQ ID NO:28) with 150 nM probe (SEQ ID NO:21 and SEQ ID NO:23). The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from various adenovirus serotypes was added per reaction. The amplification cycles used were as follows: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0206] result [Table 7] The result is C T / The values ​​are presented as RFU values. The Fam channel shows the detection results of the template nucleic acid. The Cy5 channel shows the detection results of the internal control nucleic acid.

[0207] conclusion With the exception of serotype 4, this primer and probe combination successfully detected all of the serotypes tested.

[0208] Example 8: Analysis of additional primer and probe combinations for detecting adenovirus material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 3 units of Fast Start Taq DNA polymerase (Roche), and either: (i) 150 nM forward amplification primers (SEQ ID NOs: 33 and 34) and 150 nM reverse amplification primers (SEQ ID NOs: 27 and 28) with 150 nM probe (SEQ ID NOs: 21 and 23); (ii) 150 nM forward amplification primers (SEQ ID NOs: 33 and 35) and 150 nM reverse amplification primers (SEQ ID NOs: 27 and 28) with 150 nM probe (SEQ ID NOs: 21 and 23); or (iii) 150 nM forward amplification primers (SEQ ID NOs: 34 and 35) and 150 nM reverse amplification primers (SEQ ID NOs: 27 and 28) with 150 nM probe (SEQ ID NOs: 21 and 23). The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from various adenovirus serotypes was added per reaction. The amplification cycles used were as follows: 95°C for 600 seconds with the optics off; 95°C for 30 seconds with the optics off and 55°C for 60 seconds with the optics on (5 cycles); 95°C for 10 seconds with the optics off and 55°C for 60 seconds with the optics on (40 cycles).

[0209] result Table 8a-8c. Amplification and detection of various adenovirus serotypes using primer and probe combinations [Table 8a] [Table 8b] [Table 8c] The result is C T and RFU values. The Fam channel shows the detection results for the template nucleic acid. The Cy5 channel shows the detection results for the internal control nucleic acid.

[0210] conclusion The primers and probes in Table 8a successfully detected all of the serotypes tested, while Tables 8b and 8c detected the majority of the serotypes tested.

[0211] Example 9: Analysis of additional primer and probe combinations for detecting adenovirus material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 3 units of Fast Start Taq DNA Polymerase (Roche), and 150 nM forward amplification primers (SEQ ID NOs: 25 and 26) and 150 nM reverse amplification primers (SEQ ID NOs: 27 and 28) with 150 nM probe (SEQ ID NOs: 36 and 37). The total reaction volume was 20 microliters, and 5 microliters of template nucleic acid extracted from an adenovirus 19 serotype positive control plasmid was added per reaction at six different concentrations. The amplification cycles used were: 95°C for 600 seconds with optics off; 95°C for 30 seconds with optics off and 55°C for 60 seconds with optics on (5 cycles); 95°C for 10 seconds with optics off and 55°C for 60 seconds with optics on (40 cycles).

[0212] result [Table 9] The result is C T / Presented as RFU values.

[0213] conclusion These primers and probes successfully detected the control tested adenovirus 19 serotypes.

[0214] Example 10: Further analysis of primer and probe combinations from Example 9 material and method The following reagents were used: 1x Fast Start Master Buffer (Roche), 3 units of Fast Start Taq DNA polymerase (Roche), and 150 nM of forward amplification primers (SEQ ID NOs: 25 and 26) and 150 nM of reverse amplification primers (SEQ ID NOs: 27 and 28) with 150 nM of probe (SEQ ID NOs: 36 and 37). The total reaction volume was 20 microliters with 5 microliters of template nucleic acid extracted from various adenovirus serotypes, resulting in 3 x 10 0 The amplification cycles used were: 95°C with optics off for 600 seconds; 95°C with optics off for 30 seconds and 55°C with optics on for 60 seconds (5 cycles); 95°C with optics off for 10 seconds and 55°C with optics on for 60 seconds (40 cycles).

[0215] result [Table 10-1] [Table 10-2] The result is C T and presented as RFU values.

