Compositions and methods for detection of hepatitis A virus nucleic acid
View Patent ↗Nucleic acid oligomeric sequences and in vitro nucleic acid amplification and detection methods for detecting the presence of HAV RNA sequences in samples are disclosed. Kits comprising nucleic acid oligomers for amplifying and detecting HAV nucleic acid sequences are disclosed.
1. A combination of at least two oligomers for amplifying a HAV nucleic acid containing a target sequence around nucleotide position 5800 of a HAV genome, comprising:
a first amplification oligomer consisting of 19 to 31 nt contained in the sequence consisting of SEQ ID NO:149 and that includes at least the sequence consisting of SEQ ID NO:150, or a promoter primer oligomer in a size range of 51 to 56 nt that includes a HAV target-specific portion consisting of bases 28 to 54 in SEQ ID NO:36 joined to a 5′ promoter sequence; and
a second amplification oligomer.
2. The combination of at least two oligomers of claim 1 for wherein the second amplification oligomer is selected from the group consisting of: SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97.
3. The combination of at least two oligomers of claim 1 wherein the first amplification oligomer consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer consists of SEQ ID NO:97.
4. The combination of at least two oligomers of claim 1 , further comprising at least one capture probe oligomer made up of a target-specific sequence consisting of SEQ ID NO:11 which is optionally covalently attached to a tail sequence or moiety that binds to an immobilized probe.
5. The combination of at least two oligomers of claim 1 , further comprising at least one detection probe oligomer consisting of SEQ ID NO:121.
6. The combination of at least two oligomers according to claim 1 wherein the combination of oligonucleotides is packaged in a kit.
7. The combination of at least two oligomers of claim 1 , wherein the second amplification oligomer is 18 to 20 nt in size and includes the sequence consisting of SEQ ID NO:159.
8. The combination of at least two oligomers of claim 1 wherein the first amplification oligomer consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer consists of SEQ ID NO:94.
9. The combination of at least two oligomers of claim 1 wherein the first amplification oligomer consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer consists of SEQ ID NO:95.
10. The combination of at least two oligomers of claim 1 wherein the first amplification oligomer consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer consists of SEQ ID NO:96.
11. A method of detecting the presence of HAV in a sample comprising the steps of:
purifying HAV nucleic acid that contains a target sequence around nucleotide position 5800 of a HAV genome from other components in a sample containing HAV to produce a purified HAV nucleic acid;
amplifying the target sequence in the purified HAV nucleic acid, or a cDNA made therefrom, by using an in vitro amplification reaction that includes at least two amplification oligomers to produce an amplified product of the target sequence, wherein:
a first amplification oligomer consists of 19 to 31 nt contained in the sequence consisting of SEQ ID NO:149 and includes at least the sequence of SEQ ID NO:150, or consists of SEQ ID NO:66, or is a promoter primer oligomer in a size range of about 51 to 56 nt that includes a HAV target-specific portion consisting of bases 28 to 54 in SEQ ID NO:36 joined to a 5′ promoter sequence,
and a second amplification oligomer; and
detecting the amplified product by using a detection probe that hybridizes specifically with at least a portion of the amplified product.
12. The method of claim 11 , wherein the purifying step contacts the sample with at least one capture probe oligomer made up of a target-specific sequence consisting of SEQ ID NO:11 which is optionally covalently attached to a tail sequence or moiety that binds to an immobilized probe wherein the target-specific sequence consisting of SEQ ID NO:11 hybridizes specifically to a sequence in HAV RNA to form a hybridization complex with the HAV RNA, and wherein the purifying step separates the hybridization complex that contains the HAV RNA from the other components in the sample.
13. The method of claim 11 , wherein the amplifying step uses the second amplification oligomer selected from the group consisting of: SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product made from the HAV nucleic acid.
14. The method of claim 11 , wherein the amplifying step uses the first amplification oligomer that consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer that consists of SEQ ID NO:97; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product made from the HAV nucleic acid.
15. The method of claim 11 , wherein the detecting step uses a detection oligomer consisting of SEQ ID NO:121.
16. The method of claim 11 , wherein the amplifying step uses a second amplification oligomer that is in a size range of 18 to 20 nt that includes the sequence consisting of SEQ ID NO:159.
17. The method of claim 11 , wherein the amplifying step uses the first amplification oligomer that consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer that consists of any one of SEQ ID NO:94; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product made from the HAV nucleic acid.
18. The method of claim 11 , wherein the amplifying step uses the first amplification oligomer that consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer that consists of any one of SEQ ID NO:95; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product made from the HAV nucleic acid.
19. The method of claim 11 , wherein the amplifying step uses the first amplification oligomer that consists of SEQ ID NO:36 or SEQ ID NO:66 and the second amplification oligomer that consists of any one of SEQ ID NO:96; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product made from the HAV nucleic acid.