Compositions and methods for detecting or quantifying parainfluenza virus

Abstract

To provide compositions and methods for detecting or quantifying parainfluenza viruses.SOLUTION: Compositions, methods, kits and uses are provided for detecting or quantifying a Human Parainfluenza virus 1 (HPIV-1), HPIV-2, HPIV-3 and / or HPIV-4 nucleic acid, e.g., using nucleic acid amplification and hybridization assays. In some embodiments, the compositions, methods, kits and uses target the HN gene of HPIV-1, HPIV-2 and / or HPIV-3, and / or the NP gene of HPIV-4.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 476,435, filed March 24, 2017, which is incorporated by reference herein for all purposes.

[0002] Embodiments herein relate more particularly to the field of pathogen detection by using compositions and methods for detecting viruses, including human parainfluenza viruses 1, 2, 3, and 4 (HPIV-1, HPIV-2, HPIV-3, and HPIV-4). [Background technology]

[0003] Human parainfluenza viruses are members of the Paramyxoviridae family and are negative-sense single-stranded RNA viruses. HPIV types 1-4 have been identified, with type 4 including subtypes 4a and 4b. HPIVs can cause cold-like symptoms, airway illness, croup (particularly HPIV-1 and 2), and bronchiolitis, bronchitis, and / or pneumonia (particularly HPIV-3). HPIVs spread in ways common to respiratory viruses, such as through the air, including through coughing or sneezing, by close interpersonal contact, including shaking hands or other contact, or by contact with objects or surfaces exposed to the virus.

[0004] Although there is no known vaccine or specific antiviral treatment for HPIV infection, symptoms can be managed. Given the variety of symptoms that can be caused by HPIV, and their similarities to other respiratory infections such as the common cold, influenza, and bacterial pneumonia, rapid identification of HPIV infection is important to facilitate appropriate treatment and avoid administration of unnecessary or ineffective medications.

[0005] Nucleic acid-based detection techniques, including amplification-based approaches such as TaqMan™ RT-PCR, provide rapid detection and quantification of HPIV. However, the heterogeneity of HPIV sequences across and within various types and subtypes can cause problems with the sensitivity and / or specificity of such techniques. Thus, there is a need for compositions and methods that allow for sensitive and specific detection and quantification of HPIV. The present disclosure is intended to meet these needs, provide other benefits, or at least provide the public with a useful choice. Summary of the Invention [Means for solving the problem]

[0006] Thus, provided herein is a composition or kit comprising at least a first and a second amplification oligomer, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a human parainfluenza virus 3 (HPIV-3) amplicon of at least about 50 nucleotides in length that includes at least one HPIV-3 position located in the range of positions 1295-1305, 1350-1360, and / or 1380-1390.

[0007] Provided herein are compositions or kits comprising at least a first and a second amplification oligomer, where (a) the first amplification oligomer is configured to hybridize to a site including position 1270 of HPIV-3 and the second amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon, or (b) the first amplification oligomer is configured to hybridize to a site including position 1355 of HPIV 3 and the second amplification oligomer is configured to hybridize to a site including position 1437 of HPIV 3, and the first and second amplification oligomers are configured to generate an HPIV 3 amplicon.

[0008] 1. A method for detecting HPIV-3 in a sample, comprising: contacting a sample with at least first and second amplification oligomers, thereby forming a composition; conducting a nucleic acid amplification reaction in the composition that, in the presence of HPIV-3 nucleic acid, produces an HPIV-3 amplicon of at least about 50 nucleotides in length; and detecting the presence or absence of at least one amplicon; Also provided herein are methods, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a human parainfluenza virus 3 (HPIV-3) amplicon that includes at least one HPIV-3 position located within positions 1295-1305, 1350-1360, and / or 1380-1390.

[0009] 1. A method for detecting HPIV-3 in a sample, comprising: contacting the sample with at least first and second amplification oligomers, thereby forming a composition; and conducting a nucleic acid amplification reaction in the composition, in the presence of HPIV-3 nucleic acid, to generate an HPIV-3 amplicon; detecting the presence or absence of at least one amplicon; Also provided herein are methods in which (a) a first amplification oligomer is configured to hybridize to a site including position 1270 of HPIV-3 and a second amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon, or (b) a first amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3 and a second amplification oligomer is configured to hybridize to a site including position 1437 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon.

[0010] In some embodiments, the amplicon comprises HPIV-3 position 1305. In some embodiments, the amplicon comprises HPIV-3 position 1355. In some embodiments, the amplicon comprises HPIV-3 position 1380.

[0011] In some embodiments, the first oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:25 for hybridization to HPIV-3 nucleic acid under stringent conditions. In some embodiments, the first oligomer comprises the sequence of SEQ ID NO:25 with up to two mismatches. In some embodiments, the first oligomer comprises the sequence of SEQ ID NO:25.

[0012] In some embodiments, the second oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 36 for hybridization to HPIV-3 nucleic acid under stringent conditions. In some embodiments, the second oligomer comprises the sequence of SEQ ID NO: 36 with up to two mismatches. In some embodiments, the second oligomer comprises the sequence of SEQ ID NO: 36.

[0013] In some embodiments, the first oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 28 for hybridization to HPIV-3 nucleic acid under stringent conditions. In some embodiments, the first oligomer comprises the sequence of SEQ ID NO: 28 with up to two mismatches. In some embodiments, the first oligomer comprises the sequence of SEQ ID NO: 28.

[0014] In some embodiments, under stringent conditions, the second oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 37 for hybridization to HPIV-3 nucleic acid. In some embodiments, the second oligomer comprises the sequence of SEQ ID NO: 37 with up to two mismatches. In some embodiments, the second oligomer comprises the sequence of SEQ ID NO: 37.

[0015] In some embodiments, the first oligomer comprises at least one 5-methylcytosine residue. In some embodiments, at least about half of the C residues in the first oligomer are 5-methylated. In some embodiments, substantially all of the C residues in the first oligomer are 5-methylated. In some embodiments, the second oligomer comprises at least one 5-methylcytosine. In some embodiments, at least about half of the C residues in the second oligomer are 5-methylated. In some embodiments, substantially all of the C residues in the second oligomer are 5-methylated.

[0016] In some embodiments, the compositions or kits disclosed herein further comprise a third oligomer configured to hybridize to the amplicon, and the third oligomer comprises a detectable label. In some embodiments of the methods disclosed herein, the presence or absence of the amplicon is detected according to the occurrence or absence of hybridization of the third oligomer to the amplicon.

[0017] In some embodiments of the methods disclosed herein, HPIV-3 nucleic acid is quantified by monitoring the production of HPIV-3 amplicons.

[0018] In some embodiments, the third oligomer competes under stringent conditions for hybridization to HPIV-3 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO:31, 32, 33, 34, or 35. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:31 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:31. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:32 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:32. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:33 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:33. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:34 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:34. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO: 35 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO: 35.

[0019] In some embodiments, the third oligomer competes under stringent conditions for hybridization to HPIV-3 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO:26, 27, 28, 29, or 30. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:26 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:26. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:27 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:27. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:28 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:28. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:29 with up to two mismatches. In some embodiments, the third oligomer comprises the sequence of SEQ ID NO:29.

[0020] In some embodiments, the third oligomer comprises at least one 5-methylcytosine. In some embodiments, at least about half of the C residues in the third oligomer are 5-methylated. In some embodiments, substantially all of the C residues in the third oligomer are 5-methylated.

[0021] In some embodiments, the composition or kit further comprises a fourth oligomer different from the third oligomer, the fourth oligomer configured to hybridize to the amplicon and comprises a detectable label. In some embodiments, the fourth oligomer comprises a sequence of SEQ ID NO: 31, 32, 33, 34, or 35. In some embodiments, the fourth oligomer comprises a sequence of SEQ ID NO: 26, 27, 28, 29, or 30. In some embodiments, the fourth oligomer comprises at least one 5-methylcytosine. In some embodiments, at least about half of the C residues in the fourth oligomer are 5-methylated. In some embodiments, substantially all of the C residues in the fourth oligomer are 5-methylated.

[0022] In some embodiments, a composition or kit disclosed herein further comprises a first human parainfluenza virus 1 (HPIV-1) amplification oligomer and a second HPIV-1 amplification oligomer, wherein the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-1 position located within positions 330-490 or 960-1100 of HPIV-1.

[0023] Also provided herein are compositions or kits comprising at least a first HPIV-1 amplification oligomer and a second HPIV-1 amplification oligomer, wherein the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-1 position located within positions 330-490 or 960-1100 of HPIV-1.

[0024] 1. A method for detecting HPIV-1 in a sample, comprising: contacting the sample with at least first and second amplification oligomers, thereby forming a composition; and conducting a nucleic acid amplification reaction in the composition, in the presence of HPIV-1 nucleic acid, to generate an HPIV-1 amplicon; Also provided herein are methods comprising: detecting the presence or absence of at least one amplicon, wherein the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-1 position located within HPIV-1 positions 330-490 or 960-1100.

[0025] In some embodiments, a first HPIV-1 amplification oligomer is configured to hybridize to a site including position 970 of HPIV-1, and a second HPIV-1 amplification oligomer is configured to hybridize to a site including position 1098 of HPIV-1.

[0026] In some embodiments, the first HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:6 for hybridization to HPIV-1 nucleic acid under stringent conditions. In some embodiments, the first HPIV-1 oligomer comprises the sequence of SEQ ID NO:6 with up to two mismatches. In some embodiments, the first HPIV-1 oligomer comprises the sequence of SEQ ID NO:6.

[0027] In some embodiments, the second HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 24 for hybridization to HPIV-1 nucleic acid under stringent conditions. In some embodiments, the second HPIV-1 oligomer comprises the sequence of SEQ ID NO: 24 with up to two mismatches. In some embodiments, the second HPIV-1 oligomer comprises the sequence of SEQ ID NO: 24.

[0028] In some embodiments, a first HPIV-1 amplification oligomer is configured to hybridize to a site including HPIV-1 position 331, and a second HPIV-1 amplification oligomer is configured to hybridize to a site including HPIV-1 position 486.

[0029] In some embodiments, the first HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 17 for hybridization to HPIV-1 nucleic acid under stringent conditions. In some embodiments, the first HPIV-1 oligomer comprises the sequence of SEQ ID NO: 17 with up to two mismatches. In some embodiments, the first HPIV-1 oligomer comprises the sequence of SEQ ID NO: 17.

[0030] In some embodiments, the second HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:22 for hybridization to HPIV-1 nucleic acid under stringent conditions. In some embodiments, the second HPIV-1 amplification oligomer comprises the sequence of SEQ ID NO:22 with up to two mismatches. In some embodiments, the second HPIV-1 amplification oligomer comprises the sequence of SEQ ID NO:22.

[0031] In some embodiments, the composition or kit further comprises a third HPIV-1 oligomer configured to hybridize to the HPIV-1 amplicon, wherein the third oligomer comprises a detectable label.

[0032] In some embodiments, the third HPIV-1 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125. In some embodiments, 75 or 76, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125 with up to two mismatches. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 5, 7, 21, 23, or 125. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 8, 9, 20, or 125. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 12, 13, 14, or 15. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 16, 18, or 19.

[0033] In some embodiments, the third HPIV-1 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 10 for hybridization to HPIV-3 nucleic acid under stringent conditions. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 10 with up to two mismatches. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 10.

[0034] In some embodiments, the third HPIV-1 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 11 for hybridization to HPIV-3 nucleic acid under stringent conditions. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 11 with up to two mismatches. In some embodiments, the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 11.

[0035] In some embodiments, at least one, at least two, or each of the first, second, and third HPIV-1 oligomers contains at least one 5-methylcytosine. In some embodiments, at least about half of the C residues in the first, second, and / or third HPIV-1 oligomer are 5-methylated. In some embodiments, substantially all of the C residues in the first, second, and / or third HPIV-1 oligomer are 5-methylated.

[0036] In some embodiments, the kit or composition further comprises a first human parainfluenza virus 1 (HPIV-2) amplification oligomer and a second HPIV-2 amplification oligomer, wherein the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-2 position located within positions 1600-1700 of HPIV-2.

[0037] A composition or kit comprising at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, Also provided herein are compositions or kits, wherein the first HPIV 2 amplification oligomer and the second HPIV 2 amplification oligomer are configured to amplify an HPIV 2 amplicon of at least about 50 nucleotides in length that includes at least one HPIV 2 position located within positions 1600-1700 of HPIV 2.

[0038] 1. A method for detecting HPIV-2 in a sample, comprising: contacting the sample with at least first and second amplification oligomers, thereby forming a composition; and conducting a nucleic acid amplification reaction in the composition, in the presence of HPIV-2 nucleic acid, to generate an HPIV-2 amplicon; detecting the presence or absence of at least one amplicon; Also provided herein are methods, wherein the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-2 position located within positions 1600-1700 of HPIV-2.

[0039] In some embodiments, a first HPIV-2 amplification oligomer is configured to hybridize to a site including position 1603 of HPIV-2, and a second HPIV-2 amplification oligomer is configured to hybridize to a site including position 1698 of HPIV-2.

[0040] In some embodiments, the first HPIV-2 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 44 for hybridization to HPIV-2 nucleic acid under stringent conditions. In some embodiments, the first HPIV-2 oligomer comprises the sequence of SEQ ID NO: 44 with up to two mismatches. In some embodiments, the first HPIV-2 oligomer comprises the sequence of SEQ ID NO: 44.

[0041] In some embodiments, the second HPIV-2 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 40 for hybridization to HPIV-2 nucleic acid under stringent conditions. In some embodiments, the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40 with up to two mismatches. In some embodiments, the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40.

[0042] In some embodiments, the composition or kit further comprises a third HPIV-2 oligomer configured to hybridize to the HPIV-2 amplicon, wherein the third oligomer comprises a detectable label.

[0043] In some embodiments, the third HPIV-2 oligomer competes under stringent conditions for hybridization to HPIV-3 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO: 38, 39, 41, 42, 43, or 45. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 38, 39, 41, 42, 43, or 45 with up to two mismatches. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 45. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 43. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 42. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 41. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 39. In some embodiments, the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 38.