[0216] Consideration All of the serotypes tested were detected using this primer and probe concentration, except for serotype 6, which was detected at 3 × 10 1 TCID 50 / mL or higher was successfully detected.

[0217] Example 11. Multiplex amplification and detection of adenovirus and human metapneumovirus and rhinovirus material and method Analytical Sensitivity and Reactivity: PCR formulations containing primers and probes for the amplification and detection of adenovirus, human metapneumovirus, and rhinovirus were prepared to contain (per reaction): 15 uL Supermix (11.1 units Taq), 1.2 uL MMLV Reverse Transcriptase (RT) (24 units), 2.0 uL Primer Probe Mix, 0.084 uL of 0.5 M EDTA, and 1.716 uL of water (AMR formulation). Target nucleic acids were analyzed using a number of stock microorganisms. 1 Extracted from each microorganism, 0.1 TCID 50 / mL, 1TCID 50 / mL, and 10 TCID 50 The target nucleic acid eluate was diluted to 10 μL / mL. 10 μL of the target nucleic acid eluate from each dilution was individually combined with the PCR formulation in the reaction volume for a total reaction volume of 30 μL. The primers and probes used in this experiment are shown in SEQ ID NOs: 48-49 and 51-74, including the internal control. The probe used in this example was a dual-labeled probe containing a quencher and a fluorophore. 1 Target nucleic acids were isolated from previously characterized stock microorganisms obtained from TriCore Reference Laboratories (Albuquerque, NM), ZeptoMetrix Corporation (Buffalo, NY), and ATCC (Manassas, VA).

[0218] Multiplex real-time PCR reactions were set up using a Panther instrument (Hologic, Inc., San Diego, CA) for sample preparation and a benchtop PCR thermal cycler for real-time amplification and detection. Taqman™ (Roche Molecular Systems, Inc., Pleasanton, CA) chemistry was used for detection reactions. In the thermal cycler, adenovirus target nucleic acids were detected in the ROX channel, hMPV nucleic acids in the HEX channel, HRV nucleic acids in the FAM channel, and the internal control in the RED677 channel. Assays were performed using 10 replicates of known concentrations of each viral target. Each individual sample preparation was evaluated as one real-time RT-PCR replicate on the benchtop PCR thermal cycler. Positive or negative determinations were made using background-subtracted curves. Tables 11-13 show the results for 10 TCID 50 This shows a 100% hit rate at virus concentrations of 1 / mL or less.

[0219] Following these studies, two additional bases were added to the 5' end of primer SEQ ID NO: 75 to generate primer SEQ ID NO: 50. Rhinovirus sensitivity assays were performed as described above using SEQ ID NO: 50 in place of SEQ ID NO: 75, and the results are presented in Table 14. [Table 11] [Table 12] [Table 13] [Table 14]

[0220] Conclusion: The multiplexed combination of amplification and detection oligonucleotides presented in this review was used to detect 10 TCID 50 It can detect virus concentrations of 0.1 μg / mL or less.

[0221] Example 12. Clinical specimen detection and clinical specificity using multiplex amplification and detection assays material and method A PCR formulation (AMR formulation) containing all of the primers and probes listed above in Example 11 (using SEQ ID NO: 74) was used to test clinical specimens identified by commercially available assays as rhinovirus positive, hMPV positive, and / or adenovirus positive, or negative for all three viruses. FilmArray RVP Respiratory Panel(BioFire The AMR assays used included the GenMark eSensor Respiratory Virus Panel (RVP) (GenMark Diagnostics, Salt Lake City, UT), the GenMark eSensor Respiratory Virus Panel (RVP) (GenMark Diagnostics, Inc., Carlsbad, CA), and the Luminex xTAG Respiratory Virus Panel (Luminex Corporation, Austin, TX). All samples were extracted using the Panther instrument, and PCR cycling was performed on the benchtop PCR thermal cycling instrument described above. The AMR assay detected HRV, hMPV, and adenovirus in previously characterized clinical specimens, with concordance of 94.8% (164 / 173) for HRV, 97.2% (279 / 287) for hMPV, and 93.2% (466 / 500) for adenovirus. The AMR assay identified 86 of 88 clinical specimens as negative, with 97.7% concordance with the reference assay (Luminex xTAG Respiratory Virus Panel). Additionally, the internal control was validated for all clinically negative specimens. Two "false-positive" results obtained using the AMR formulation assay were determined to be true-positive using the GenMark eSensor RVP assay and the Prodesse ProAdeno+ Assay (Hologic, Inc., San Diego, CA). Thus, after removing the clinical specimens falsely identified as negative by the Luminex assay, the AMR assay demonstrated 100% concordance (86 / 86).

