Diagnostic assays for parvovirus B19
View Patent ↗Human parvovirus B19 primers and probes derived from conserved regions of the parvovirus B19 genome are disclosed. Also disclosed are nucleic acid-based assays using the primers and probes.
1. A method for detecting human parvovirus B19 in a biological sample, the method comprising:
isolating nucleic acids from a biological sample suspected of containing human parvovirus wherein the nucleic acids are isolated from the biological sample by a method comprising (i) contacting a solid support comprising magnetic beads that comprise capture nucleic acids associated therewith with the biological sample under hybridizing conditions wherein target nucleic acid strands, if present in the biological sample, hybridize with the capture nucleic acids, and (ii) separating the solid support from the sample;
amplifying the isolated nucleic acids using a sense and an antisense primer wherein
(a) the sense primer consists of SEQ ID NO:88;
(b) the antisense primer consists of SEQ ID NO:89; and
detecting the presence of the amplified nucleic acids as an indication of the presence of human parvovirus B19 in the sample,
wherein amplifying uses a fluorogenic 5′ nuclease assay using the sense primer and the antisense primer and detecting is done using at least one probe comprising a detectable label.
2. The method of claim 1 , wherein the capture nucleic acids further comprise a homopolymer chain to link the capture nucleic acids to the solid support.
3. The method of claim 2 , wherein the homopolymer chain is a polyA chain.
4. The method of claim 3 , wherein the capture nucleic acids comprise the oligonucleotide of SEQ ID NO:55 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:55.
5. The method of claim 1 , wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′, 4′, 5′, 7′,-tetrachloro-4-7-dichlorofluorescein (TET).
6. The method of claim 5 , wherein the probe further comprises detectable labels at the 5′-end and at the 3′-end.
7. The method of claim 1 , wherein an internal control sequence is present.
8. The method of claim 7 , wherein the internal control sequence is derived from the nucleotide sequence of SEQ ID NO:92.
9. The method of claim 8 , further comprising a detectably labeled probe sequence for the internal control sequence.
10. The method of claim 4 , wherein the capture nucleic acids comprise the oligonucleotide of SEQ ID NQ:55.
11. The method of claim 10 , wherein the capture nucleic acids comprise the oligonucleotide of SEQ ID NO:55 and one or more of the oligonucleotides of SEQ ID NOS:49-54.
12. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:49 and 55.
13. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:50 and 55.
14. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:51 and 55.
15. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:52 and 55.
16. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:53 and 55.
17. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:54 and 55.
18. The method of claim 11 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:49, 52, 53 and 55.
19. The method of claim 1 , wherein the capture nucleic acids comprises the oligonucleotide of SEQ ID NO:55 and one or more of the oligonucleotides of SEQ ID NOS:49-54.
20. The method of claim 19 , wherein the capture nucleic acids comprises the oligonucleotides of SEQ ID NOS:49, 52, 53 and 55.