Assay for detection of human parvovirus B19 nucleic acid
View Patent ↗Nucleic acid oligomers specific for human parvovirus B19 genomic DNA are disclosed. An assay for amplifying and detecting human parvovirus B19 nucleic acid in biological specimens is disclosed. Compositions for detecting the presence of parvovirus B19 genomic DNA in human biological specimens are disclosed.
1. A combination of at least two separate nucleic acid oligomers wherein the first oligomer is selected from the group consisting of:
a) SEQ ID NO: 24,
b) the complete complement of SEQ ID NO: 24, and
c) the RNA equivalent of the sequence of a) or b),
wherein the first oligomer optionally includes a promoter sequence joined to the 5′ terminus of the sequence;
and wherein the second oligomer is selected from the group consisting of:
d) SEQ ID NO: 13,
e) the complete complement of SEQ ID NO: 13, and
f) the RNA equivalent of the sequence of d) or e).
2. The combination of oligomers of claim 1 wherein the oligomer that consists of SEQ ID NO: 24 and a promoter sequence joined to the 5′ terminus of SEQ ID NO: 24 consists of SEQ ID NO: 23.
3. The combination of oligomers of claim 1 wherein the promoter sequence joined to the 5′ terminus of the sequence is the promoter sequence of SEQ ID NO: 19.
4. The combination of oligomers of claim 1 , further comprising a third nucleic acid oligomer that contains a target complementary sequence of 27 to 33 nucleotides and which includes a target specific sequence consisting of a) SEQ ID NO: 41, b) the complete complement of SEQ ID NO: 41, or c) the RNA equivalent of the sequence of a) or b); and wherein the third oligomer optionally includes a 3′ tail portion that is non specific for a parvovirus target sequence, and wherein the target specific sequence optionally includes at least one 2′-methoxy substituted RNA group.
5. The combination of oligomers of claim 4 , wherein the third oligomer contains the target specific sequence consisting of a) SEQ ID NO: 20, b) the complete complement of SEQ ID NO: 20, or c) the RNA equivalent of the sequence of a) or b), and optionally a 3′ tail portion that is non specific for a parvovirus target.
6. The combination of oligomers of claim 4 wherein the third oligomer contains the target specific sequence consisting of a) SEQ ID NO: 21, b) the complete complement of SEQ ID NO: 21, or c) the RNA equivalent of the sequence of a) or b), and optionally a 3′ tail portion that is non specific for a parvovirus target.
7. The combination of oligomers of claim 4 wherein the target specific sequence of the third oligomer includes at least one 2′-methoxy substituted RNA group.
8. The combination of oligomers of claim 1 , further comprising a third nucleic acid oligomer consisting of a target specific sequence of at least 25 contiguous bases contained in a) SEQ ID NO: 1, b) the complete complement of SEQ ID NO: 1, or c) the RNA equivalent of the sequence of a) or b), and optionally a 3′ tail portion that is non specific for a parvovirus target sequence.
9. The combination of oligomers of claim 1 , further comprising a third nucleic acid oligomer consisting of a) SEQ ID NO: 28, b) the complete complement of SEQ ID NO: 28, c) SEQ ID NO: 31, d) the complete complement of SEQ ID NO: 31, e) SEQ ID NO: 32, f) the complete complement of SEQ ID NO: 32, g) SEQ ID NO: 39, h) the complete complement of SEQ ID NO: 39, i) SEQ ID NO: 40, j) the complete complement of SEQ ID NO: 40, or k) the RNA equivalent of the sequence of any one of a)–j); wherein the third oligomer optionally includes at least one 2′-methoxy substituted RNA group.
10. The combination of oligomers of claim 9 wherein the third oligomer consists of a) the sequence of SEQ ID NO: 40, b) the complete complement of SEQ ID NO: 40, or c) the RNA equivalent of the sequence of a) or b).
11. The combination of oligomers of claim 9 wherein the third oligomer consists of a) the sequence of SEQ ID NO: 28, b) the complete complement of SEQ ID NO: 28, c) the sequence of SEQ ID NO: 32, d) the complete complement of SEQ ID NO: 32, or e) the RNA equivalent of the sequence of any one of a)–d).
12. The combination of oligomers of claim 9 wherein the third oligomer consists of a) the sequence of SEQ ID NO: 39, b) the complete complement of SEQ ID NO: 39, or c) the RNA equivalent of the sequence a) or b).
13. The combination of oligomers of claim 9 wherein the third oligomer consists of a) the sequence of SEQ ID NO: 31, b) the complete complement of SEQ ID NO: 31, or c) the RNA equivalent of the sequence of a) or b).
14. The combination of oligomers of claim 9 wherein the third oligomer includes at least one 2′-methoxy substituted RNA group.
15. A method of detecting human parvovirus B19 nucleic acid in a biological sample, comprising the steps of:
providing a biological sample containing parvovirus B19 nucleic acid;
amplifying in vitro a portion of the parvovirus B19 nucleic acid by using at least one nucleic acid polymnerase activity and at least one first amplification oligomer and one second amplification oligomer, selected from the group consisting of
a first amplification oligomer consisting of SEQ ID NO:23 or SEQ ID NO:24 optionally including a promoter sequence joined to a 5′ terminus of SEQ ID NO:24, and
a second amplification oligomer consisting of SEQ ID NO:13; and
detecting an amplified product of the parvovirus B19 nucleic acid by using a labeled detection probe that hybridizes specifically with the amplified product, thereby indicating presence of parvovirus B19 nucleic acid in the biological sample.
16. The method of claim 15 , wherein the amplifying step uses the oligomer consisting of SEQ ID NO: 24 including a promoter sequence joined to the 5′ terminus of SEQ ID NO: 24, and the oligomer consisting of the sequence of SEQ ID NO: 13.
17. The method of claim 15 , wherein the amplifying step uses an amplification reaction that is substantially isothermal.
18. The method of claim 15 , wherein the detecting step uses a labeled detection probe selected from the group consisting of SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:39, and SEQ ID NO:40.
19. The method of claim 15 , wherein the detecting step uses a labeled detection probe consisting of SEQ ID NO:28.
20. The method of claim 15 , wherein the detecting step uses a labeled detection probe comprising at least one 2′-methoxy substituted RNA group.
21. The method of claim 15 , wherein the detecting step uses a labeled detection probe that includes a label that is detected in a homogeneous reaction.
22. The method of claim 15 , wherein the detecting step uses a labeled detection probe that includes a chemiluminescent label attached to the oligomer via a linker compound.
23. The method of claim 15 , further comprising the steps of:
contacting the biological sample with at least one capture oligomer comprising a target-specific sequence that hybridizes to a parvovirus B19 target sequence, thus forming a complex comprising the capture oligomer and parvovirus B19 nucleic acid; and
separating the complex from the biological sample before the amplifying step.
24. The method of claim 23 , wherein the capture oligomer contains a target complementary sequence of 27 to 33 nucleotides that contains a target specific sequence consisting of a) SEQ ID NO: 41, b) SEQ ID NO: 20, c) SEQ ID NO: 21, or d) the RNA equivalent of the sequence of any one of a)–c), and wherein the capture oligomer optionally includes a 3′ tail sequence that is non specific for a parvovirus target sequence.
25. The method of claim 23 , wherein the capture oligomer comprises at least one 2′-methoxy substituted RNA group.