Evaluation of PCR-based methods for the quantitation of integrated HIV-1 DNA.
Integration of HIV-1 DNA is essential both for productive viral replication and for viral persistence in patients. