Compositions and assays to detect influenza virus A and B nucleic acids
View Patent ↗Methods for detecting influenza virus A and influenza virus B nucleic acids in biological samples by using in vitro amplification and detection are disclosed. Compositions that are target-specific nucleic acid sequences and kits comprising target-specific nucleic acid oligomers for amplifying in vitro influenza virus A or influenza virus B nucleic acid and detecting amplified nucleic acid sequences are disclosed.
1. A method for amplifying and detecting influenza virus A in a sample comprising the steps of:
(a) contacting a sample suspected of containing influenza virus A nucleic acid with a combination of oligomers, said combination of oligomers comprising:
(i) a first amplification oligomer having a target specific sequence consisting essentially of SEQ ID NO:23 or the reverse complement of SEQ ID NO:23; and
(ii) a second amplification oligomer that is sufficiently complementary to the influenza A nucleic acid to hybridize and participate in a nucleic acid amplification reaction, the second amplification oligomer having a target specific sequence 21 to 23 bases long, wherein the target specific sequence contains SEQ ID NO:17 or the reverse complement of SEQ ID NO:17;
(b) performing a nucleic acid amplification reaction; and
(c) detecting an amplification product generated in step (b), thereby determining the presence or absence of influenza A virus in the sample.
2. The method of claim 1 , wherein the second amplification oligomer has a target specific sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and the reverse complement of any one of the aforementioned sequences.
3. The method of claim 1 , wherein either the first amplification oligomer or the second amplification oligomer contains a promoter sequence.
4. The method of claim 1 , wherein the detecting step uses an oligonucleotide detection probe.
5. The method of claim 4 , wherein the contacting step further comprises contacting the sample with the detection probe.
6. The method of claim 4 , wherein the detection probe is added to the reaction after step (a).
7. The method of claim 4 , wherein the target specific sequence of the detection probe is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and the reverse complement of any one of the aforementioned sequences.
8. The method of claim 4 , wherein the detection probe comprises at least one detectable label.
9. The method of claim 8 , wherein the detection probe is a TaqMan probe, a molecular torch or a molecular beacon.
10. The method of claim 8 , wherein the detectable label is a chemiluminescent compound.
11. The method of claim 1 , wherein detecting step is a real-time detection reaction.
12. The method of claim 1 , wherein the detecting step uses a homogenous detection reaction.
13. The method of claim 1 , wherein the nucleic acid amplification reaction is a multiplex reaction.
14. The method of claim 13 , wherein the nucleic acid amplification reaction is a multiplex reaction comprising a target nucleic acid that is an internal control target sequence.
15. The method of claim 14 , wherein the internal control target sequence is amplified using one or more amplification oligomers having a target specific sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, and combinations thereof.
16. The method of claim 13 , wherein the nucleic acid amplification reaction is a multiplex reaction and wherein the sample further contains a nucleic acid from influenza virus B.
17. The method of claim 1 further comprising a target capture step to separate or purify the influenza virus A nucleic acid prior to the contacting step wherein the target capture step uses a target capture probe.
18. The method of claim 17 , wherein the target specific sequence of the target capture probe is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, reverse complement of SEQ ID NO:3, and the reverse complement of SEQ ID NO:4.
19. The method of claim 18 , wherein the target capture probe is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6.
20. The method of claim 1 , wherein the amplification reaction uses thermal cycling.
21. The method of claim 1 , wherein the amplification reaction is isothermal.