Compositions and assays to detect swine H1N1 influenza a virus nucleic acids
Methods for detecting the presence or absence of the swine H1N1 influenza A virus, seasonal H1 influenza A virus and/or seasonal H3 influenza A virus nucleic acids in biological samples are disclosed. Compositions that are target-specific nucleic acid sequences and kits comprising target-specific nucleic acid oligomers for amplifying in vitro the swine H1N1 influenza A virus, seasonal H1 influenza A virus and/or seasonal H3 influenza A virus nucleic acid and detecting amplified nucleic acid sequences are disclosed.
1. A composition for detecting the presence of swine H1N1 influenza A virus comprising:
(a) a first amplification primer, a second amplification primer, and a probe oligonucleotide, wherein
the first amplification primer comprises a swine H1N1 influenza A-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 42;
(ii) the second amplification primer comprises a swine H1N1 influenza A-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 43; and
(c) a swine H1N1 influenza A-specific oligonucleotide detection probe having a swine H1N1 influenza A-specific sequence of at least 18 contiguous nucleotides, wherein the detection probe is complementary to a swine H1N1 influenza A nucleic acid sequence that is amplifiable by the first and second amplification primers, and wherein the detection probe comprises at least a first detectable label operably linked to the oligonucleotide detection probe.
2. The composition of claim 1 , wherein the swine H1N1 influenza A-specific oligonucleotide detection probe comprises a swine H1N1 influenza A-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 44, or a complement thereof.
3. The composition according to claim 1 , further comprising at least one nucleic acid oligomer specific for seasonal H1 influenza A virus and/or at least one nucleic acid oligomer specific for seasonal H3 influenza A virus.
4. A kit comprising, in a suitable container, the composition of claim 1 , and instructions for use of said composition in an assay for amplification of polynucleotides in a biological sample.
5. A method for the detection of a swine H1N1 influenza A virus from a sample, comprising the steps of:
(a) contacting the sample with the composition of claim 1 , wherein the sample contains or is suspected of containing a swine H1N1 influenza A virus nucleic acid;
(b) providing conditions for amplifying the nucleic acid, if present, from step (a) by a polymerase chain reaction to generate an amplification product from the nucleic acid; and
(c) detecting the presence or absence of the amplification product from step (b), wherein the presence of the amplification product indicates that the sample contains the swine H1N1 influenza A virus.
6. The method according to claim 5 , wherein the detecting step is selected from the group consisting of: a real-time detecting step, and a taqman PCR detecting step.
7. The method according to claim 5 , comprising: further contacting the sample with a composition comprising: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 102.
8. The method according to claim 7 , wherein the detecting step is a real-time detecting step.
9. The method according to claim 8 , wherein the detecting step is a taqman PCR detecting step.
10. The method according to claim 5 , comprising: further contacting the sample with a primer pair selected from the group consisting of: SEQ ID NOs: 1 and 2, SEQ ID NOs: 5 and 6, SEQ ID NOs: 8 and 9, SEQ ID NOs: 12 and 13, SEQ ID NOs: 17 and 18, SEQ ID NOs: 21 and 22, SEQ ID NOs: 26 and 27, SEQ ID NOs: 30 and 31, SEQ ID NOs: 34 and 35, SEQ ID NOs: 38 and 39, SEQ ID NOs: 45 and 46, SEQ ID NOs: 50 and 51, SEQ ID NOs: 54 and 55, and SEQ ID NOs: 59 and 60.
11. The method according to claim 10 , wherein the detecting step is a real-time detecting step.
12. The method according to claim 11 , wherein the detecting step is a taqman PCR detecting step.
13. The method according claim 10 , wherein the detecting step uses a probe selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 61, and SEQ ID NO: 62.
14. The method according to claim 5 , comprising further contacting the sample with:
(a) at least one primer pair selected from the group consisting of: SEQ ID NOs: 63 and 64, SEQ ID NOs: 68 and 69, and SEQ ID NOs: 72 and 73;
(b) at least one primer pair selected from the group consisting of: SEQ ID NOs: 77 and 78, SEQ ID NOs: 82 and 83, SEQ ID NOs: 85 and 86, SEQ ID NOs: 88 and 89, SEQ ID NOs: 92 and 93, SEQ ID NOs: 96 and 97, and SEQ ID NOs: 99 and 100; or
(c) at least one primer pair selected from (a) and at least one primer pair selected from (b);
wherein the amplifying comprises a multiplex amplification reaction.
15. The method according to claim 14 , wherein the detecting step further comprises:
(a) a probe selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76,
(b) a probe selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 98, SEQ ID NO: 101, and SEQ ID NO: 102; or
(c) a probe selected from (a) and a probe selected from (b).
16. The method according to claim 5 , comprising further contacting the sample with a composition comprising: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 99, SEQ ID NO:100, and SEQ ID NO:102.
17. The method according to claim 5 , comprising further contacting the sample with a composition comprising:
(i) a primer pair consisting of SEQ ID NOs: 72 and 73 and a probe selected from the group consisting of: SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO:76; and
(ii) a primer pair consisting of SEQ ID NOs: 99 and 100, and a probe selected from the group consisting of: SEQ ID NO: SEQ ID NO: 101 and SEQ ID NO: 102.
18. The composition of claim 3 , wherein the swine H1N1 influenza A-specific oligonucleotide detection probe comprises a swine H1N1 influenza A-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 44, or a complement thereof.
19. The kit of claim 4 , wherein the swine H1N1 influenza A-specific oligonucleotide detection probe comprises a swine H1N1 influenza A-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 44, or a complement thereof.
20. A method of claim 5 , wherein the swine H1N1 influenza A-specific oligonucleotide detection probe comprises a swine H1N1 influenza A-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 44, or a complement thereof.