[0222] Conclusion: The multiplexed combination of amplification and detection oligonucleotides presented in this example is capable of detecting viral targets in clinical specimens and is useful in competitive assays. It shows good agreement with

[0223] specificity material and method A PCR formulation (AMR formulation) containing all of the primers and probes listed in Example 11 (using SEQ ID NO: 74) was evaluated for cross-reactivity with other microorganisms. These microorganisms are commonly found in specimen types (nasopharyngeal and lower respiratory tract specimens) tested in clinics for the presence or absence of one or more of adenovirus, hMPV, and HRV. Microorganisms were tested pooled or individually (see AMR Panels 1-26 in Table 15). Three replicates from each panel were processed individually on the Panther instrument, and PCR cycling was performed on the benchtop PCR thermal cycling instrument described above. Table 15 shows that only viruses targeted by the AMR formulations (AMR24-26) were detected. No cross-reactivity was observed with microorganisms not targeted by the assay (AMR1-23). [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6]

[0224] Conclusion: The multiplexed combination of amplification and detection oligonucleotides presented in this review exhibits target specificity for AdV / hMPV / RV assays.

[0225] SEQ ID NO:50 was then substituted with SEQ ID NO:75 in the multiplexing reagents, as described above in Example 11. This modified PCR reaction formulation was tested in amplification and detection reactions using clinical samples and challenge microorganisms. The modified AMR formulation produced results similar to those shown in this Example 12 (data not shown).

[0226] Exemplary Nucleic Acid Sequences Table 16 provides exemplary sequences useful in this disclosure. This table is not intended to limit the scope of the disclosure. The sequences are presented in accordance with the World Intellectual Property Organization (WIPO) Handbook on Industrial Property Information and Documentation, Standard ST.25 (1998), including Tables 1-6 in Appendix 2. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6] The sequence symbols are in accordance with Table 1 of the World Intellectual Property Organization (WIPO) Handbook on Industrial Property Information and Documentation, Standard ST.25(1998) ("WIPO ST.25(1998)").

[0227] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated by reference herein in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicant reserves the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0228] The methods illustratively described herein may suitably be practiced in the absence of any element(s) or limitation(ies) not specifically disclosed herein. It is recognized that various modifications are possible within the scope of the claimed disclosure. Thus, although the present disclosure has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the present disclosure embodied in the present disclosure disclosed herein may be employed by those skilled in the art, and such modifications and variations are deemed to be within the scope of the present disclosure.

[0229] The present disclosure is broadly and generically described herein. Each of the narrower species and subgeneric groupings included in the generic disclosure also form part of the present method. This includes the generic description of the method with a condition or negative limitation that excludes any subject matter from the genus, regardless of whether the excluded material is specifically recited herein.

[0230] Other embodiments are within the scope of the following claims. Additionally, when features or aspects of the methods are described in terms of a Markush group, those skilled in the art will recognize that the disclosure also describes each individual member or subgroup of members of the Markush group.

