Retrovirus detection
Provided are methods and compositions useful for detecting viral infection or contamination in a biological sample.
1. An isolated oligonucleotide primer pair and probe, the primer pair consists of SEQ ID NO:10 and 11, or a sequence that is at least 95% identical to SEQ ID NO:10 and 11 and which can hybridize to an MLV-related polynucleotide.
2. A method of determining viral content in a subject prior to or after undergoing a retroviral gene delivery therapy using an MLV-related virus, comprising:
obtaining a sample from the subject;
contacting the sample with a primer pair as set forth in claim 1 under conditions suitable for nucleic acid amplification to obtain amplified products;
contacting the sample with a one or more probes that hydridizes to the amplified product;
detecting a hybridized product;
indicating that the subject has viral content comprising an MLV-related virus.
3. The method of claim 2 , wherein the MLV-related virus is a recombinant retroviral vector used in gene delivery.
4. The method of claim 2 , wherein the MLV-related virus is an XMRV virus.
5. The method of claim 2 , wherein the MLV-related virus comprises a 5′ LTR, gag, pol, env genes, a regulatory domain 3′ of the env gene linked to a heterologous polynucleotide to be delivered and a 3′ LTR.
6. The method of claim 5 , wherein the regulatory domain is an internal ribosome entry site (IRES).
7. The method of claim 5 , wherein the heterologous polynucleotide encodes a polypeptide having cytosine deaminase activity.
8. A method for detecting the presence of a viral agent in a sample comprising:
measuring the amount of a polynucleotide in a sample using a quantitative polymerase chain reaction or other amplification process comprising oligonucleotide primer/probe combination of claim 1 .
9. The method of claim 8 , wherein the polynucleotide is DNA.
10. The method of claim 8 , wherein the polynucleotide is RNA.
11. The method of claim 8 , wherein the quantitative polymerase chain reaction is RT-qPCR.
12. The method of claim 8 , measuring detects a single copy of a viral agent related nucleic acid.
13. The method of claim 8 , wherein the viral agent comprises a MLV related virus and/or XMRV.
14. The method of claim 8 , wherein the sample is mammalian tissue or mammalian blood.
15. The method of claim 13 , wherein the viral agent is a gene therapy vector.
16. The method of claim 15 , wherein the gene therapy vector is a replication-competent vector.
17. The method of claim 8 , wherein the method is performed prior to and/or subsequent to a therapeutic regimen comprising a gene therapy vector treatment.
18. The method of claim 8 , wherein the method is performed to monitor the dosage of a therapeutic regimen comprising a gene therapy vector in a subject.
19. The method of claim 15 , wherein the gene therapy vector comprises a replication competent MLV vector.
20. A kit comprising the isolated oligonucleotide primer pair and a probe of claim 1 and one or more reagents for (i) obtaining a sample from the subject; (ii) reverse transcribing RNA, and/or (iii) amplifying RNA or DNA.
21. A method of claim 2 for detecting <100 copies of MLV related DNA in a sample extracted from fixed histopathological sections.
22. A method of claim 2 for detecting <100 copies of MLV related RNA in a sample extracted from fixed histopathological sections.
23. A method of claim 2 or 8 , wherein the method detects both MLV related virus and XMRV.
24. A method of claim 2 or 8 , wherein the method detects only MLV related virus and does not detect XMRV.
25. A method of claim 2 or 8 , wherein the method detects XMRV gag and MLV gag.
26. A method of claim 2 or 8 , wherein the method detects XMRV pol and MLV pol.
27. A method of claim 2 or 8 , wherein the method detects XMRV Env and MLV Env.
28. A method of screening a blood supply or tissue bank for infection by an MLV, MLV-variant or MLV-related virus comprising performing an amplification reaction on the blood supply or tissue bank utilizing the primers of claim 1 , and detecting an amplified product.
29. The isolated oligonucleotide primer pair and a probe of claim 1 , wherein the probe comprises the sequence of SEQ ID NO:12.
30. The isolated oligonucleotide primer pair and a probe of claim 29 , wherein the probe is fluorescently labeled and comprises a sequence of SEQ ID NO:12.