[0044] In some embodiments, at least one, at least two, or each of the first, second, and third HPIV-2 oligomers contains at least one 5-methylcytosine. In some embodiments, at least about half of the C residues in the first, second, and / or third HPIV-2 oligomers are 5-methylated. In some embodiments, substantially all of the C residues in the first, second, and / or third HPIV-2 oligomers are 5-methylated.

[0045] In some embodiments, a composition or kit disclosed herein further comprises a first human parainfluenza virus 1 (HPIV-4) amplification oligomer and a second HPIV-4 amplification oligomer, wherein the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-4 position located within positions 620-740, 2130-2410, 2520-3040, or 10090-11980 of HPIV-4.

[0046] Also provided herein are compositions or kits comprising at least a first HPIV-4 amplification oligomer and a second HPIV-4 amplification oligomer, wherein: The first HPIV 4 amplification oligomer and the second HPIV 4 amplification oligomer are configured to amplify an HPIV 4 amplicon of at least about 50 nucleotides in length that includes at least one HPIV 4 position located within HPIV 4 positions 620-740, 2130-2410, 2520-3040, or 10090-11980.

[0047] 1. A method for detecting HPIV-4 in a sample, comprising: contacting the sample with at least first and second amplification oligomers, thereby forming a composition; and conducting a nucleic acid amplification reaction in the composition, in the presence of HPIV-4 nucleic acid, to generate an HPIV-4 amplicon; detecting the presence or absence of at least one amplicon; Also provided herein are methods, wherein the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-4 position located within HPIV-4 positions 620-740, 2130-2410, 2520-3040, or 10090-11980.

[0048] In some embodiments, a first HPIV-4 amplification oligomer is configured to hybridize to a site including HPIV-4 position 10094, 10205, or 10248, and a second HPIV-4 amplification oligomer is configured to hybridize to a site including HPIV-4 position 11685, 11739, or 11981.

[0049] In some embodiments, the first HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100. In some embodiments, the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100 with up to two mismatches. In some embodiments, the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100.

[0050] In some embodiments, the second HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 54, 63, 64, 113, 114, or 115. In some embodiments, the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 54, 63, 64, 113, 114, or 115 with up to two mismatches. In some embodiments, the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 54, 63, 64, 113, 114, or 115.

[0051] In some embodiments, the composition or kit further comprises a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, wherein the third oligomer comprises a detectable label.

[0052] In some embodiments, the third HPIV-4 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 53, 55, 58, 60, 62, 65, 67, 68, 76, 83, 84, 85, 86, 92, 94, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112. In some embodiments, the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 53, 55, 58, 60, 62, 65, 67, 68, 76, 83, 84, 85, 86, 92, 94, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112 with up to two mismatches. In some embodiments, the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 53, 55, 58, 60, 62, 65, 67, 68, 76, 83, 84, 85, 86, 92, 94, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112.

[0053] In some embodiments, a first HPIV-4 amplification oligomer is configured to hybridize to a site including position 625 of HPIV-4, and a second HPIV-4 amplification oligomer is configured to hybridize to a site including position 738 of HPIV-4.

[0054] In some embodiments, the first HPIV-4 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 78 for hybridization to HPIV-4 nucleic acid under stringent conditions. In some embodiments, the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 78 with up to two mismatches. In some embodiments, the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 78.

[0055] In some embodiments, the second HPIV-4 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 56 for hybridization to HPIV-4 nucleic acid under stringent conditions. In some embodiments, the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 56 with up to two mismatches. In some embodiments, the second HPIV-4 amplification oligomer comprises the sequence of SEQ ID NO: 56.

[0056] In some embodiments, the composition or kit further comprises a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, wherein the third oligomer comprises a detectable label.

[0057] In some embodiments, the third HPIV-4 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 71 for hybridization to HPIV-3 nucleic acid under stringent conditions. In some embodiments, the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 71 with up to two mismatches. In some embodiments, the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 71.

[0058] In some embodiments, the first HPIV-4 amplification oligomer is configured to hybridize to a site including HPIV-4 positions 2128, 2352, 2386, 2388, 2404, or 2524, and the second HPIV-4 amplification oligomer is configured to hybridize to a site including HPIV-4 positions 2907, 3006, or 3039. In some embodiments, the first HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 72 or 124. In some embodiments, the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 72 or 124 with up to two mismatches. In some embodiments, the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 72 or 124.

[0059] In some embodiments, the second HPIV-4 amplification oligomer competes under stringent conditions for hybridization to HPIV-4 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO:50, 51, 52, 93, 95, or 96. In some embodiments, the second HPIV-4 oligomer comprises the sequence of SEQ ID NO:50, 51, 52, 93, 95, or 96 with up to two mismatches. In some embodiments, the second HPIV-4 oligomer comprises the sequence of SEQ ID NO:50, 51, 52, 93, 95, or 96.

[0060] In some embodiments, the composition or kit further comprises a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, wherein the third oligomer comprises a detectable label.

[0061] In some embodiments, the third HPIV-4 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 46, 47, 48, 49, 59, 61, 69, 70, 72, 73, 74, 75, 77, 88, 90, 91, 116, 117, 118, 119, 121, 122, or 123. In some embodiments, the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 46, 47, 48, 49, 59, 61, 69, 70, 72, 73, 74, 75, 77, 88, 90, 91, 116, 117, 118, 119, 121, 122, or 123 with up to two mismatches. In some embodiments, the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 46, 47, 48, 49, 59, 61, 69, 70, 72, 73, 74, 75, 77, 88, 90, 91, 116, 117, 118, 119, 121, 122, or 123.

[0062] In some embodiments, at least one, at least two, or each of the first, second, and third HPIV-4 oligomers contains at least one 5-methylcytosine. In some embodiments, at least about half of the C residues in the first, second, and / or third HPIV-4 oligomer are 5-methylated. In some embodiments, substantially all of the C residues in the first, second, and / or third HPIV-4 oligomer are 5-methylated.

[0063] In some embodiments of the methods disclosed herein, the nucleic acid amplification reaction comprises thermal cycling. In some embodiments of the methods disclosed herein, the nucleic acid amplification reaction comprises reverse transcription. In some embodiments of the methods disclosed herein, the nucleic acid amplification reaction comprises RT-PCR. In some embodiments of the methods disclosed herein, at least one detectable label is fluorescent. In some embodiments, the at least one oligomer comprising a fluorescent label further comprises a quencher. In some embodiments, the at least one oligomer comprising a fluorescent label is a non-extendable oligomer. In some embodiments of the methods disclosed herein, the nucleic acid amplification reaction comprises PCR using a polymerase having 5' to 3' (5'-to-3') exonuclease activity, and detection of the amplicon using a probe oligomer comprising a fluorophore and a quencher, wherein exonuclease degradation of the probe by the polymerase reduces quenching of the fluorescence by the quencher.

[0064] In some embodiments, at least one, at least two, or at least three oligomers contain at least one non-Watson-Crick (NWC) position, hi some embodiments, at least one, two, three, or four of the oligomers configured to hybridize to HPIV-1, HPIV-2, HPIV-3, or HPIV-4 each contain at least one NWC position.

[0065] In some embodiments, at least one, at least two, or at least three oligomers contain a degenerate position, hi some embodiments, at least one, two, three, or four of the oligomers configured to hybridize to HPIV-1, HPIV-2, HPIV-3, or HPIV-4, respectively, contain at least one degenerate position.

[0066] In some embodiments, the first and second amplification oligomers, the first and second HPIV-1 amplification oligomers, the first and second HPIV-2 amplification oligomers, and / or the first and second HPIV-4 amplification oligomers comprise a target hybridizing region that is about 10-60 bases in length, about 14-50 bases in length, about 14-40 bases in length, about 14-35 bases in length, or about 15-30 bases in length.

[0067] In some embodiments, the HPIV-3 amplicon, HPIV-1 amplicon, HPIV-2 amplicon, and / or HPIV-4 amplicon is about 50-1000 nucleotides in length, about 50-100 nucleotides in length, about 100-200 nucleotides in length, about 200-300 nucleotides in length, about 300-400 nucleotides in length, about 400-500 nucleotides in length, about 500-600 nucleotides in length, about 600-700 nucleotides in length, about 700-800 nucleotides in length, about 800-900 nucleotides in length, about 900-1000 nucleotides in length, or about 1000-2000 nucleotides in length.

[0068] In some embodiments, the compositions disclosed herein are aqueous, frozen, or lyophilized.