Claims

1. 1. A composition comprising a combination of amplification oligomers configured to amplify an adenovirus target nucleic acid and at least one additional target nucleic acid selected from the group consisting of a metapneumovirus target nucleic acid and a rhinovirus target nucleic acid, (A) for the adenovirus target nucleic acid, the first adenovirus amplification oligomer and the second adenovirus amplification oligomer are selected from the group consisting of nucleotide positions 52-74 of SEQ ID NO:47, nucleotide positions 76-99 of SEQ ID NO:47, nucleotide positions 40-56 of SEQ ID NO:47, nucleotide positions 65-87 of SEQ ID NO:47, nucleotide positions 1-18 of SEQ ID NO:47, nucleotide positions 7-23 of SEQ ID NO:47, nucleotide positions 28-45 of SEQ ID NO:47, nucleotide positions 27-45 of SEQ ID NO:47, nucleotide positions 30-32 of SEQ ID NO:47, nucleotide positions 33-34 of SEQ ID NO:47, nucleotide positions 35-36 of SEQ ID NO:47, nucleotide positions 37-39 of SEQ ID NO:47, nucleotide positions 38-40 of SEQ ID NO:47, nucleotide positions 39-41 of SEQ ID NO:47, nucleotide positions 39-42 of SEQ ID NO:47, nucleotide positions 39-43 of SEQ ID NO:47, nucleotide positions 39-44 of SEQ ID NO:47, nucleotide positions 39-45 of SEQ ID NO:47, nucleotide positions 40-46 of SEQ ID NO:47, nucleotide positions 41-42 of SEQ ID NO:47, nucleotide positions 42-44 of SEQ ID NO:47, nucleotide positions 43-45 of SEQ ID NO:47, nucleotide positions 44-45 of SEQ ID NO:47, nucleotide positions 45-46 of SEQ ID NO:47, nucleotide positions 46-47 of SEQ ID NO:47, nucleotide positions 47-49 of SEQ ID NO:47, nucleotide positions 48-50 nucleotide positions 26-45 of SEQ ID NO:47, nucleotide positions 139-155 of SEQ ID NO:47, nucleotide positions 103-123 of SEQ ID NO:47, nucleotide positions 159-175 of SEQ ID NO:47, nucleotide positions 83-99 of SEQ ID NO:47, and / or nucleotide positions 83-98 of SEQ ID NO:47, wherein the first adenoviral amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs:71-74; and the second adenovirus amplification oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NOs: 61 and 62; and (B) for the at least one additional target nucleic acid, (1) the first metapneumovirus amplification oligomer and the second metapneumovirus amplification oligomer are selected from the group consisting of nucleotide positions 966 to 1147 of SEQ ID NO: 150, nucleotide positions 844 to 1027 of SEQ ID NO: 159, nucleotide positions 1000 to 1040 of SEQ ID NO: 150, nucleotide positions 880 to 915 of SEQ ID NO: 159, nucleotide positions 1027 to 1080 of SEQ ID NO: 150, nucleotide positions 913 to 958 of SEQ ID NO: 159, nucleotide positions 1073 to 1115 of SEQ ID NO: 150, and and / or configured to amplify a metapneumovirus target nucleic acid of at least 50 nucleotides in length comprising at least one position ranging from nucleotide positions 953 to 995 of SEQ ID NO: 159, wherein the first metapneumovirus amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 52 and 53; and the second metapneumovirus amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 56 and 68; and / or (2) The first rhinovirus amplification oligomer and the second rhinovirus amplification oligomer are selected from the group consisting of nucleotide positions 230 to 556 of SEQ ID NO: 120, nucleotide positions 199 to 525 of SEQ ID NO: 101, nucleotide positions 80 to 410 of SEQ ID NO: 76, nucleotide positions 263 to 303 of SEQ ID NO: 120, nucleotide positions 231 to 264 of SEQ ID NO: 101, nucleotide positions 106 to 156 of SEQ ID NO: 76, nucleotide positions 312 to 346 of SEQ ID NO: 120, nucleotide positions 279 to 314 of SEQ ID NO: 101, and nucleotide positions 455 to 50 of SEQ ID NO:

76.

6. A rhinovirus amplification oligomer configured to amplify a rhinovirus target nucleic acid of at least 50 nucleotides in length comprising at least one position ranging from nucleotide positions 480 to 533 of SEQ ID NO:120, nucleotide positions 455 to 506 of SEQ ID NO:101, and / or nucleotide positions 338 to 397 of SEQ ID NO:76, wherein the first rhinovirus amplification oligomer comprises a target hybridizing sequence selected from the group consisting of SEQ ID NOs:50, 51, 59, 60, and 65; and the second rhinovirus amplification oligomer comprises the target hybridizing sequence of SEQ ID NO:

57. composition.

