Compositions and methods for detection of hepatitis A virus nucleic acid
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:
for a second HAV target region,
a first amplification oligomer selected from (i) a promoter primer oligomer that includes a HAV target-specific portion of 21 to 27 contiguous nt contained in the sequence of SEQ ID NO:60, or (ii) a promoter primer oligomer in a size range of 48 to 54 at that includes a HAV target-specific portion of any one SEQ ID NOS:29 to 32; and
a second amplification oligomer of contiguous nt contained in the sequence of SEQ ID NO:86 that includes at least the sequence of SEQ ID NO:156.
2. The combination of at least two oligomers of claim 1 for the second HAV target region selected from the group consisting of: SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:156.
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 to 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:113, and SEQ ID NO:114.
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 second HAV target region,
a first amplification oligomer selected from (i) a promoter primer oligomer that includes of HAV target-specific portion of 21 to 27 contiguous nt contained in the sequence of SEQ ID NO:60, or (ii) a promoter primer oligomer in a size range of 48 to 54 nt that includes a HAV target-specific portion of any one of SEQ ID NOS:29 to 32; and
a second amplification oligomer of contiguous nt contained in the sequence of SEQ ID NO:86 that includes at least the sequence of SEQ ID NO:156;
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 (i) SEQ ID NOS:1 to 7 and (ii) ID NOS:8 to 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 he 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 second HAV target region by using at least two oligomers specific for the second HAY target region selected from the group consisting of: SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:156; and wherein the detecting step uses at least one detection probe that hybridizes specifically to the amplified product of the second 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; optionally 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:113, and SEQ ID NO:114; optionally 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.
10. An amplification reagent comprising the combination of at least two oligomers of claim 1 .
11. The amplification reagent of claim 10 , wherein the reagent is a solution.
12. The amplification reagent of claim 11 , wherein the solution further comprises dinucleotide triphosphates.