[0069] Also disclosed herein is the use of a composition or kit disclosed herein for detecting or quantifying HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 nucleic acid in a sample. In certain embodiments, for example, the following are provided: (Item 1) A composition or kit comprising at least a first and a second amplification oligomer, The composition or kit, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a human parainfluenza virus 3 (HPIV-3) amplicon of at least about 50 nucleotides in length that includes at least one HPIV-3 position located in the range of positions 1295-1305, 1350-1360, and / or 1380-1390. (Item 2) A composition or kit comprising at least a first and a second amplification oligomer, (a) the first amplification oligomer is configured to hybridize to a site including position 1270 of HPIV-3 and the second amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon; or (b) a composition or kit, wherein the first amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3 and the second amplification oligomer is configured to hybridize to a site including position 1437 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon. (Item 3) 1. A method for detecting HPIV-3 in a sample, comprising: contacting the sample with at least a first and a second amplification oligomer, thereby forming a composition; conducting a nucleic acid amplification reaction in said composition in the presence of HPIV-3 nucleic acid to produce an HPIV-3 amplicon of at least about 50 nucleotides in length; detecting the presence or absence of at least one of said amplicons; The method of claim 1, wherein the first amplification oligomer and the second amplification oligomer are configured to amplify a human parainfluenza virus 3 (HPIV-3) amplicon that includes at least one HPIV-3 locus located within positions 1295-1305, 1350-1360, and / or 1380-1390. (Item 4) 1. A method for detecting HPIV-3 in a sample, comprising: contacting the sample with at least a first and a second amplification oligomer, thereby forming a composition; conducting a nucleic acid amplification reaction in said composition in the presence of HPIV-3 nucleic acid to produce an HPIV-3 amplicon; detecting the presence or absence of at least one of said amplicons; (a) the first amplification oligomer is configured to hybridize to a site including position 1270 of HPIV-3 and the second amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon; or (b) the first amplification oligomer is configured to hybridize to a site including position 1355 of HPIV-3 and the second amplification oligomer is configured to hybridize to a site including position 1437 of HPIV-3, and the first and second amplification oligomers are configured to generate an HPIV-3 amplicon. (Item 5) 5. The composition, kit or method of any one of items 1 to 4, wherein the amplicon comprises position 1305 of HPIV-3. (Item 6) 6. The composition, kit or method of any one of items 1 to 5, wherein the amplicon comprises position 1355 of HPIV-3. (Item 7) 7. The composition, kit or method of any one of items 1 to 6, wherein the amplicon comprises position 1380 of HPIV-3. (Item 8) 8. The composition, kit, or method of any one of items 1 to 7, wherein the first oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 25. (Item 9) 9. The composition, kit, or method according to any one of items 1 to 8, wherein the first oligomer comprises a sequence of SEQ ID NO: 25 with up to two mismatches. (Item 10) 10. The composition, kit, or method according to any one of items 1 to 9, wherein the first oligomer comprises the sequence of SEQ ID NO: 25. (Item 11) The composition, kit, or method of any one of items 1 to 6 or 8 to 10, wherein the second oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 36. (Item 12) 12. The composition, kit, or method of any one of items 1 to 6 or 8 to 11, wherein the second oligomer comprises a sequence of SEQ ID NO: 36 with up to two mismatches. (Item 13) 13. The composition, kit, or method of any one of items 1 to 6 or 8 to 12, wherein the second oligomer comprises the sequence of SEQ ID NO: 36. (Item 14) The composition, kit, or method of any one of claims 1 to 4, 6 to 7, or 11 to 13, wherein the first oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 28. The composition, kit, or method of any one of items 1 to 4, 6 to 7, or 11 to 14, wherein the first oligomer comprises a sequence of SEQ ID NO: 28 with up to two mismatches. (Item 16) 16. The composition, kit, or method of any one of items 1 to 4, 6 to 7, or 11 to 15, wherein the first oligomer comprises the sequence of SEQ ID NO:28. (Item 17) 17. The composition, kit, or method of any one of items 1 to 10 or 14 to 16, wherein the second oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 37. (Item 18) 18. The composition, kit, or method of any one of items 1 to 10 or 14 to 17, wherein the second oligomer comprises a sequence of SEQ ID NO: 37 with up to two mismatches. (Item 19) 19. The composition, kit, or method of any one of items 1 to 10 or 14 to 18, wherein the second oligomer comprises the sequence of SEQ ID NO: 37. (Item 20) 20. The composition, kit, or method of any one of items 1 to 19, wherein the first oligomer comprises at least one 5-methylcytosine residue. (Item 21) 21. The composition, kit, or method of any one of items 1 to 20, wherein at least about half of the C residues in the first oligomer are 5-methylated. (Item 22) 22. The composition, kit or method of any one of items 1 to 21, wherein substantially all of the C residues in the first oligomer are 5-methylated. (Item 23) 23. The composition, kit, or method of any one of items 1 to 22, wherein the second oligomer comprises at least one 5-methylcytosine. (Item 24) 24. The composition, kit, or method of any one of items 1 to 23, wherein at least about half of the C residues in the second oligomer are 5-methylated. (Item 25) 25. The composition, kit or method of any one of items 1 to 24, wherein substantially all of the C residues in the second oligomer are 5-methylated. (Item 26) 26. The composition, kit, or method of any one of items 1 to 25, wherein the composition or kit further comprises a third oligomer configured to hybridize to the amplicon, the third oligomer comprising a detectable label. (Item 27) 27. The method of claim 26, wherein the presence or absence of the amplicon is detected by the occurrence or absence of hybridization of the third oligomer to the amplicon. (Item 28) 28. The method of any one of items 3 to 27, wherein the HPIV-3 nucleic acid is quantified by monitoring the production of the HPIV-3 amplicon. (Item 29) 29. The composition, kit, or method of any one of items 26 to 28, wherein the third oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 31, 32, 33, 34, or 35. (Item 30) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises a sequence of SEQ ID NO: 31 having up to two mismatches. (Item 31) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises the sequence of SEQ ID NO: 31. (Item 32) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises a sequence of SEQ ID NO: 32 having up to two mismatches. (Item 33) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises the sequence of SEQ ID NO: 32. (Item 34) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises a sequence of SEQ ID NO: 33 having up to two mismatches. (Item 35) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises the sequence of SEQ ID NO: 33. (Item 36) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises a sequence of SEQ ID NO: 34 having up to two mismatches. (Item 37) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises the sequence of SEQ ID NO: 34. (Item 38) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises a sequence of SEQ ID NO: 35 having up to two mismatches. (Item 39) 30. The composition, kit, or method according to any one of items 26 to 29, wherein the third oligomer comprises the sequence of SEQ ID NO: 35. (Item 40) 29. The composition, kit, or method of any one of items 26 to 28, wherein the third oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 26, 27, 28, 29, or 30. (Item 41) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises a sequence of SEQ ID NO: 26 with up to two mismatches. (Item 42) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises the sequence of SEQ ID NO: 26. (Item 43) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises a sequence of SEQ ID NO: 27 with up to two mismatches. (Item 44) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises the sequence of SEQ ID NO: 27. (Item 45) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises a sequence of SEQ ID NO: 28 with up to two mismatches. (Item 46) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises the sequence of SEQ ID NO: 28. (Item 47) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises a sequence of SEQ ID NO: 29 with up to two mismatches. (Item 48) 41. The composition, kit, or method of any one of items 26 to 28 or 40, wherein the third oligomer comprises the sequence of SEQ ID NO: 29. (Item 49) 49. The composition, kit, or method of any one of items 26 to 48, wherein the third oligomer comprises at least one 5-methylcytosine. (Item 50) 50. The composition, kit, or method of any one of items 26 to 49, wherein at least about half of the C residues in the third oligomer are 5-methylated. (Item 51) 51. The composition, kit, or method of any one of items 26 to 50, wherein substantially all of the C residues in the third oligomer are 5-methylated. (Item 52) 52. The composition, kit, or method of any one of items 26 to 51, wherein the composition or kit further comprises a fourth oligomer different from the third oligomer, the fourth oligomer configured to hybridize to the amplicon and comprising a detectable label. (Item 53) 53. The composition, kit, or method of item 52, wherein the fourth oligomer comprises the sequence of SEQ ID NO: 31, 32, 33, 34, or 35. (Item 54) 53. The composition, kit, or method of item 52, wherein the fourth oligomer comprises the sequence of SEQ ID NO: 26, 27, 28, 29, or 30. (Item 55) 55. The composition, kit, or method of any one of items 52 to 54, wherein the fourth oligomer comprises at least one 5-methylcytosine. (Item 56) 56. The composition, kit, or method of any one of items 52 to 55, wherein at least about half of the C residues in the fourth oligomer are 5-methylated. (Item 57) 57. The composition, kit, or method of any one of items 52 to 56, wherein substantially all of the C residues in the fourth oligomer are 5-methylated. (Item 58) further comprising a first human parainfluenza virus 1 (HPIV-1) amplification oligomer and a second HPIV-1 amplification oligomer; 58. The composition, kit, or method of any one of items 1 to 57, wherein the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-1 position located within positions 330 to 490 or 960 to 1100 of HPIV-1. (Item 59) A composition or kit comprising at least a first HPIV-1 amplification oligomer and a second HPIV-1 amplification oligomer, A composition or kit, wherein the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-1 position located within positions 330-490 or 960-1100 of HPIV-1. (Item 60) 1. A method for detecting HPIV-1 in a sample, comprising: contacting the sample with at least a first and a second amplification oligomer, thereby forming a composition; conducting a nucleic acid amplification reaction in said composition in the presence of HPIV-1 nucleic acid to produce an HPIV-1 amplicon; detecting the presence or absence of at least one of said amplicons; The method, wherein the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-1 position located within positions 330-490 or 960-1100 of HPIV-1. (Item 61) 61. The composition, kit, or method of any one of items 58 to 60, wherein the first HPIV-1 amplification oligomer is configured to hybridize to a site including position 970 of HPIV-1 and the second HPIV-1 amplification oligomer is configured to hybridize to a site including position 1098 of HPIV-1. (Item 62) 62. The composition, kit, or method of any one of items 58 to 61, wherein the first HPIV-1 amplification oligomer competes for hybridization to HPIV-1 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO:6. (Item 63) 63. The composition, kit, or method of any one of items 58 to 62, wherein the first HPIV-1 oligomer comprises the sequence of SEQ ID NO: 6 with up to two mismatches. (Item 64) 63. The composition, kit, or method of any one of items 58 to 62, wherein the first HPIV-1 oligomer comprises the sequence of SEQ ID NO:6. (Item 65) 65. The composition, kit, or method of any one of items 58 to 64, wherein the second HPIV-1 amplification oligomer competes for hybridization to HPIV-1 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 24. (Item 66) Item 67. The composition, kit, or method of any one of items 58 to 65, wherein the second HPIV-1 oligomer comprises the sequence of SEQ ID NO: 24 with up to two mismatches. 66. The composition, kit, or method of any one of items 58 to 65, wherein the second HPIV-1 oligomer comprises the sequence of SEQ ID NO:24. (Item 68) 60. The composition, kit, or method of any one of items 58 to 59, wherein the first HPIV-1 amplification oligomer is configured to hybridize to a site including position 331 of HPIV-1 and the second HPIV-1 amplification oligomer is configured to hybridize to a site including position 486 of HPIV-1. (Item 69) 69. The composition, kit, or method of any one of items 58-59 or 68, wherein the first HPIV-1 amplification oligomer competes for hybridization to HPIV-1 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 17. (Item 70) 70. The composition, kit, or method of any one of items 58-59 or 68-69, wherein the first HPIV-1 oligomer comprises the sequence of SEQ ID NO: 17 with up to two mismatches. (Item 71) 70. The composition, kit, or method of any one of items 58-59 or 68-69, wherein the first HPIV-1 oligomer comprises the sequence of SEQ ID NO:17. (Item 72) 72. The composition, kit, or method of any one of items 58-59 or 68-71, wherein the second HPIV-1 amplification oligomer competes for hybridization to HPIV-1 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO:22. (Item 73) The composition, kit, or method of any one of items 58-59 or 68-72, wherein the second HPIV-1 oligomer comprises the sequence of SEQ ID NO: 22 with up to two mismatches. (Item 74) 73. The composition, kit, or method of any one of items 58-59 or 68-72, wherein the second HPIV-1 oligomer comprises the sequence of SEQ ID NO:22. (Item 75) 75. The composition, kit, or method of any one of items 58 to 74, wherein the composition or kit further comprises a third HPIV-1 oligomer configured to hybridize to the HPIV-1 amplicon, the third oligomer comprising a detectable label. (Item 76) 76. The composition, kit, or method of item 75, wherein the third HPIV-1 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125. (Item 77) 77. The composition, kit, or method of item 75 or 76, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125 with up to two mismatches. (Item 78) 78. The composition, kit, or method of item 77, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 5, 7, 21, 23, or 125. (Item 79) 78. The composition, kit, or method of item 77, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 8, 9, 20, or 125. (Item 80) 78. The composition, kit, or method of item 77, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 12, 13, 14, or 15. (Item 81) 78. The composition, kit, or method of item 77, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 16, 18, or 19. (Item 82) 76. The composition, kit, or method of item 75, wherein the third HPIV-1 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 10 or 11 for hybridization to HPIV-3 nucleic acid under stringent conditions. (Item 83) 84. The composition, kit, or method of claim 75 or 82, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 10 or 11 with up to two mismatches. 83. The composition, kit, or method of item 75 or 82, wherein the third HPIV-1 oligomer comprises the sequence of SEQ ID NO: 10 or 11. (Item 85) 85. The composition, kit, or method of any one of items 58-84, wherein at least one, at least two, or each of the first, second, and third HPIV-1 oligomers contains at least one 5-methylcytosine. (Item 86) 85. The composition, kit, or method of any one of items 58 to 84, wherein at least about half of the C residues in the first, second, and / or third HPIV-1 oligomers are 5-methylated. (Item 87) 85. The composition, kit, or method of any one of items 58 to 84, wherein substantially all of the C residues in the first, second, and / or third HPIV-1 oligomers are 5-methylated. (Item 88) further comprising a first human parainfluenza virus 1 (HPIV-2) amplification oligomer and a second HPIV-2 amplification oligomer; 88. The composition, kit, or method of any one of paragraphs 1 to 87, wherein the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-2 position located within positions 1600 to 1700 of HPIV-2. (Item 89) A composition or kit comprising at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, A composition or kit, wherein the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-2 position located within positions 1600 to 1700 of HPIV-2. (Item 90) 1. A method for detecting HPIV-2 in a sample, comprising: contacting the sample with at least a first and a second amplification oligomer, thereby forming a composition; conducting a nucleic acid amplification reaction in said composition in the presence of HPIV-2 nucleic acid to produce an HPIV-2 amplicon; detecting the presence or absence of at least one of said amplicons; The method of claim 1, wherein the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-2 position located within positions 1600 to 1700 of HPIV-2. (Item 91) 91. The composition, kit, or method of any one of items 88 to 90, wherein the first HPIV-2 amplification oligomer is configured to hybridize to a site including position 1603 of HPIV-2 and the second HPIV-2 amplification oligomer is configured to hybridize to a site including position 1698 of HPIV-2. (Item 92) 92. The composition, kit, or method of any one of items 88 to 91, wherein the first HPIV-2 amplification oligomer competes for hybridization to HPIV-2 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 44. (Item 93) Item 94. The composition, kit, or method of any one of items 88 to 92, wherein the first HPIV-2 oligomer comprises the sequence of SEQ ID NO: 44 with up to two mismatches. 93. The composition, kit, or method of any one of items 88 to 92, wherein the first HPIV-2 oligomer comprises the sequence of SEQ ID NO: 44. (Item 95) 95. The composition, kit, or method of any one of items 88 to 94, wherein the second HPIV-2 amplification oligomer competes for hybridization to HPIV-2 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 40. (Item 96) 96. The composition, kit, or method of any one of items 88 to 95, wherein the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40 with up to two mismatches. (Item 97) 96. The composition, kit, or method of any one of items 88 to 95, wherein the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40. (Item 98) 98. The composition, kit, or method of any one of items 88 to 97, wherein the composition or kit further comprises a third HPIV-2 oligomer configured to hybridize to the HPIV-2 amplicon, the third oligomer comprising a detectable label. (Item 99) 99. The composition, kit, or method of item 98, wherein the third HPIV-2 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 38, 39, 41, 42, 43, or 45. (Item 100) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 38, 39, 41, 42, 43, or 45 with up to two mismatches. (Item 101) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:45. (Item 102) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:43. (Item 103) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:42. (Item 104) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:41. (Item 105) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:39. (Item 106) 100. The composition, kit, or method of item 98 or 99, wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:38. (Item 107) 107. The composition, kit, or method of any one of items 88-106, wherein at least one, at least two, or each of the first, second, and third HPIV-2 oligomers contains at least one 5-methylcytosine. (Item 108) 108. The composition, kit, or method of any one of items 88-107, wherein at least about half of the C residues in the first, second, and / or third HPIV-2 oligomers are 5-methylated. (Item 109) 108. The composition, kit, or method of any one of items 88 to 107, wherein substantially all of the C residues in the first, second, and / or third HPIV-2 oligomers are 5-methylated. (Item 110) further comprising a first human parainfluenza virus 1 (HPIV-4) amplification oligomer and a second HPIV-4 amplification oligomer; 109. The composition, kit, or method of any one of paragraphs 1-109, wherein the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-4 position located within positions 620-740, 2130-2410, 2520-3040, or 10090-11980 of HPIV-4. (Item 111) A composition or kit comprising at least a first HPIV-4 amplification oligomer and a second HPIV-4 amplification oligomer, A composition or kit, wherein the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-4 position located within positions 620-740, 2130-2410, 2520-3040, or 10090-11980 of HPIV-4. (Item 112) 1. A method for detecting HPIV-4 in a sample, comprising: contacting the sample with at least a first and a second amplification oligomer, thereby forming a composition; conducting a nucleic acid amplification reaction in said composition in the presence of HPIV-4 nucleic acid to produce an HPIV-4 amplicon; detecting the presence or absence of at least one said amplicon; The method of claim 1, wherein the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon of at least about 50 nucleotides in length that includes at least one HPIV-4 position located within HPIV-4 positions 620-740, 2130-2410, 2520-3040, or 10090-11980. (Item 113) 13. The composition, kit, or method of any one of items 110 to 112, wherein the first HPIV-4 amplification oligomer is configured to hybridize to a site including position 10094, 10205, or 10248 of HPIV-4, and the second HPIV-4 amplification oligomer is configured to hybridize to a site including position 11685, 11739, or 11981 of HPIV-4. (Item 114) 114. The composition, kit, or method of any one of claims 110 to 113, wherein the first HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100. (Item 115) 115. The composition, kit, or method of any one of items 110 to 114, wherein the first HPIV-4 oligomer comprises a sequence of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100 having up to two mismatches. (Item 116) 115. The composition, kit, or method of any one of items 110 to 114, wherein the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100. (Item 117) 117. The composition, kit, or method of any one of claims 110 to 116, wherein the second HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 54, 63, 64, 113, 114, or 115. (Item 118) 118. The composition, kit, or method of any one of items 110 to 117, wherein the second HPIV-4 oligomer comprises a sequence of SEQ ID NO: 54, 63, 64, 113, 114, or 115 having up to two mismatches. (Item 119) 118. The composition, kit, or method of any one of items 110 to 117, wherein the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 54, 63, 64, 113, 114, or 115. (Item 120) 120. The composition, kit, or method of any one of claims 110 to 119, wherein the composition or kit further comprises a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, the third oligomer comprising a detectable label. (Item 121) 121. The composition, kit, or method of item 120, wherein the third HPIV-4 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 53, 55, 58, 60, 62, 65, 67, 68, 76, 83, 84, 85, 86, 92, 94, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112. (Item 122) 122. The composition, kit, or method of any one of items 120 to 121, wherein the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 53, 55, 58, 60, 62, 65, 67, 68, 76, 83, 84, 85, 86, 92, 94, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112 having up to two mismatches. (Item 123) 122. The composition, kit, or method of any one of items 120 to 121, wherein the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 53, 55, 58, 60, 62, 65, 67, 68, 76, 83, 84, 85, 86, 92, 94, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112. (Item 124) 13. The composition, kit, or method of any one of items 110 to 112, wherein the first HPIV-4 amplification oligomer is configured to hybridize to a site including position 625 of HPIV-4 and the second HPIV-4 amplification oligomer is configured to hybridize to a site including position 738 of HPIV-4. (Item 125) 125. The composition, kit, or method of any one of paragraphs 110 to 112 or 124, wherein the first HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 78. (Item 126) The composition, kit, or method of any one of items 110 to 112 or 124 to 125, wherein the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 78 with up to two mismatches. (Item 127) The composition, kit, or method of any one of items 110 to 112 or 124 to 125, wherein the first HPIV-4 oligomer comprises the sequence of SEQ ID NO:78. (Item 128) 128. The composition, kit, or method of any one of items 110 to 112 or 124 to 127, wherein the second HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 56. (Item 129) The composition, kit, or method of any one of items 110 to 112 or 124 to 128, wherein the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 56 with up to two mismatches. (Item 130) The composition, kit, or method of any one of items 110 to 112 or 124 to 128, wherein the second HPIV-4 oligomer comprises the sequence of SEQ ID NO:56. (Item 131) The composition, kit, or method of any one of items 110-112 or 124-130, wherein the composition or kit further comprises a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, the third oligomer comprising a detectable label. (Item 132) 132. The composition, kit, or method of claim 131, wherein the third HPIV-4 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO:71. (Item 133) 133. The composition, kit, or method of item 131 or 132, wherein the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 71 with up to two mismatches. (Item 134) 133. The composition, kit, or method of item 131 or 132, wherein the third HPIV-4 oligomer comprises the sequence of SEQ ID NO:71. (Item 135) 13. The composition, kit, or method of any one of items 110 to 112, wherein the first HPIV-4 amplification oligomer is configured to hybridize to a site including position 2128, 2352, 2386, 2388, 2404, or 2524 of HPIV-4, and the second HPIV-4 amplification oligomer is configured to hybridize to a site including position 2907, 3006, or 3039 of HPIV-4. (Item 136) 136. The composition, kit, or method of any one of paragraphs 110 to 112 or 135, wherein the first HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 72 or 124. (Item 137) The composition, kit, or method of any one of items 110 to 112 or 135 to 136, wherein the first HPIV-4 oligomer comprises a sequence of SEQ ID NO: 72 or 124 having up to two mismatches. (Item 138) The composition, kit, or method of any one of items 110 to 112 or 135 to 136, wherein the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 72 or 124. (Item 139) 139. The composition, kit, or method of any one of paragraphs 110 to 112 or 135 to 138, wherein the second HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 50, 51, 52, 93, 95, or 96. (Item 140) The composition, kit, or method of any one of items 110 to 112 or 135 to 139, wherein the second HPIV-4 oligomer comprises a sequence of SEQ ID NO: 50, 51, 52, 93, 95, or 96, having up to two mismatches. (Item 141) The composition, kit, or method of any one of items 110-112 or 135-139, wherein the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 50, 51, 52, 93, 95, or 96. (Item 142) 142. The composition, kit, or method of any one of paragraphs 110-112 or 135-141, wherein the composition or kit further comprises a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, the third oligomer comprising a detectable label. (Item 143) 143. The composition, kit, or method of claim 142, wherein the third HPIV-4 oligomer competes for hybridization to HPIV-3 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 46, 47, 48, 49, 59, 61, 69, 70, 72, 73, 74, 75, 77, 88, 90, 91, 116, 117, 118, 119, 121, 122, or 123. (Item 144) 144. The composition, kit, or method of item 142 or 143, wherein the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 46, 47, 48, 49, 59, 61, 69, 70, 72, 73, 74, 75, 77, 88, 90, 91, 116, 117, 118, 119, 121, 122, or 123 with up to two mismatches. (Item 145) 144. The composition, kit, or method of item 142 or 143, wherein the third HPIV-4 oligomer comprises the sequence of SEQ ID NO: 46, 47, 48, 49, 59, 61, 69, 70, 72, 73, 74, 75, 77, 88, 90, 91, 116, 117, 118, 119, 121, 122, or 123. (Item 146) 146. The composition, kit, or method of any one of paragraphs 110-145, wherein at least one, at least two, or each of the first, second, and third HPIV-4 oligomers contains at least one 5-methylcytosine. (Item 147) 147. The composition, kit, or method of any one of items 110 to 146, wherein at least about half of the C residues in the first, second, and / or third HPIV-4 oligomers are 5-methylated. (Item 148) 148. The composition, kit, or method of any one of items 110 to 147, wherein substantially all of the C residues in the first, second, and / or third HPIV-4 oligomers are 5-methylated. (Item 149) The method of any one of items 3 to 58, 60 to 88, or 90 to 148, wherein the nucleic acid amplification reaction comprises thermal cycling. (Item 150) 150. The method of any one of items 3 to 58, 60 to 88, or 90 to 149, wherein the nucleic acid amplification reaction comprises reverse transcription. (Item 151) The method according to any one of items 3 to 58, 60 to 88, or 90 to 150, wherein the nucleic acid amplification reaction comprises RT-PCR. (Item 152) The composition, kit, or method of any one of items 26-58, 61-88, or 91-151, wherein at least one detectable label is fluorescent. (Item 153) 153. The composition, kit, or method of item 152, wherein at least one oligomer comprising a fluorescent label further comprises a quencher. (Item 154) 154. The composition, kit, or method of item 152 or 153, wherein at least one oligomer comprising a fluorescent label is a non-extendable oligomer. (Item 155) 5. The method of any one of items 3 to 58, 60 to 88, or 90 to 154, wherein the nucleic acid amplification reaction comprises PCR using a polymerase having 5' to 3' exonuclease activity, and detecting the amplicon using a probe oligomer comprising a fluorophore and a quencher, wherein exonucleolytic degradation of the probe by the polymerase reduces quenching of fluorescence by the quencher. (Item 156) 156. The composition, kit, or method of any one of items 1-155, wherein at least one, at least two, or at least three oligomers comprise at least one non-Watson-Crick (NWC) position. (Item 157) 157. The composition, kit, or method of item 156, wherein at least one, two, three, or four of the oligomers configured to hybridize to HPIV-1, HPIV-2, HPIV-3, or HPIV-4 each contain at least one NWC position. (Item 158) 158. The composition, kit or method of any one of items 1-157, wherein at least one, at least two, or at least three oligomers comprise degenerate positions. (Item 159) 159. The composition, kit, or method of item 158, wherein at least one, two, three, or four of the oligomers configured to hybridize to HPIV-1, HPIV-2, HPIV-3, or HPIV-4 each contain at least one degenerate position. (Item 160) 161. The composition, kit, or method of any one of claims 1 to 159, wherein the first and second amplification oligomers, the first and second HPIV-1 amplification oligomers, the first and second HPIV-2 amplification oligomers, and / or the first and second HPIV-4 amplification oligomers comprise a target hybridizing region of about 10 to 60 bases in length, about 14 to 50 bases in length, about 14 to 40 bases in length, about 14 to 35 bases in length, or about 15 to 30 bases in length. 161. The composition, kit, or method of any one of items 1 to 160, wherein the HPIV-3 amplicon, the HPIV-1 amplicon, the HPIV-2 amplicon, and / or the HPIV-4 amplicon is about 50 to 1000 nucleotides in length, about 50 to 100 nucleotides in length, about 100 to 200 nucleotides in length, about 200 to 300 nucleotides in length, about 300 to 400 nucleotides in length, about 400 to 500 nucleotides in length, about 500 to 600 nucleotides in length, about 600 to 700 nucleotides in length, about 700 to 800 nucleotides in length, about 800 to 900 nucleotides in length, about 900 to 1000 nucleotides in length, or about 1000 to 2000 nucleotides in length. (Item 162) The kit according to any one of items 1 to 2, 5 to 59, 61 to 89, 91 to 111, 113 to 148, or 153 to 161. (Item 163) The composition according to any one of items 1-2, 5-59, 61-89, 91-111, 113-148, or 153-161. (Item 164) 164. The composition according to item 163, which is aqueous, frozen or lyophilized. (Item 165) 165. Use of the composition or kit according to any one of items 162 to 164 for detecting or quantifying HPIV-1, HPIV-2, HPIV-3 and / or HPIV-4 nucleic acids in a sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] definition Before describing the present teachings in detail, it is to be understood that the present disclosure is not limited to particular compositions or process steps, which may vary. As used in this specification and the appended claims, it is noted that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an oligomer" includes a plurality of oligomers, and so forth. The conjunction "or" should be construed in an inclusive sense, ie, equivalent to "and / or," unless an inclusive sense is unreasonable in the context.

