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 target region comprising:
a first amplification oligomer that is a length of 23 to 26 contiguous nucleotides contained in the sequence of SEQ ID NO:138 that includes at least the sequence of SEQ ID NO:139 or SEQ ID NO:140, and
a second amplification oligomer that is a length of 19 to 25 contiguous nucleotides in contained in the sequence of SEQ ID NO:141 and that includes at least one sequence of SEQ ID NOS: 142 to 146.
2. The combination of at least two oligomers of claim 1 , wherein the first amplification oligomer is selected from the group consisting of: SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56 and wherein the second amplification oligomer is 19 to 25 nt in length and is contained in the sequence of SEQ ID NO: 141 and contains at least one sequence of SEQ ID NOS: 142 to 146.
3. The combination of at least two oligomers of claim 1 , further comprising at least one capture probe oligomer selected from the group consisting of SEQ ID NOS: 1 to 7 and SEQ ID NOS: 8-14 linked to a moiety that allows it to bind a solid support.
4. The combination of at least two oligomers of claim 1 , further comprising at least one detection probe oligomer selected from the group consisting of SEQ ID NO:109, and SEQ ID NO:111.
5. The combination of at least two oligomers according to claim 1 , wherein said combination is packaged in a kit.
6. A method of detecting the presence of HAV in a sample comprising the steps of:
purifying HAV nucleic acid from other components in a sample containing HAV;
amplifying a HAV 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 specific for a selected HAV target region, which include:
for a first HAV target region, a first amplification oligomer that is a length of about 23 to 26 contiguous nucleotides contained in the sequence of SEQ ID NO:138 and that includes at least the sequence of SEQ ID NO:139 or SEQ ID NO:140; and
a second amplification oligomer that is a length of 19 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO:141 and that includes at least one sequence of SEQ ID NOS: 142 to 146;
to produce an amplified product of the selected HAV target region; and
detecting the amplified product.
7. The method of claim 6 , wherein the purifying step contacts the sample with at least one capture probe oligomer selected from the group consisting of SEQ ID NOS: 1 to 7 and SEQ ID NOS: 8-14 linked to a moiety that allows it to bind a solid support, wherein a target hybridization sequence of said at least one capture probe oligomer hybridizes specifically to a sequence in HAV RNA to form a hybridization complex with the HAV RNA, and can be used with a solid support to separate the HAV RNA from other sample components.
8. The method of claim 6 , wherein the amplifying step amplifies a sequence in the first HAV target region by using at least two oligomers specific for the first HAV target region selected from the group consisting of: SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product of the first HAV target region.
9. The method of claim 6 , wherein said detecting step uses a nucleic acid detection oligomer, wherein said nucleic acid detection oligomer comprises a target hybridizing sequence that specifically hybridizes with at least one amplified product generated in said amplifying step.
10. The method of claim 9 , wherein said nucleic acid detection oligomer comprises a target hybridizing region nucleic acid sequence consisting essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NO:109 and SEQ ID NO:111.
11. The method of claim 9 , wherein said nucleic acid detection oligomer further comprises a 2′-O-methyl linkages, a chemiluminescent label, an acridinium ester label, a synthetic linker or combinations thereof.
12. The method of claim 6 , wherein the first amplification oligomer further comprises a promoter sequence joined at its 5′ end, and wherein the promoter sequence is SEQ ID NO:19.
13. The method of claim 12 , wherein the first amplification oligomer is selected from the group consisting of SEQ ID NOS: 21-26, and wherein the second amplification oligomer is selected from the group consisting of SEQ ID NOS: 15 to 18 and 80 to 85.
14. The method of claim 6 , wherein the first amplification oligomer is SEQ ID NO:52 further comprising SEQ ID NO:19 joined to its 5′ end, the second amplification oligomer is SEQ ID NO:16 and amplified product is detected using detection probes that are SEQ ID NO:109, SEQ ID NO:111 or both.
15. The method of claim 6 , wherein the sample is a biological sample collected from humans.
16. The method of claim 15 , wherein the sample collected from a human is a plasma sample.
17. The method of claim 15 , wherein the sample collected from a human is a sample used for the preparation of therapeutic factors isolated from humans.
18. The method of claim 14 , wherein the sample collected from a human is a sample used for the preparation of therapeutic factors isolated from humans.
19. The method of claim 6 , further comprising detecting the presence of parvovirus B19 in the sample.
20. The method of claim 14 , further comprising detecting the presence of parvovirus B19 in the sample.