Methods and compositions for detecting BK virus
The invention provides methods and compositions for rapid, sensitive, and highly specific nucleic acid-based (e.g., DNA based) detection of a BK virus in a sample. In general, the methods involve detecting a target nucleic acid having a target sequence of a conserved region of BK viral genomes. The invention also features compositions, including primers, probes, and kits, for use in the methods of the invention.
1. A kit for detecting the presence of BK virus (BKV) nucleic acid in a sample comprising at least two oligonucleotide primers designed to hybridize to SEQ ID NO: 1 or a complement thereof but not to JC virus nucleic acid under conditions which discriminate between BKV and JCV nucleic acid, and at least one probe; wherein the at least two oligonucleotide primers comprise a first primer comprising SEQ ID NO: 6 and a second primer comprising SEQ ID NO: 7, and wherein the at least one probe comprises 8-44 nucleotides and hybridizes to nucleotides 57-90 of SEQ ID NO: 1 or a full complement thereof and comprises a detectable label selected from among a radioactive isotope, a fluorophore, a chemiluminescent molecule, a chromophore, an electron dense label, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a dye, a metal ion, a metal sol, biotin, avidin, strepavidin or a hapten.
2. The kit of claim 1 , further comprising a second primer pair selected from the group consisting of
(a) a first primer comprising SEQ ID NO: 9 and a second primer comprising SEQ ID NO: 10;
(b) a first primer comprising SEQ ID NO: 12 and a second primer comprising SEQ ID NO: 13;
(c) a first primer comprising SEQ ID NO: 15 and a second primer comprising SEQ ID NO: 16; and
(d) a first primer comprising SEQ ID NO: 18 and a second primer comprising SEQ ID NO: 19.
3. The kit of claim 1 , wherein the detectable label is a fluorophore.
4. The kit of claim 3 , wherein the at least one probe further comprises a quencher molecule.
5. The kit of claim 4 , wherein the fluorophore and quencher molecule are attached to same probe.
6. The kit of claim 1 , wherein the one or more oligonucleotide primers comprise a degenerate oligonucleotide.
7. The kit of claim 2 , further comprising one or more additional probes selected from:
(a) a probe comprising nucleotides 52-80 of SEQ ID NO: 2 or a complement thereof;
(b) a probe comprising nucleotides 51-75 of SEQ ID NO: 57 or a complement thereof;
(c) a probe comprising nucleotides 32-62 of SEQ ID NO: 4 or a complement thereof;
(c) a probe comprising nucleotides 19-41 of SEQ ID NO: 5 or a complement thereof.
8. The kit of claim 7 , wherein the one or more additional probes comprises a detectable label selected from among a radioactive isotope, a fluorophore, a chemiluminescent molecule, a chromophore, an electron dense label, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a dye, a metal ion, a metal sol, biotin, avidin, strepavidin or a hapten.
9. The kit of claim 8 , wherein the detectable label is a fluorophore.
10. The kit of claim 8 , wherein the one or more additional probes further comprises a quencher molecule.
11. The kit of claim 9 , wherein the fluorophore and quencher molecule are attached to same probe.
12. The kit of claim 1 , wherein the at least one probe comprises nucleotides 57-90 of SEQ ID NO: 1 or a complement thereof.