[0071] Temperatures, concentrations, times, etc. discussed in this disclosure are preceded by an implicit "about" whereby minor and non-substantial deviations are understood to be within the scope of the teachings herein. In general, the term "about" refers to non-substantial variations in the amounts of components of a composition that do not significantly affect the activity or stability of the composition. All ranges should be interpreted as including the endpoints unless there is an express exclusion, such as "not including the endpoints." Thus, for example, "within 10 to 15" includes the values ​​10 and 15. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It should be understood that both the summary and detailed description above are exemplary and explanatory only, and not limiting of the teachings. To the extent that any material incorporated by reference conflicts with the express content of this disclosure, the express content controls.

[0072] Unless otherwise stated, embodiments herein that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments herein that recite "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components, and embodiments herein that recite "consisting essentially of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional components, compositions, or method steps that do not substantially alter the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics include, but are not limited to, the specificity of HPIV nucleic acid that distinguishes it from other known respiratory pathogens, and, optionally, the ability to detect approximately 1-100 TCID copies of the virus. 50 / ml, and, optionally, including the ability to detect HPIV nucleic acid sequences present in a sample within about 60 minutes and / or within about 40 cycles of the initiation of the amplification reaction if a cycle amplification reaction is used.

[0073] A "sample" or "specimen," including a "biological" or "clinical" sample, is, for example, a nasopharyngeal or throat swab, a nasal or bronchial or bronchoalveolar wash, ), nasal aspirate, sputum, other respiratory tissues or exudates, biopsy tissues including lymph nodes, or bodily fluids such as blood or urine, derived from living or dead humans or animals that may contain parainfluenza virus target nucleic acid. Samples may be treated to prepare the sample for analysis by physically or mechanically disrupting the tissue or cellular structure and releasing intracellular nucleic acids into solutions that may contain enzymes, buffers, salts, detergents, etc.

[0074] "Nucleic acid" and "polynucleotide" refer to polymeric compounds that contain nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs linked together to form polynucleotides, including polymers that are conventional RNA, DNA, mixed RNA-DNA, and analogs thereof. The nucleic acid "backbone" can be composed of a variety of linkages, including sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA; PCT No. WO95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, such as 2' methoxy or 2' halide substitutions. The nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., inosine or others; The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11 th ed., 1992), purine or pyrimidine derivatives (e.g., N 4-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5- or 6-position, purine bases with substituents at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4 -alkyl-pyrimidines; U.S. Patent No. 5,378,825 and PCT WO 93 / 13121). Nucleic acids may contain one or more "abasic" residues, where the backbone does not contain a nitrogenous base relative to the position of the polymer (U.S. Patent No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases and linkages, or may contain both conventional building blocks and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), analogs that contain one or more LNA nucleotide monomers with bicyclic furanose units locked into the RNA that mimic the sugar configuration, which enhances hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry, 1999). 43(42):13233-41). Examples of oligomers that may affect the stability of a hybridization complex include PNA oligomers, oligomers containing 2'-methoxy or 2'-fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or steric association of a hybridization complex, including oligomers containing charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates). Unless otherwise indicated, 5-methylcytosine may be used with any of the above backbones / sugars / linkages, including RNA or DNA backbones (or mixtures thereof). When referring to a range for the length of an oligonucleotide, amplicon, or other nucleic acid, it is understood that the range includes all integers (e.g., a length of 19-25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0075] "Antisense," "negative sense," or "negative strand" HPIV nucleic acid refers to a sequence of HPIV present in infectious virus (or a fragment thereof). "Sense," "positive sense," or "positive strand" HPIV nucleic acid is the complement of an HPIV sequence present in infectious virus (or a fragment thereof). Exemplary portions of HPIV-1, HPIV-2, HPIV-3, and HPIV-4 are provided in the sequence listing below (for brevity, the complete HPIV genome known in the art is not included). Positions or nucleotides of HPIV sequences are defined with reference to the following positions in SEQ ID NOs: 1-4, respectively: The first nucleotide of SEQ ID NO: 1 is position 201 of HPIV-1. The first nucleotide of SEQ ID NO: 2 is position 1401 of HPIV-2. The first nucleotide of SEQ ID NO: 3 is position 1101 of HPIV-3. The first nucleotide of SEQ ID NO: 4 is position 501 of HPIV-4. Unless otherwise indicated, "hybridizing to an HPIV nucleic acid" includes hybridizing to either the sense or antisense strand of an HPIV nucleic acid, e.g., to either the genomic (antisense) strand, the sense transcript, or a dsDNA HPIV sequence.

[0076] A "C residue" includes methylated and unmethylated cytosine, unless the context indicates otherwise.

[0077] "RNA and DNA equivalents" refers 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. Differences between RNA and DNA equivalents do not contribute to differences in homology, since the equivalents have the same degree of complementarity to a particular sequence. Unless otherwise indicated, reference to HPIV nucleic acid includes HPIV RNA and its DNA equivalents.

[0078] "Oligomer" or "oligonucleotide" generally refers to a nucleic acid less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 2-5 nt and an upper limit of about 500-900 nt. Some specific embodiments are oligomers in a size range having a lower limit of about 5-15, 16, 17, 18, 19, or 20 nt and an upper limit of about 50-600 nt, and other specific embodiments are oligomers in a size range having a lower limit of about 10-20 nt and an upper limit of about 22-100 nt. Oligomers may be purified from naturally occurring sources, but may also be synthesized by using any well-known enzymatic or chemical method. Oligomers may be referred to by their functional name (e.g., capture probe, primer, or promoter primer), although one of skill in the art will understand that such terms refer to oligomers.

