Compositions and methods for detecting or quantifying human parainfluenza virus 2
Compositions, methods, kits, and uses are provided for detecting or quantifying an 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.
1. A composition comprising at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, wherein:
the first HPIV-2 amplification oligomer and second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 target nucleic acid sequence of at least about 50 nucleotides in length comprising at least one HPIV-2 position located within HPIV-2 positions 1600-1700, wherein the first HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 44, and
wherein the composition further comprises a third HPIV-2 oligomer configured to hybridize to the HPIV-2 target nucleic acid sequence, and the third oligomer comprises a chemiluminescent label or a fluorescent label.
2. The composition or kit of claim 1 , wherein the second HPIV-2 amplification oligomer is configured to hybridize to a site comprising HPIV-2 position 1698.
3. The composition of claim 2 , wherein the second HPIV-2 amplification oligomer competes for hybridization to an HPIV-2 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 40.
4. The composition of claim 3 , wherein the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40 or a sequence with one or two mismatches to SEQ ID NO: 40.
5. The composition of claim 4 , wherein the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40.
6. The composition of claim 1 , wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 39, 41, 42, 43, or 45 or a sequence with one or two mismatches to SEQ ID NO: 39, 41, 42, 43, or 45.
7. The composition of claim 6 , wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 39.
8. The composition of claim 7 , wherein the third HPIV-2 oligomer comprises the sequence of SEQ ID NO: 38.
9. The composition or kit of claim 1 , wherein at least one of the first, second, and third HPIV-2 oligomers comprises at least one 5-methylcytosine.
10. A method of detecting HPIV-2 in a sample, comprising:
contacting the sample with at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, wherein the first HPIV-2 amplification oligomer and second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 target nucleic acid sequence of at least about 50 nucleotides in length comprising at least one HPIV-2 position located within HPIV-2 positions 1600-1700, wherein the first HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 44,
performing a nucleic acid amplification reaction which produces an HPIV-2 amplicon in the presence of an HPIV-2 nucleic acid, and
detecting the presence or absence of the HPIV-2 amplicon.
11. The method of claim 10 , wherein the second HPIV-2 amplification oligomer is configured to hybridize to a site comprising HPIV-2 position 1698.
12. The method of claim 11 , wherein the second HPIV-2 amplification oligomer competes for hybridization to an HPIV-2 nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 40.
13. The method of claim 12 , wherein the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40 or a sequence with one or two mismatches to SEQ ID NO: 40.
14. The method of claim 13 , wherein the second HPIV-2 oligomer comprises the sequence of SEQ ID NO: 40.
15. The method of claim 10 , wherein the nucleic acid amplification reaction comprises PCR with a polymerase with 5′-to-3′ exonuclease activity, and detecting the HPIV-2 amplicon using a probe oligomer comprising a fluorophore and a quencher, wherein exonucleolysis of the probe by the polymerase reduces quenching of fluorescence by the quencher.
16. A reaction mixture for detecting HPIV-2 in a sample, comprising:
(i) at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, wherein the first HPIV-2 amplification oligomer and second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 target nucleic acid sequence of at least about 50 nucleotides in length comprising at least one HPIV-2 position located within HPIV-2 positions 1600-1700, wherein the first HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 44 and/or the second HPIV-2 amplification oligomer comprise the sequence of SEQ ID NO: 40, and
(ii) a third HPIV-2 oligomer configured to hybridize to the HPIV-2 target nucleic acid sequence, wherein the third oligomer comprises a chemiluminescent label or a fluorescent label,
optionally wherein the reaction mixture further comprises at least one enzyme selected from the group consisting of a DNA polymerase, a reverse transcriptase, and an RNA polymerase, and
wherein the reaction mixture is aqueous or lyophilized.
17. A composition comprising at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, wherein:
the first HPIV-2 amplification oligomer and second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 target nucleic acid sequence of at least about 50 nucleotides in length comprising at least one HPIV-2 position located within HPIV-2 positions 1600-1700, wherein the second HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 40, and
wherein the composition further comprises a third HPIV-2 oligomer configured to hybridize to the HPIV-2 target nucleic acid sequence, and the third oligomer comprises a chemiluminescent label or a fluorescent label.
18. The composition of claim 17 , wherein the first HPIV-2 amplification oligomer is configured to hybridize to a site comprising HPIV-2 position 1603 and competes for hybridization to an HPIV-2 nucleic acid under stringent conditions with an oligomer having the sequence consisting of SEQ ID NO: 44.
19. The composition of claim 18 , wherein the first HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 44 or a sequence with one or two mismatches to SEQ ID NO: 44.
20. A method of detecting HPIV-2 in a sample, comprising:
contacting the sample with at least a first HPIV-2 amplification oligomer and a second HPIV-2 amplification oligomer, wherein the first HPIV-2 amplification oligomer and second HPIV-2 amplification oligomer are configured to amplify an HPIV-2 target nucleic acid sequence of at least about 50 nucleotides in length comprising at least one HPIV-2 position located within HPIV-2 positions 1600-1700, wherein the second HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 40,
performing a nucleic acid amplification reaction which produces an HPIV-2 amplicon in the presence of an HPIV-2 nucleic acid, and
detecting the presence or absence of the HPIV-2 amplicon.
21. The method of claim 20 , wherein the first HPIV-2 amplification oligomer is configured to hybridize to a site comprising HPIV-2 position 1603 and competes for hybridization to an HPIV-2 nucleic acid under stringent conditions with an oligomer having the sequence consisting of SEQ ID NO: 44.
22. The method of claim 21 , wherein the first HPIV-2 amplification oligomer comprises the sequence of SEQ ID NO: 44 or a sequence with one or two mismatches to SEQ ID NO: 44.