2. and further comprising an adenovirus detection probe oligomer configured to hybridize to a region of said adenovirus target nucleic acid, said adenovirus detection probe oligomer comprising a target hybridizing sequence consisting of 22 or 24 contiguous nucleotides of SEQ ID NO: 138, said adenovirus detection probe oligomer comprising a target hybridizing sequence selected from the group consisting of SEQ ID NOs: 63, 64, 139, and 140, and said adenovirus detection probe oligomer comprising: (i) a detectable label; (ii) a 2′ methoxy backbone, (ii) a blocking moiety at or near the 3′ end of the detection probe oligomer, the blocking moiety being configured to prevent enzyme-mediated extension of the detection probe in an amplification reaction; and (iii) target-specific sequences and other sequences that contribute to the three-dimensional structure of the detection probe oligomer; The composition of claim 1 , comprising at least one of:

3. 3. The composition of claim 2, wherein the first and second adenovirus detection probe oligomers each independently comprise a sequence selected from the group consisting of SEQ ID NOs: 63, 64, 139, and 140.

4. (a) a metapneumovirus detection probe oligomer comprising a target hybridization sequence selected from the group consisting of SEQ ID NOs: 67, 69, and 70; and / or (b) a rhinovirus detection probe oligomer comprising a target hybridization sequence selected from the group consisting of SEQ ID NOs: 48, 49, and 54.

3. The composition of claim 1 or 2, further comprising:

5. the composition comprises the first and second metapneumovirus amplification oligomers; the first metapneumovirus amplification oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NOs: 52 and 53; and the second metapneumovirus amplification oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NOs: 56 and 68; The composition according to any one of claims 1 to 4.

6. the composition comprises the first and second rhinovirus amplification oligomers; the first rhinovirus amplification oligomer comprises a target-hybridizing sequence selected from the group consisting of SEQ ID NOs: 50, 51, 59, 60, and 65; and the second rhinovirus amplification oligomer comprises the target-hybridizing sequence of SEQ ID NO:57; The composition according to any one of claims 1 to 5.

7. A dry composition comprising a combination of amplification oligomers according to any one of claims 1 to 6.

8. The dry composition of claim 7 , wherein the dry composition further comprises an enzyme, a dNTP, or both.

9. The dry composition of claim 8 , wherein the enzyme has 5′ to 3′ exonuclease activity.

10. 10. The dry composition of claim 8 or 9, wherein the enzyme is a polymerase enzyme.

11. 11. The dry composition of any one of claims 7 to 10, wherein the dry composition has an inorganic salt concentration of 10 mM or less, or 7 mM or less, or 5 mM or less, or between 0.5 mM and 10 mM.

12. 12. The composition of any of claims 1-11, wherein the composition comprises at least six oligonucleotides independently comprising a sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 71, 72, 73, 74, and 139.

13. 1. A method for determining the presence or absence of an adenovirus target nucleic acid and at least one additional target nucleic acid in a sample, wherein the at least one additional target nucleic acid is selected from a metapneumovirus target nucleic acid and a rhinovirus target nucleic acid, the method comprising: (A) contacting the sample with a combination of amplification oligomers according to any one of claims 1 to 6; (B) performing an in vitro nucleic acid amplification reaction, wherein the adenoviral target nucleic acid and at least one additional target nucleic acid, if either is present in the sample, are used by the combination of amplification oligomers to amplify the target nucleic acid to generate an amplification product; (C) detecting the amplification product; thereby determining the presence or absence of said adenovirus target nucleic acid and said at least one additional target nucleic acid in said sample.

14. A kit comprising the composition according to any one of claims 1 to 6 or the dry composition according to any one of claims 7 to 12.

15. Use of the composition of any one of claims 1 to 6, the dry composition of any one of claims 7 to 12, or the kit of claim 14 for determining the presence or absence of an adenovirus target nucleic acid and at least one additional target nucleic acid selected from the group consisting of a metapneumovirus target nucleic acid and a rhinovirus target nucleic acid in a sample.

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