[0079] "Amplicon" or "amplification product" refers to a nucleic acid molecule produced in a nucleic acid amplification reaction and derived from a target nucleic acid. The amplicon or amplification product contains a target nucleic acid sequence that can be of the same or opposite sense as the target nucleic acid.

[0080] "Amplification oligonucleotide" or "amplification oligomer" refers to an oligonucleotide that hybridizes to a target nucleic acid or its complement and participates in a nucleic acid amplification reaction, e.g., serves as a primer or promoter-primer. Particular amplification oligomers contain at least about 10 contiguous bases, and optionally at least 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous bases, that are complementary to a region of a target nucleic acid sequence or its complement. The contiguous bases can be at least about 80%, at least about 90%, or fully complementary to the target sequence to which the amplification oligomer binds. One of skill in the art will understand that the ranges described include all integers and rational numbers within the range (e.g., 92% or 98.377%). Particular amplification oligomers are about 10 to about 60 bases in length and can optionally include modified nucleotides.

[0081] "Primer" refers to an oligomer that hybridizes to a template nucleic acid and has a 3' end that is extended by polymerization. A primer may be modified as needed, for example by including a 5' region that is non-complementary to the target sequence. Such modifications may include functional additions such as tags, promoters, or other sequences that are used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0082] Within the context of transcription-mediated amplification, a primer modified with a 5' promoter sequence may be referred to as a "promoter-primer." Those skilled in the art of molecular biology or biochemistry will understand that an oligomer capable of functioning as a primer can be modified to include a 5' promoter sequence and then function as a promoter-primer, and similarly, any promoter-primer can function as a primer with or without its 5' promoter sequence.

[0083] "Nucleic acid amplification" refers to any in vitro procedure that generates multiple copies of a target nucleic acid sequence, or its complementary sequence, or fragments thereof (i.e., an amplified sequence that contains less than the entire target nucleic acid). Examples of nucleic acid amplification procedures include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NSAID), and nucleic acid sequence-based amplification (MSA). Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chain reaction (LCR) (e.g., European Patent Application No. 0320308), helicase-dependent amplification (e.g., U.S. Pat. No. 7,282,328), and strand-displacement amplification (STA) (e.g., U.S. Pat. No. 5,282,328). These include transcription-related methods such as sequence-dependent amplification (SDA) (e.g., U.S. Pat. No. 5,422,252). Amplification can be linear or exponential. Replicase-mediated amplification uses self-replicating RNA molecules and a replicase, such as QB replicase. PCR amplification uses DNA polymerase, primers, and thermal cycling steps to synthesize multiple copies of two complementary strands of DNA or cDNA. LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation. Helicase-dependent amplification uses a helicase to separate the two strands of a DNA duplex to generate a single-stranded template, followed by hybridization of a sequence-specific primer that hybridizes to the template and extension by DNA polymerase to amplify the target sequence.SDA uses primers that contain recognition sites for restriction endonucleases that nick one strand of a hemimodified DNA duplex containing the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Although certain embodiments use PCR or TMA, it will be apparent to one of skill in the art that the oligomers disclosed herein can readily be used as primers in other amplification methods.

[0084] Transcription-associated amplification uses DNA polymerase, RNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, promoter-containing oligonucleotides, and optionally other oligonucleotides, to ultimately generate multiple RNA transcripts from a nucleic acid template (see U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacian et al., U.S. Pat. No. 5,437,990, Burg et al., PCT Nos. WO 88 / 01302 and WO 88 / 10315, Gingeras et al., U.S. Pat. No. 5,130,238, Malek et al., U.S. Pat. Nos. 4,868,105 and 5,124,246, Urdea et al., PCT No. WO 94 / 03472, McDonough et al., PCT No. WO 95 / 03430, and Ryder et al., PCT No. WO 95 / 03430). al.) Methods using TMA have been described in detail previously (U.S. Patent Nos. 5,399,491 and 5,554,516).

[0085] In cyclic amplification methods that detect amplicons in real time, the term "threshold cycle" (Ct) is a measure of the time of 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 the 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 analytical procedures.

[0086] As used herein, the term "relative fluorescence units" ("RFU") is a unit of measurement for fluorescence intensity. RFU varies with the properties of the detection means used in the measurement and can be used as a measure to compare relative intensities between samples and controls. The analytical sensitivity (limit of detection or LoD) is determined by the 50% median tissue culture infective dose (TCID 50 / ml) is determined. 50 / ml is the amount of pathogen that produces pathological changes in 50% of the seeded cell cultures.

[0087] "Detection probe" or "probe" refers to an oligomer that specifically hybridizes to a target sequence, including an amplification sequence, under conditions that promote nucleic acid hybridization for the detection of a target nucleic acid. Detection can be either direct (i.e., a probe that hybridizes directly to the target) or indirect (i.e., a probe that hybridizes to an intermediate structure that links the probe to the target). The target sequence of a probe generally refers to a specific sequence within a larger sequence to which the probe specifically hybridizes. Detection probes can include target-specific sequences and non-target-complementary sequences. Such non-target-complementary sequences can include sequences that confer desired secondary or tertiary structures, such as hairpin structures, that can be used to facilitate detection and / or amplification (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412). Probes of defined sequences can be produced by techniques known to those of skill in the art, such as by chemical synthesis and by in vitro or in vivo expression from recombinant nucleic acid molecules.

[0088] "Hybridization" or "hybridize" refers to the ability of two fully or partially complementary nucleic acid strands to come together, in parallel or antiparallel orientation, under certain hybridization assay conditions to form a stable structure having a double-stranded region. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. These hydrogen bonds are most commonly formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid strand, although base pairing can also form between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, e.g., RLP Adams et al., The Biochemistry of the Nucleic Acids (11th ed.1992).

[0089] "Preferentially hybridize" means that under stringent hybridization conditions, an amplification or detection probe oligomer can hybridize to its target nucleic acid to form a stable oligomer:target hybrid, but does not form a sufficient number of stable oligomer:non-target hybrids. Amplification and detection oligomers that hybridize preferentially to a target nucleic acid are useful for amplifying and detecting the target nucleic acid, but not to non-target organisms, especially organisms closely related phylogenetically. Thus, the oligomer hybridizes to a sufficiently higher degree to the target nucleic acid than to the non-target nucleic acid, allowing the skilled artisan to amplify and / or accurately detect the presence (or absence) of a nucleic acid derived from a particular influenza virus, as desired. In general, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence will reduce the degree or rate of hybridization of the oligonucleotide to its target region. However, the inclusion of one or more non-complementary nucleosides or nucleic acid bases may facilitate the ability of the oligonucleotide to discriminate between non-target organisms.

[0090] Preferential hybridization can be measured using techniques known in the art and described herein, such as in the examples provided below. In some embodiments, there is at least a 10-fold difference, at least a 100-fold difference, or at least a 1,000-fold difference between target and non-target hybridization signals in the test sample. In some embodiments, the non-target hybridization signals in the test sample are at or below background signal levels.

[0091] "Stringent hybridization conditions" or "stringent conditions" refer to conditions that allow an oligomer to hybridize preferentially to a target nucleic acid (such as an HPIV nucleic acid) but not to nucleic acids derived from closely related non-target nucleic acids. While the definition of stringent hybridization conditions remains the same, the actual reaction environment that can be used for stringent hybridization can vary depending on factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and non-target nucleic acid sequences that may be present in the test sample, and the target sequence. Hybridization conditions include temperature and the composition of the hybridization reagent or solution. Exemplary hybridization assay conditions for amplifying and / or detecting target nucleic acids derived from one or more HPIV strains using oligomers of the present disclosure correspond to a temperature of about 60° C. with a salt concentration of a monovalent salt, such as KCl, in the range of about 0.6-0.9 M. Specific hybridization assay conditions are described below in the Examples section. Other acceptable stringent hybridization conditions can be readily ascertained by one of ordinary skill in the art.

[0092] By "competes under stringent conditions for hybridization to HPIV nucleic acid" with a reference oligomer is meant that the oligomer substantially reduces binding of the reference oligomer to its target HPIV sequence under stringent conditions, the competing oligomer when provided in excess is capable of reducing binding of the reference oligomer by about 20%, 30%, 40%, 50% or more at subsaturating concentrations, or the Tm of the competing oligomer is higher or within about 5° C., 4° C., 3° ​​C., 2° C., or 1° C. than the Tm of the reference oligomer with respect to the target. Suitable oligonucleotide competition assay conditions and procedures are known in the art.

[0093] "Assay conditions" refers to conditions that allow for 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.

[0094] "Label" or "detectable label" refers to a moiety or compound directly or indirectly attached to the probe to be detected or to produce a detectable signal. Direct attachment can use covalent or non-covalent interactions (e.g., hydrogen bonds, hydrophobic or ionic interactions, and chelate or coordinate complex formation), while indirect attachment can use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide), which amplifies the detectable signal. Any detectable moiety can use, for example, radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles (e.g., latex or metal beads) that impart detectable color, fluorescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds), and fluorescent compounds (i.e., fluorophores). Embodiments of fluorophores 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 be used in combination with a quencher molecule that absorbs 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™) or TAMRA™ compounds. Certain embodiments include "homogeneous detectable labels" that are detectable in homogeneous systems in which bound labeled probes in a mixture exhibit a detectable change compared to unbound labeled probes, allowing the label to be detected without physically removing hybridized labeled probes from unhybridized labeled probes (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Certain homogeneous detectable labels include chemiluminescent compounds, including acridinium ester ("AE") compounds, such as the well-known standard AE or AE derivatives (U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604).Methods for synthesizing labels, binding labels to nucleic acids, and detecting signals from labels are well known (e.g., 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, and European Patent Application No. 0747706). Certain methods for binding AE compounds to nucleic acids are known (e.g., see U.S. Patent Nos. 5,585,481 and 5,639,604, column 10, line 6 to column 11, line 3, and Example 8). Specific AE labeling locations are the central region of the probe and near the region of A / T base pairs, the 3' or 5' end of the probe, or at or near the mismatch site with a known sequence where the probe should not be detected compared to the desired target sequence. Other detectably labeled probes include TaqMan™ probes, molecular torches, and molecular beacons. TaqMan™ probes contain donor and acceptor labels, where fluorescence is detected upon enzymatic degradation of the probe during amplification to release the fluorophore from the presence of the quencher. Molecular torches and beacons exist in open and closed configurations, where the closed configuration quenches the fluorophore and the open position separates the fluorophore from the quencher to allow fluorescence. Hybridization to the target opens the otherwise closed probe.

[0095] Although sequences do not need to be completely complementary, a sequence is "sufficiently complementary" if it allows stable hybridization of two nucleic acid sequences, such as a stable hybrid of a probe and a target sequence. That is, a "sufficiently complementary" sequence hybridizes to another sequence by hydrogen bonding between a subset of a series of complementary nucleotides by using standard base pairing (e.g., G:C, A:T, or A:U), but the two sequences may contain one or more residues (including abasic positions) that are not complementary, as long as the entire sequence forms a stable hybridization complex under appropriate hybridization conditions. Sufficiently complementary sequences may be at least about 80%, at least about 90%, or completely complementary in the sequences that hybridize to each other. Appropriate hybridization conditions are well known to those skilled in the art and can be predicted based on sequence composition or can be empirically determined by using routine testing (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2001). nd ed. at §§ 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).

[0096] A "non-extendable" oligomer contains a blocking moiety at or near its 3' end to prevent extension. The blocking group near the 3' end is, in some embodiments, within 5 residues of the 3' end and large enough to restrict binding of a polymerase to the oligomer, while other embodiments contain a blocking group 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. Further examples of blocking moieties include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other labels that prevent extension; inverted nucleotides (e.g., attached to the previous nucleotide via a 3' to 3' phosphodiester, optionally with an exposed 5'-OH or phosphate); or proteins or peptides attached to the oligonucleotide to prevent further extension of the nascent nucleic acid chain by a polymerase. Non-extendable oligonucleotides of the present disclosure can be at least 10 bases in length and can be up to 15, 20, 25, 30, 35, 40, 50, or more nucleotides in length. Non-extendable oligonucleotides that contain a detectable label can be used as probes.

[0097] In particular, references in the claims to a "sequence of SEQ ID NO:X" refer to the corresponding base sequence set forth in the 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).

[0098] A "degenerate" position in an oligomer refers to a position where more than one base pair is present in a population of oligomers. For example, in SEQ ID NO:5, the third nucleotide is Y, which represents C or T / U. Oligomers with degenerate positions can be synthesized by providing a mixture of nucleotide precursors corresponding to the desired combination of degeneracy at the synthesis step where introduction of the degenerate position is desired.

[0099] 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 HPIV target sequence through a 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 through a wobble (GU) or purine-purine (GA) pair. In some embodiments, the NWC position is configured to hybridize through a GT pair. In some embodiments, the NWC position is configured to hybridize through a GU pair. In some embodiments, the NWC position is configured to hybridize through a GA pair.

[0100] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Common definitions can be found in technical texts related to the field of molecular biology, such as DICTIONARY OF MICROBIOLOGY. AND MOLECULAR BIOLOGY, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY) or THE HARPER COLLINS DICTIONARY OF BIOLOGY (Hale & Marham, 1991, Harper Perennial, New York, NY).

[0101] Exemplary Compositions, Kits, Methods, and Uses The present disclosure provides oligomers, compositions, and kits useful for amplifying, detecting, or quantitating HPIV-1, HPIV-2, HPIV-3, and / or HPIV-4 from a sample.

[0102] In some embodiments, the oligomer is provided, e.g., in a kit or composition. The oligomer generally includes a target-hybridizing region configured to specifically hybridize, e.g., to an HPIV nucleic acid. While oligomers of different lengths and base compositions can be used to amplify HPIV nucleic acids, in some embodiments, the oligomers of the present disclosure have a target-hybridizing region that is about 10-60 bases long, about 14-50 bases long, about 14-40 bases long, about 14-35 bases long, or about 15-30 bases long. In some embodiments, the oligomer includes a second sequence region in addition to the target hybridizing region, such as a promoter, which can be located 5' of the target hybridizing region. In some embodiments, the oligomer does not include a second sequence region.

