Compositions and assays to detect swine H1N1 influenza A virus, seasonal H1 influenza A virus and seasonal H3 influenza A virus nucleic acids
View Patent ↗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 method for the detection of an influenza A virus from a sample, comprising the steps of:
(a) contacting an influenza A virus nucleic acid from a sample with a composition comprising at least two seasonal H1 influenza A-specific amplification primers, wherein the at least two primers comprise nucleic acid sequences in combination(s) selected from the group consisting of Combination 1: SEQ ID NO:63 and SEQ ID NO:64; Combination 2: SEQ ID NO:68 and SEQ ID NO:69; and Combination 3: SEQ ID NO:72 and SEQ ID NO:73, or RNA equivalents thereof;
(b) providing conditions for amplifying the nucleic acid 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 amplification product from step b, wherein the presence of the amplification product indicates that the sample contained an influenza A virus.
2. The method according to claim 1 , wherein the detecting step is a real-time detecting step, or a taqman real-time PCR detecting step.
3. The method according to claim 1 , wherein the sample contains an influenza A virus nucleic acid selected from the group consisting of: a seasonal H1 influenza A virus nucleic acid, an influenza A virus nucleic acid that is substantially identical to a seasonal H1 influenza A virus nucleic acid, an influenza A virus nucleic acid that is at least 90% identical to a seasonal H1 influenza A virus nucleic acid an influenza A virus nucleic acid that has an H1 gene that is substantially identical to the H1 gene of a seasonal H1 influenza A virus nucleic acid, and an influenza A virus nucleic acid that has an H1 gene that is at least 90% identical to the H1 gene of a seasonal H1 influenza A virus nucleic acid; and wherein in step (b) an amplification product is generated from said influenza A virus nucleic acid.
4. The method of claim 1 , wherein at the contacting step the influenza A virus nucleic acid from a sample is contacted with a composition according to one of Mixture 1 to Mixture 21.
5. The method according to claim 4 , wherein the detecting step is a real-time detecting step or a taqman PCR real-time detecting step.
6. The method according to claim 1 , wherein said detection step c uses a seasonal H1 influenza A-specific detection probe oligonucleotide comprising at least a first detectable label operably linked to said detection probe oligonucleotide.
7. The method according to claim 6 , wherein said primer sequences are SEQ ID NO:63 and SEQ ID NO:64 and said detection probe sequence is SEQ ID NO:66; wherein said primer sequences are SEQ ID NO:68 and SEQ ID NO:69 and said detection probe sequence is SEQ ID NO:70; or wherein said primer sequences are SEQ ID NO:72 and SEQ ID NO:73 and said detection probe sequence is SEQ ID. NO. 74.
8. A method for the detection of a seasonal H1 Influenza A Virus from a sample, comprising the steps of:
(a) contacting a seasonal H1 Influenza A Virus nucleic acid from a sample with primer pair each comprising oligonucleotide primer sequences consisting of sequences selected from Table 2;
(b) providing conditions for amplifying the nucleic acid 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 amplification product from step b, wherein the detecting step uses a detection probe oligonucleotide comprising a nucleic acid probe sequence selected from Table 2, and wherein the presence of the amplification product indicates that the sample contained a seasonal H1 Influenza A Virus.
9. The method according to claim 8 , wherein the detecting step is a real-time detecting step or a taqman real-time PCR detecting step.
10. The method according to claim 8 , wherein the sample further contains an influenza A virus nucleic acid selected from the group consisting of: a H1N1 influenza A virus nucleic acid, a seasonal H3 influenza A virus, and a combination thereof, and an amplification product is generated therefrom.
11. The method according to claim 8 , wherein the amplifying step is a multiplex amplification reaction that further comprises a primer pair comprising nucleic acid sequences selected from Table 1, a primer pair comprising nucleic acid sequences selected from Table 3 or a primer pair comprising nucleic acid sequences selected from Table 1 and a primer pair comprising nucleic acid sequences selected from Table 3.
12. The method according to claim 11 , wherein the detecting step further comprises a probe comprising a nucleic acid sequence selected from Table 1, a probe comprising a nucleic acid sequence selected from Table 3 or a probe comprising a nucleic acid sequence selected from Table 1 and a probe comprising a nucleic acid sequence selected from Table 3.
13. The method according to claim 11 , wherein the amplifying step generates a detectable amplification product from an influenza A virus nucleic acid that is at least 90% identical to an influenza A virus nucleic acid selected from the group consisting of: a H1N1 influenza A virus nucleic acid, a seasonal H3 influenza A virus, and a combination thereof.