[0103] In some embodiments, a pair of oligomers is provided, where one oligomer is configured to hybridize with the sense strand of an HPIV nucleic acid and the other is configured to hybridize with the antisense strand of an HPIV nucleic acid. Such oligomers include primer pairs for PCR or other forms of amplification.

[0104] In some embodiments, one or more oligomers, e.g., a primer pair, or a primer pair and an optionally labeled third oligomer (e.g., for use as a probe), are configured to hybridize to the HPIV hemagglutinin-neuraminidase (HN) gene. In some embodiments, the HN gene is an HPIV-1, HPIV-2, or HPIV-3 HN gene. In some embodiments, a plurality of oligomers, e.g., a plurality of primer pairs, or a plurality of primer pairs and an optionally labeled third oligomer (e.g., for use as a probe), are provided that collectively hybridize to the HN gene of HPIV-1 and HPIV-2; HPIV-1 and HPIV-3; HPIV-2 and HPIV-3; or HPIV-1, HPIV-2, and HPIV-3.

[0105] In some embodiments, one or more oligomers, such as a primer pair, or a primer pair and an optionally labeled third oligomer (e.g., for use as a probe), are configured to hybridize to the HPIV nucleocapsid (NP) gene. In some embodiments, the NP gene is HPIV-4a or HPIV-4b In some embodiments, the one or more oligomers are configured to hybridize with either or both of the HPIV-4a or HPIV-4b NP genes.

[0106] In some embodiments, one or more oligomers comprise a degenerate position. In some embodiments, the HPIV-1 primers, HPIV-1 primer pairs, and / or HPIV-1 probes comprise a degenerate position. In some embodiments, the HPIV-3 primers, HPIV-3 primer pairs, and / or HPIV-3 probes comprise a degenerate position. In some embodiments, the HPIV-4 primers, HPIV-4 primer pairs, and / or HPIV-4 probes comprise a degenerate position. In some embodiments, the degenerate position is Y, R, S, W, K, or M. Exemplary oligomers having degenerate positions (e.g., Y, R, S, W, K, or M) are listed in the sequence listing below.

[0107] In some embodiments, one or more oligomers comprise a non-Watson-Crick (NWC) position. In some embodiments, the HPIV-1 primers, HPIV-1 primer pairs, and / or HPIV-1 probes comprise an NWC position. In some embodiments, the HPIV-2 primers, HPIV-2 primer pairs, and / or HPIV-2 probes comprise a degenerate position. In some embodiments, the HPIV-2 primers, HPIV-2 primer pairs, and / or HPIV-2 probes comprise an NWC position. In some embodiments, the HPIV-3 primers, HPIV-3 primer pairs, and / or HPIV-3 probes comprise an NWC position. In some embodiments, the HPIV-4 primers, HPIV-4 primer pairs, and / or HPIV-4 probes comprise an NWC position.

[0108] Exemplary NWC positions include U residues in the various exemplary oligomers in the sequence listing. Further exemplary NWC positions include G at position 11 of SEQ ID NO:38 and G at position 8 of SEQ ID NO:78.

[0109] In some embodiments, one or more oligomers in a set, kit, composition, or reaction mixture contain a methylated cytosine (e.g., 5-methylcytosine). In some embodiments, at least about half of the cytosines in the oligomer are methylated. In some embodiments, all or substantially all (e.g., all but one or two) of the cytosines in the oligomer are methylated. In some embodiments, the cytosines at the 3' end or within 2, 3, 4, or 5 bases of the 3' end are unmethylated.

[0110] Exemplary primer pairs and optional third oligomers are set forth in the table below. [Table A-1] [Table A-2] [Table A-3]

[0111] In some embodiments, oligomers containing a label are provided. Such oligomers can be used as probes. In some embodiments, the labeled oligomer has a sequence corresponding to the SEQ ID NO listed in the column of Oligomer 3 of Table A. In some embodiments, the label is a non-nucleotide label. Suitable labels include compounds that emit detectable light signals, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogenous mixture. More than one label and more than one type of label can be present on a particular probe, or detection can rely on the use of a mixture of probes, each of which is labeled with a compound that produces a detectable signal (see, for example, U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). Labels can be attached to probes by a variety of means, including covalent binding, chelation, and ionic interactions, although in some embodiments, the labels are attached covalently. For example, in some embodiments, the detection probe has a chemiluminescent label attached, such as, for example, an acridinium ester (AE) compound (see, for example, U.S. Patent Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744). Labels, such as fluorescent or chemiluminescent labels, can be attached to the probe by non-nucleotidic linkers (see, for example, U.S. Patent Nos. 5,585,481; 5,656,744; and 5,639,604).

[0112] In some embodiments, the probe (e.g., comprises fluorescent label) further comprises a second label that interacts with the first label. For example, the second label can be a quencher. Such a probe can be used in TaqMan™ assays, where, for example, the probe is hybridized to a target or amplicon, followed by nucleic acid degradation by a polymerase that comprises 5' to 3' exonuclease activity, releases fluorescent label, thereby increasing fluorescence or resulting in fluorescence that is independent of interaction with the second label.

[0113] In some applications, one or more probes that exhibit at least some degree of self-complementarity are used to facilitate the detection of probe:target duplexes in test samples without first requiring the removal of unhybridized probes prior to detection. Specific embodiments of such detection probes include probes that form conformations that are held by intramolecular hybridization, such as the conformation commonly referred to as hairpin. Suitable hairpin probes include "molecular torches" (see, for example, U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945) and "molecular beacons" (see, for example, U.S. Pat. Nos. 5,118,801 and 5,312,728). Molecular torches contain distinct regions of self-complementarity (coined "target binding domain" and "target closing domain") that are connected by a linking region (e.g., -(CH2CHO)3- linker) and hybridize to each other under predetermined hybridization assay conditions. Upon exposure to the appropriate target or denaturing conditions, the two complementary regions of the molecular torch (which may be fully or partially complementary) melt, making the target binding domain available for hybridization to the target sequence when the predetermined hybridization assay conditions are restored. Molecular torches are designed such that the target binding domain promotes hybridization to the target sequence more than the target closing domain. The target binding domain and the target closing domain of the molecular torch contain interactive labels (e.g., fluorescent / quencher) positioned to produce a different signal when the molecular torch self-hybridizes as opposed to when the molecular torch hybridizes to the target nucleic acid, thereby allowing detection of the probe:target duplex in a test sample in the presence of an unhybridized probe having a viable label associated therewith.

[0114] Examples of interacting donor / acceptor label pairs that may be used in connection with the present disclosure include, without attempting to distinguish between FRET and non-FRET pairs, fluorescein / tetramethylrhodamine, IAEDANS / fluororescein, EDANS / DABCYL, coumadin ... Examples of suitable dyes include phosphorus / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, lucifer yellow / DABCYL, BODIPY / DABCYL, eosin / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, Texas Red / DABCYL, CY5 / BH1, CY5 / BH2, CY3 / BH1, CY3 / BH2, and fluorescein / QSY7 dyes. Those skilled in the art will appreciate 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. 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. Exemplary fluorophore moieties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, and the CY dyes (e.g., CY5). Exemplary quencher moieties that can be used as the other member of a donor-acceptor pair include DABCYL and the BLACK HOLE QUENCHER moiety available from Biosearch Technologies, Inc. (Novato, Calif.).

[0115] In some embodiments, the labeled oligomer (e.g., probe) is non-extendable. For example, the labeled oligomer can be made non-extendable by 3'-phosphorylation, with a 3'-terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxynucleotide), a 3'-terminal inverted nucleotide (e.g., the last nucleotide is inverted so that it is attached to the penultimate nucleotide by a 3' to 3' phosphodiester bond or its analog, e.g., phosphorothioate), or with an attached fluorophore, quencher, or other label that prevents extension (possibly, but not necessarily, attached through the 3' position of the terminal nucleotide). In some embodiments, the 3'-terminal nucleotide is not methylated.

[0116] Also provided by the present disclosure is a reaction mixture for determining the presence or absence or quantifying the amount of HPIV target nucleic acid in a sample. The reaction mixture according to the present disclosure includes at least one or more of the following: a combination of oligomers described herein for amplifying HPIV target nucleic acid, and a detection probe oligomer described herein for determining the presence or absence of HPIV amplification product. The reaction mixture may further include a number of optional components, such as, for example, a capture probe, for example, a poly(k) capture probe as described in US2013 / 0209992, which is incorporated herein by reference. For amplification reaction mixtures, the reaction mixture will typically include other reagents suitable for performing in vitro amplification, such as buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP, and / or ATP, CTP, GTP, and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or a reverse transcriptase, and / or an RNA polymerase), and will typically include test sample components that may or may not be present with HPIV target nucleic acid. A reaction mixture may contain amplification oligomers for only one target region of the HPIV genome, or may contain amplification oligomers for multiple HPIV target regions of the same or different types or subtypes, e.g., one, two, three, or four of HPIV-1, HPIV-2, HPIV-3, and HPIV-4. In addition, for reaction mixtures that include detection probes along with a combination of amplification oligomers, the selection of amplification oligomers and detection probe oligomers for the reaction mixture will be linked by a common target region (i.e., the reaction mixture will contain a probe that binds to a sequence amplifiable by the combination of amplification oligomers in the reaction mixture).

[0117] In some embodiments, the reaction mixture comprises KCl. In some embodiments, the KCl concentration is about 50 mM. In some embodiments, the KCl concentration is greater than about 50 mM, e.g., about 60-150 mM, about 75-125 mM, about 80-120 mM, about 85-115 mM, or about 90-110 mM. In some embodiments, the KCl concentration is 55-65, 65-75, 75-85, 85-95, 95-105, 105-115, 115-125, 125-135, or 135-145, each of the above being in mM and modified by "about" as appropriate. In some embodiments, a composition according to the present disclosure comprises KCl, e.g., at any of the above concentrations. In some embodiments, a method according to the present disclosure comprises performing an amplification reaction in the presence of KCl, e.g., at any of the above concentrations.

[0118] Kits for carrying out the methods described herein are also provided by the present disclosure. Kits according to the present disclosure include at least one or more of the following: a combination of amplification oligomers described herein for amplifying HPIV target nucleic acid, and at least one detection probe oligomer described herein for determining the presence or absence of HPIV amplification products. In some embodiments, any combination of oligomers described herein is present in the kit. The kit may further include a number of optional components, such as, for example, a capture probe, e.g., a poly(k) capture probe as described in US2013 / 0209992. Other reagents that may be present in the kit include reagents suitable for carrying out in vitro amplification, such as buffers, salt solutions, suitable nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and / or ATP, CTP, GTP, and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or reverse transcriptase, and / or an RNA polymerase), and the like. The oligomers described herein may be packaged in a variety of different embodiments, and one of skill in the art will understand that the present disclosure encompasses many different kit configurations. For example, the kit may include amplification oligomers for only one target region of the HPIV genome, or may include amplification oligomers for multiple HPIV target regions of the same or different types or subtypes, e.g., one, two, three, or four of HPIV-1, HPIV-2, HPIV-3, and HPIV-4. In addition, for kits that include a detection probe along with a combination of amplification oligomers, the selection of amplification oligomers and detection probe oligomers for the kit will be linked by a common target region (i.e., the kit will include a probe that binds to a sequence that is amplifiable by the combination of amplification oligomers of the kit). In certain embodiments, the kit further includes a set of instructions for carrying out a method according to the present disclosure, which instructions may be associated with a package insert and / or packaging of the kit or its components.

[0119] Any method disclosed herein should also be understood as a disclosure of the corresponding use of the materials involved in the method directed to the purpose of the method. Any of the oligomers comprising HPIV sequences, and any combinations (e.g., kits and compositions) comprising such oligomers, should also be understood as disclosed for use in detecting or quantitating HPIV, and in preparing compositions for detecting or quantitating HPIV.

[0120] Broadly speaking, the method can include one or more of the following components: target capture, where HPIV nucleic acid (e.g., from a sample, such as a clinical sample) is annealed to a capture oligomer; isolation, e.g., washing, to remove material not associated with the capture oligomer; amplification; and amplicon detection, e.g., amplicon quantification, which can be performed in real time with amplification. Certain embodiments involve each of the above steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the components listed above. Certain embodiments involve any two of the components listed immediately above, e.g., washing and amplification or amplification and detection.

[0121] In some embodiments, amplification comprises: (1) contacting the sample with at least two oligomers to amplify an HPIV nucleic acid target region corresponding to the HPIV target nucleic acid, the oligomers including at least two amplification oligomers described above (e.g., one or more oriented in the sense orientation and one or more oriented in the antisense orientation for exponential amplification); (2) performing an in vitro nucleic acid amplification reaction in which any HPIV target nucleic acid present in the sample is used as a template to generate an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of HPIV in the sample or quantifying the amount of HPIV nucleic acid in the sample.

[0122] Detection methods according to the present disclosure can further include obtaining a sample to be subjected to a subsequent step of the method. In certain embodiments, "obtaining" the sample to be used includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed, and / or removing the sample from a location within the facility where one or more steps of the method are performed (e.g., from a storage facility or other depository).

[0123] In certain embodiments, the method further includes purifying the HPIV target nucleic acid from other components in the sample prior to amplification, e.g., prior to the capture step. Such purification may include methods to separate and / or concentrate organisms contained in the sample from other sample components, or to remove or degrade non-nucleic acid sample components, e.g., proteins, carbohydrates, salts, lipids, etc. In some embodiments, DNA in the sample is degraded, e.g., with DNase, and optionally removing or inactivating the DNase or removing the degraded DNA.

[0124] In certain embodiments, purifying the target nucleic acid includes capturing the target nucleic acid and specifically or non-specifically separating the target nucleic acid from other sample components. Non-specific target capture methods may involve selective precipitation of the nucleic acid from a substantially aqueous mixture, attachment of the nucleic acid to a support that is washed to remove other sample components, or other means of physically separating the nucleic acid from a mixture containing HPIV nucleic acid and other sample components.

[0125] Target capture typically occurs in a liquid phase mixture containing one or more capture probe oligomers that hybridize to HPIV target sequences under hybridization conditions. For embodiments that include a capture probe tail, the HPIV target:capture probe complex is captured by adjusting the hybridization conditions such that the capture probe tail hybridizes to the immobilized probe. Certain embodiments use particulate solid supports such as paramagnetic beads.

[0126] Isolation can follow capture, with the complex on the solid support being separated from other sample components. Isolation can be accomplished by any suitable technique, for example, by washing the support associated with the HPIV target sequence one or more times (e.g., two or three times) to remove other sample components and / or unbound oligomers. In embodiments using particulate solid supports such as paramagnetic beads, the particles associated with the HPIV target can be suspended in a wash solution and, in some embodiments, retrieved from the wash solution by using magnetic forces. To limit the number of handling steps, the HPIV target nucleic acid can be amplified by simply mixing the HPIV target sequence in the complex on the support with the amplification oligomers and proceeding with the amplification step.

[0127] Exponentially amplifying the HPIV target sequence utilizes an in vitro amplification reaction that uses at least two amplification oligomers that flank the target region to be amplified. In some embodiments, at least a first and a second oligomer as described above are provided. In some embodiments, a plurality of oligomer pairs are provided, including oligomer pairs configured to hybridize to at least two, three, or four of the HPIV-1, HPIV-2, HPIV-3, or HPIV-4 nucleic acids. The amplification reaction can be cyclic or isothermal. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA).

[0128] The detection step can be performed using any of a variety of known techniques for detecting signals specifically associated with the amplified target sequence, such as by hybridizing the amplification product with a labeled detection probe and detecting a signal resulting from the labeled probe (including, in some embodiments, from a label released from the probe after hybridization). In some embodiments, the labeled probe includes a second moiety, such as a quencher or other moiety that interacts with the first label, as described above. The detection step can also provide additional information about the amplified sequence, such as all or a portion of its nucleic acid base sequence. Detection can be performed after the amplification reaction is completed, or can be performed simultaneously with amplifying the target region, for example, in real time. In one embodiment, the detection step allows homogeneous detection, for example, detection of hybridized probes without removing unhybridized probes from the mixture (see, for example, U.S. Patent Nos. 5,639,604 and 5,283,174). In some embodiments, the nucleic acid is associated with a surface that results in a physical change, such as a detectable charge. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g., intercalating agents such as ethidium bromide or SYBR Green), or by detecting an increase in dyes associated with the nucleic acids in the solution phase. Other detection methods may use nucleic acid detection probes that are configured to specifically hybridize to sequences in the amplified product and detect the presence of a probe:product complex, or by using a complex of probes that can amplify a detectable signal associated with the amplified product (e.g., U.S. Patent Nos. 5,424,413, 5,451,503, and 5,849,481, each of which is incorporated herein by reference). Directly or indirectly labeled probes that specifically associate with the amplified product provide a detectable signal that indicates the presence of the target nucleic acid in the sample.In particular, the amplification product contains a target sequence in HPIV genomic RNA or a target sequence complementary to a sequence in HPIV genomic RNA, and the probe binds directly or indirectly to a sequence contained in the amplification product to indicate the presence of HPIV nucleic acid in the tested sample.

[0129] In embodiments that detect amplification products near or at the end of the amplification step, linear detection probes can be used to provide a signal indicating the hybridization of the probe to the amplification product. One example of such detection uses a luminescent label probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the non-hybridized probe. Detection is performed by chemiluminescence using a luminometer (see, for example, International Patent Application Publication No. WO89 / 002476). In other embodiments that use real-time detection, the detection probe can 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 can include a target hybridizing sequence and a non-target hybridizing sequence. Various forms of such probes are described, for example, in U.S. Pat. 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 in U.S. Patent Application Publication Nos. 2006 / 0068417A1 and 2006 / 0194240A1. EXAMPLES

[0130] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0131] General Reagents and Methods. Amplifications were performed using an ABI 7500 FAST® instrument unless otherwise indicated. Viral isolates used as amplification targets or controls were diluted in a suitable medium, such as Micro Test M4 medium (Remel Inc. Catalog No. R12500), Micro Test M5 viral transport medium (Remel, Inc. Catalog No. R12515), Micro Test M6 viral transport medium (Remel, Inc. Catalog No. R12530), Micro Test M4RT viral transport medium (Remel, Inc. Catalog No. R12505), or Copan Universal transport medium (Copan Diagnostics, Inc., Catalog No. 330C). Nucleic acid was extracted from viral isolates using a non-specific target capture procedure as described in US Patent Application Publication No. 2013 / 0209992.

[0132] The HPIV-1 through HPIV-3 results were compared with those obtained in parallel using the Prodesse® ProParaflu+™ assay as discussed in Loeffelholz et al., J. Clin. Microbiol. 49:4083-4088 (2011).

[0133] HPIV-4 (including subtypes 4a and 4b) results were compared to FDA cleared HPIV-4 assays (eSensor® Respiratory Virus Panel (GenMark Dx), xTAG™ Respiratory Virus Panel (Luminex), or Biofire FilmArray® Respiratory Panel (Biofire Diagnostics)). The FilmArray® Respiratory Panel is also discussed in Loeffelholz et al., supra. Samples previously characterized as positive for HPIV-4 by one of those three FDA approved assays were tested with the oligomers of the present disclosure. Discordant samples were analyzed using the Cepheid Comparisons were made with results obtained during development using alternative buffers and enzyme systems on the SmartCycler II.

[0134] PCR reactions were typically assembled as follows: 19.05 μL Supermix (Promega GoTaq® Supermix) 0.35 μL Roche MMLV reverse transcriptase (35 U) 0.6μL GoTaq MDX Hotstart Taq(3U) 5 μL of nucleic acid (primers, probes, and targets in appropriate dilutions) = 25 μL total reaction volume.

[0135] Example 1 - Oligomer Design Considerations For HPIV-1, HPIV-2, and HPIV-3, the hemagglutinin-neuraminidase (HN) gene was targeted. For detection of HPIV-4a and 4b, the nucleocapsid protein gene (NP) was targeted. It was determined that the same set of primers and probes could be used to detect HPIV-4a and HPIV-4b.

[0136] Due to the lack of sequence conservation between respiratory virus strains and to accommodate known mismatches, degenerate and non-Watson-Crick (NWC) bases have been considered, for example, in primers and TaqMan™ probes. Based on the review of sequence data collected by the inventors, NWC base pairing (deoxyguanosine / deoxyuridine or deoxyguanosine / deoxyadenosine) and / or degenerate nucleotides (where a population of oligomers has individual molecules with one or the other of base pairs at degenerate positions) have been introduced into certain arrangements. The use of non-Watson-Crick bases facilitates the use of single detection oligonucleotides, thus simplifying manufacturing and quality control and allowing for a more reproducible process, but may reduce the melting temperature (Tm) of annealed oligomers and assay sensitivity for targets containing non-Watson-Crick base pairs. In some oligomers, 5-methylcytosine bases were used to increase duplex stability by increasing the Tm by approximately 0.5-1.3°C for each 5-methylcytosine introduced into the oligonucleotide (compared to the corresponding unmethylated oligomer). It was found that cytosine methylation can generally improve the relatively low stability of oligomers that used non-Watson-Crick base pairing.

[0137] Example 2 - Evaluation of non-Watson-Crick base pairs To test oligomers using NWC base pairs, reference strains diluted in suitable media were extracted as above, and home-produced in vitro transcripts (IVTs) were diluted in RNase inhibitor / water. Each reference strain or IVT was tested at three annealing temperatures (57°C, 60°C, and 65°C) with target-specific oligos containing either degenerate or non-Watson-Crick bases and 5-methylcytosine. Reference strains or IVTs were selected to match the various combinations of mutations observed in the NCBI database at the positions of degenerate and non-Watson-Crick bases. For example, if A and G were observed in different strains, U was used in the NWC oligomer (resulting in either standard AU base pairing or GU wobble NWC base pairing), and if C and A were observed in different strains, G was used in the NWC oligomer (resulting in either standard CG base pairing or AG purine-purine NWC base pairing). NWC oligomers used as probes were designed to hybridize to the plus or minus strand of the target HPIV amplicon as appropriate, such that the oligomer contained U, G, or A at the NWC position. NWC oligomers used as primers were designed to hybridize at sites whose variants or complements contained predominantly either U and A, G and A, C and A, or C and U, or U and G in the strand to which the oligomer hybridized.

[0138] Testing of primers containing non-Watson-Crick base pairing demonstrated that 5-methylcytosine bases increase the Tm and binding stability of primers to DNA during PCR, thereby producing Ct values ​​similar to those found when primers with degenerate bases are used. For example, implementation of NWC base pairing and cytosine methylation resulted in increases ranging from 0 to 1.9 Ct for HPIV-2 and 0 to 3.2 Ct for HPIV-3 compared to degenerate base oligonucleotides at annealing temperatures of 57°C and 60°C. At an annealing temperature of 65°C, NWC oligomers produced Cts equal to or faster than degenerate base oligonucleotides. In many cases, at 65°C, they were not detected with degenerate base oligonucleotides.

[0139] For evaluation of non-Watson-Crick base pairs and degenerate bases, the HPIV-2 and HPIV-3 probes for the non-Watson-Crick oligonucleotides utilized license-free fluorophores and quenchers, whereas the degenerate base oligonucleotides utilized licensed fluorophores and quenchers obtained from Biosearch. This was due to the limited amount of license-free probes available. Increased Ct values ​​were observed with the license-free fluorophores when compared to the licensed fluorophores and quenchers, and this increase is likely responsible for the Ct shifts observed with the HPIV-2 and HPIV-3 oligonucleotides containing non-Watson-Crick bases. The HPIV-4 degenerate and NWC reactions utilized the same fluorophore / quencher pairs, with Ct shifts ranging from 1.4 to 3.1 Ct at 57°C and only 0.4 to 1.4 Ct at 60°C.

[0140] Where license-free fluorophores and quenchers were available for both the non-Watson Crick / 5-methylcytosine probes and the degenerate base probes for HPIV-3, they were run with a 57°C annealing step to test this possibility. Each HPIV-3 IVT strain or reference strain was tested in triplicate with both oligonucleotides. The mean Ct values ​​of samples tested with the non-Watson Crick / 5-methylcytosine oligonucleotides were consistently faster than the Ct of samples tested with the degenerate base oligonucleotides. One IVT was positive in one of three replicates with the degenerate base oligonucleotide but positive in all three replicates with the non-Watson Crick / 5-methylcytosine oligonucleotide. All other samples were positive with both mixtures. The total number of unique HPIV-3 samples tested was seven.

[0141] The introduction of non-Watson-Crick bases with 5-methylcytosine is an acceptable solution to avoid the use of degenerate bases, which may complicate manufacturing and quality control testing compared to oligomers with only non-degenerate positions. It is also possible to use oligomers containing 5-methylcytosine at one, several (e.g., about half), or all positions without degenerate bases and / or without bases designed to participate in NWC pairing, which can tolerate mismatches at one or several (e.g., two or three or four) bases (due to interstrain sequence variation within the virus) and still function as oligomers, e.g., as primers and / or probes.

[0142] Example 3 - Detection and reactivity in clinical specimens Using the exemplary primer pairs and probes of the present disclosure, the clinical sensitivity of the candidate oligomers was evaluated as described above in the General Reagents and Methods section. 73 HPIV-1, 114 HPIV-2, 192 HPIV-3, and 54 HPIV-4 samples were extracted and tested. HPIV-4 samples were also tested using a Cepheid SmartCycler II and reagents obtained from Promega to compare detection results of HPIV-4 clinical specimens and resolve discrepancies.

[0143] One HPIV-2 sample was negative in initial testing with the oligomers of the disclosure, and one HPIV-2 sample was negative in the Prodesse® ProParaflu+™ assay. Both samples were retested and positive with the oligomers of the disclosure.

[0144] Five HPIV-3 samples missed by the Prodesse® ProParaflu+™ assay were detected by the oligomers of the present disclosure.

[0145] The HPIV-4 samples were previously characterized by the FDA-approved assay as described above. During initial testing, seven samples were negative with the disclosed oligomers. The HPIV-4 detection was then modified to improve sensitivity using a lower threshold and additional 50 mM KCl (total 100 mM), with five of the seven HPIV-4 samples being positive. The hit rate for the disclosed oligomers for HPIV-4 detection was 52 / 54, or 96.3%. The two HPIV-4 negative samples were previously characterized as HPIV-4 positive during early development using the same primers and probes when tested on a Cepheid SmartCycler II with an alternative buffer / enzyme system. Nucleic acid stability may have been compromised due to multiple freeze / thaw cycles, therefore the samples were extracted again and tested in duplicate with the disclosed oligomers using additional KCl (total 100 mM). Each of these replicates was positive for HPIV-4. [Table 1]

[0146] Due to the limited number of human parainfluenza strains available, one HPIV-1, HPIV-2, HPIV-4a, and HPIV-4b, one HPIV-4 (no subtypes available), and two HPIV-3 culture and titered clinical isolates were extracted as described above and tested for reactivity. Each clinical isolate was tested using the disclosed oligomers and the Prodesse® ProParaflu™ assay (HPIV-1, HPIV-2, and HPIV-3) at two to three different concentrations in six replicates on an ABI7500 FAST®.

[0147] The LoD for additional strains between the oligomers of the present disclosure and the Prodesse® ProParaflu+™ assay was: HPIV-1 (10 1 TCID 50 / ml) and HPIV-2(10 3 TCID 50 One of the HPIV-3 strains had the same LoD (10 1 TCID 50 / ml), whereas the other had the highest test concentration of 10 2 TCID 50 Compared to the Prodesse® ProParaflu+™ assay, which detected 0 replicates at 10 / ml, the oligomers of the present disclosure were at least 2 logs better (10 1 TCID 50 / ml). LoDs were established for HPIV-4a, HPIV-4b, and one HPIV-4 strain not designated as subtype a or b. Table 2 contains the reactivity data. Target nucleic acids were isolated from previously characterized stock organisms obtained from TriCore Reference Laboratories (Albuquerque, NM), ZeptoMetrix Corporation (Buffalo, NY), and ATCC (Manassas, VA). [Table 2]

[0148] The oligomers used for HPIV-3 hybridized to positions including nucleotides 1355, 1380, and 1437 (forward primer, probe, and reverse primer, respectively). The sites to which the oligomers hybridized for HPIV-3 were positions 1349-1371, 1372-1398, and 1426-1448 (forward primer, probe, and reverse primer, respectively), and with respect to the numbering of SEQ ID NO:3, the sites to which the oligomers hybridized for HPIV-3 were positions 249-271, 272-298, and 326-348 in the forward primer, probe, and reverse primer, respectively. The sequences were SEQ ID NOs:28, 33, and 37 (forward primer, probe, and reverse primer, respectively). SEQ ID NOs:34-35 and 31-32 target the same locus as SEQ ID NO:33 and may function similarly to SEQ ID NO:33.

[0149] Example 4 - Analytical Sensitivity Reference strains were diluted in suitable media and spiked into pooled negative nasopharyngeal swab samples at four concentrations and extracted as described above. Each concentration was extracted in six replicates, and nucleic acids from replicates were not combined. From each extraction, three to four replicates were used to test the oligomers and Prodesse® ProParaflu+™ assays of the present disclosure, for a total of 20 replicates tested in each assay. Assays were performed on an ABI 7500 FAST®.

[0150] The limits of detection (LoD) for HPIV-1, HPIV-2, and HPIV-3 were similar between the oligomers of the present disclosure and the Prodesse® ProParaflu™ assay. The HPIV-1 LoD was 10 1 TCID 50 / ml, and HPIV-2 was 10 0 TCID50 / ml, and HPIV-3 was 10 1 TCID 50 / ml. The LoDs of HPIV-4a and 4b are 10 2 TCID 50 / ml and 10 0 TCID 50 / ml.

[0151] Sequence Listing In the table below, the IUPAC nucleotide code is used to identify degenerate (mixed) positions (Y=C or T, R=A or G, W=A or T, S=G or C, K=G or T, M=A or C, etc.), and individual molecules in the composition or kit may have any of the nucleotides corresponding to the IUPAC code. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

Claims

1. A composition comprising at least a first HPIV-1 amplification oligomer and a second HPIV-1 amplification oligomer, the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of at least 50 nucleotides in length that includes at least one HPIV-1 position located within HPIV-1 positions 330-490 or 960-1100, wherein the HPIV-1 positions 330-490 correspond to positions 130-290 of SEQ ID NO:1, and the HPIV-1 positions 960-1100 correspond to positions 760-900 of SEQ ID NO:1; composition.

2. A method for detecting HPIV-1 in a sample, comprising: Contacting the sample with the composition of claim 1; conducting a nucleic acid amplification reaction in the presence of HPIV-1 nucleic acid to produce an HPIV-1 amplicon; detecting the presence or absence of at least one amplicon; A method comprising:

3. The composition of claim 1 or the method of claim 2, wherein the first HPIV-1 amplification oligomer is configured to hybridize to a site including position 331 of HPIV-1 and the second HPIV-1 amplification oligomer is configured to hybridize to a site including position 486 of HPIV-1.

4. A composition described in any of claims 1 to 3, or a method described in claim 2 or claim 3, wherein the first HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:17 for hybridization to HPIV-1 nucleic acid under stringent conditions.

5. A composition described in any of claims 1 to 4 or a method described in any of claims 2 to 4, wherein the first HPIV-1 oligomer comprises the sequence of SEQ ID NO: 17 having up to two mismatches.

6. A composition described in any of claims 1 to 5, or a method described in any of claims 2 to 5, wherein the first HPIV-1 oligomer comprises the sequence of SEQ ID NO:

17.

7. A composition described in any of claims 1 to 6, or a method described in any of claims 2 to 6, wherein the second HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:22 for hybridization to HPIV-1 nucleic acid under stringent conditions.

8. The composition of claim 1, or the method of claim 2, wherein the second HPIV-1 oligomer comprises the sequence of SEQ ID NO:22 having up to two mismatches.

9. The composition of claim 1, or the method of claim 2, wherein the second HPIV-1 oligomer comprises the sequence of SEQ ID NO:

22.

10. The composition of any of claims 1 to 9, or the method of any of claims 2 to 9, wherein the composition further comprises a third HPIV-1 oligomer configured to hybridize to the HPIV-1 amplicon, wherein the third HPIV-1 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125 for hybridization to HPIV-3 nucleic acid under stringent conditions, and wherein the third oligomer comprises a detectable label.

11. The composition or method of claim 10, wherein the third HPIV-1 oligomer comprises a sequence of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125 having up to two mismatches.

12. A composition described in any of claims 1 to 11, or a method described in any of claims 2 to 11, wherein at least one, at least two, or each of the first, second, and third HPIV-1 oligomers contains at least one 5-methylcytosine.

13. The composition of any of claims 1 to 12, or the method of any of claims 2 to 12, wherein at least half of the C residues in the first, second, and / or third HPIV-1 oligomers are 5-methylated.

14. The composition of any of claims 1 to 13, or the method of any of claims 2 to 13, wherein the C residues in the first, second, and / or third HPIV-1 oligomers are 5-methylated.

15. A composition for detecting HPIV-1, and one or more of HPIV-2, HPIV-3, or HPIV-4, comprising two or more first and second amplification oligomers, the two or more first and second amplification oligomers comprising one or more of: (A) a first and second HPIV-1 amplification oligomer, and (B) a first and second HPIV-2 amplification oligomer, or (C) a first and second HPIV-3 amplification oligomer, or (D) a first and second HPIV-4 amplification oligomer; (A) I. said first HPIV-1 amplification oligomer and said second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of 100-200 nucleotides in length that includes at least one HPIV-1 locus located within SEQ ID NO:1, and said first amplification oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:17; or II. said first HPIV-1 amplification oligomer and said second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of 100-200 nucleotides in length that includes at least one HPIV-1 locus located within SEQ ID NO:1, said first HPIV-1 amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:6; or (B) the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon at least 50 nucleotides in length that includes at least one HPIV-2 position located within SEQ ID NO:2; or (C) the first HPIV-3 amplification oligomer and the second HPIV-3 amplification oligomer are configured to amplify an HPIV-3 amplicon at least 50 nucleotides in length that includes at least one HPIV-3 position located within SEQ ID NO:3; or (D) the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon at least 50 nucleotides in length that includes at least one HPIV-4 position located within SEQ ID NO:

4. composition.

16. A method for detecting HPIV-1, and one or more of HPIV-2 or HPIV-3 or HPIV-4 in a sample, comprising: (a) contacting said sample with first and second amplification oligomers for detecting HPIV-1, and one or more of HPIV-2 or HPIV-3 or HPIV-4, thereby forming a composition; (b) conducting a nucleic acid amplification reaction in the composition in the presence of a target nucleic acid to generate an amplicon; (c) detecting the presence or absence of an HPIV-1 amplicon, and one or more of an HPIV-2 amplicon, or an HPIV-3 amplicon, or an HPIV-4 amplicon; Including, (A) I. a first HPIV-1 amplification oligomer and a second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of 100-200 nucleotides in length that includes at least one HPIV-1 locus located within SEQ ID NO:1, wherein said first HPIV-1 amplification oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:17; or II. said first HPIV-1 amplification oligomer and said second HPIV-1 amplification oligomer are configured to amplify an HPIV-1 amplicon of 100-200 nucleotides in length that includes at least one HPIV-1 locus located within SEQ ID NO:1, said first HPIV-1 amplification oligomer comprising a target hybridizing sequence consisting of SEQ ID NO:6; or (B) the first HPIV-2 amplification oligomer and the second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 amplicon of at least 50 nucleotides in length that includes at least one HPIV-2 position located within SEQ ID NO:2; or (C) the first HPIV-3 amplification oligomer and the second HPIV-3 amplification oligomer are configured to amplify an HPIV-3 amplicon at least 50 nucleotides in length that includes at least one HPIV-3 position located within SEQ ID NO:3; or (D) the first HPIV-4 amplification oligomer and the second HPIV-4 amplification oligomer are configured to amplify an HPIV-4 amplicon of at least 50 nucleotides in length that includes at least one HPIV-4 position located within SEQ ID NO:4; method.

17. The method of claim 17, wherein the second HPIV-1 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:22 for hybridization to HPIV-1 nucleic acid under stringent conditions; or the second HPIV-1 oligomer comprises the sequence of SEQ ID NO:22 with up to two mismatches; or the second HPIV-1 oligomer comprises the sequence of SEQ ID NO:22; 17. The composition of claim 15 or the method of claim 16.

18. The method of claim 1 further comprising: (A) a third HPIV-1 oligomer, wherein for the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer of (A)I, the third HPIV-1 oligomer competes under stringent conditions for hybridization to an HPIV-1 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO: 5, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21, 23, or 125; and the third oligomer comprises a detectable label; or (A) for said first HPIV-1 amplification oligomer and second HPIV-1 amplification oligomer of II, said third HPIV-1 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 10 or 11 for hybridization to HPIV-1 nucleic acid under stringent conditions, and said third oligomer comprises a detectable label; A composition according to any one of claims 15 or 17, or a method according to any one of claims 16 to 17.

19. The method of claim 1 further comprising: (A) a fourth HPIV-1 oligomer, wherein for the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer of (A)I, the third HPIV-1 oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:8 and the fourth HPIV-1 oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:9; or (A) for the first HPIV-1 amplification oligomer and the second HPIV-1 amplification oligomer of II, the third HPIV-1 oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:10 and the fourth HPIV-1 oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:11; 20. The composition or method of claim 18.

20. The composition or method of claim 18, wherein the detectable label is selected from the group consisting of a fluorescent label and a quencher.

21. The method of claim 20, wherein the first HPIV-2 amplification oligomer competes for hybridization to HPIV-2 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO:44; or the first HPIV-2 oligomer comprises the sequence of SEQ ID NO:44 with up to two mismatches; or the first HPIV-2 oligomer comprises the sequence of SEQ ID NO:44; or said second HPIV-2 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:40 for hybridization to HPIV-2 nucleic acid under stringent conditions; or the second HPIV-2 oligomer comprises the sequence of SEQ ID NO:40 with up to two mismatches; or the second HPIV-2 oligomer comprises the sequence of SEQ ID NO:40; A composition according to any one of claims 15 or 17 to 20, or a method according to any one of claims 16 to 20.

22. The composition, further comprising a third HPIV-2 oligomer configured to hybridize to the HPIV-2 amplicon, the third oligomer comprising a detectable label, preferably said third HPIV-2 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO: 38, 39, 41, 42, 43, or 45 for hybridization to HPIV-3 nucleic acid under stringent conditions; A composition according to any one of claims 15 or 17 to 21, or a method according to any one of claims 16 to 21.

23. The third HPIV-2 oligomer comprises a sequence of SEQ ID NO: 38, 39, 41, 42, 43, or 45 having up to two mismatches; or the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:45; or the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:43; or the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:42; or the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:41; or the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:39; or the third HPIV-2 oligomer comprises the sequence of SEQ ID NO:38; 23. The composition or method of claim 22.

24. The first HPIV-3 oligomer comprises the sequence of SEQ ID NO:25 or SEQ ID NO:28; or said second HPIV-3 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:37 for hybridization to HPIV-3 nucleic acid under stringent conditions; or the second HPIV-3 oligomer comprises the sequence of SEQ ID NO:37; A composition according to any one of claims 15 or 17 to 23, or a method according to any one of claims 16 to 23.

25. The composition, further comprising a third HPIV-3 oligomer configured to hybridize to the amplicon, the third oligomer comprising a detectable label, preferably said third HPIV-3 oligomer competes under stringent conditions for hybridization to HPIV-3 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO:31, 32, 33, 34, or 35; or the third HPIV-3 oligomer comprises the sequence of SEQ ID NO:31 or SEQ ID NO:32 or SEQ ID NO:33; A composition according to any one of claims 15 or 17 to 24, or a method according to any one of claims 16 to 24.

26. The method of claim 25, wherein the first HPIV-4 amplification oligomer competes for hybridization to HPIV-4 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100; or the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100 with up to two mismatches; or the first HPIV-4 oligomer comprises the sequence of SEQ ID NO: 79, 80, 81, 82, 97, 98, 99, or 100; and / or the second HPIV-4 amplification oligomer competes under stringent conditions for hybridization to HPIV-4 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO:54, 63, 64, 113, 114, or 115; or the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 54, 63, 64, 113, 114, or 115 with up to two mismatches; or the second HPIV-4 oligomer comprises the sequence of SEQ ID NO: 54, 63, 64, 113, 114, or 115; A composition according to any one of claims 15 or 17 to 25, or a method according to any one of claims 16 to 25.

27. The method of claim 27, wherein the first HPIV-4 amplification oligomer competes under stringent conditions for hybridization to HPIV-4 nucleic acid with an oligomer having a sequence consisting of SEQ ID NO:78, preferably the first HPIV-4 oligomer comprises the sequence of SEQ ID NO:78 with up to two mismatches; or the first HPIV-4 oligomer comprises the sequence of SEQ ID NO:78; A composition according to any one of claims 15 or 17 to 26, or a method according to any one of claims 16 to 26.

28. The method of claim 27, wherein the second HPIV-4 amplification oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:56 for hybridization to HPIV-4 nucleic acid under stringent conditions, and preferably the second HPIV-4 oligomer comprises the sequence of SEQ ID NO:56 with up to two mismatches; or the second HPIV-4 oligomer comprises the sequence of SEQ ID NO:56; 28. The composition of claim 27.

29. The composition further comprising a third HPIV-4 oligomer configured to hybridize to the HPIV-4 amplicon, the third oligomer comprising a detectable label, preferably said third HPIV-4 oligomer competes with an oligomer having a sequence consisting of SEQ ID NO:71 for hybridization to HPIV-3 nucleic acid under stringent conditions; or the third HPIV-4 oligomer comprises the sequence of SEQ ID NO:71 with up to two mismatches; or the third HPIV-4 oligomer comprises the sequence of SEQ ID NO:71; The composition according to any one of claims 27 to 28.

30. The composition of claims 1 or 3-15 or 17-29, which is aqueous, frozen or lyophilized.

31. A kit described in any one of claims 1 or 3 to 15 or 17 to 29.

32. Use of a composition or kit described in any one of claims 1 or 3 to 15 or 17 to 31 for detecting or quantifying HPIV-1 nucleic acid, and two or more of HPIV-2 nucleic acid, HPIV-3 nucleic acid, or HPIV-4 nucleic acid in a sample.

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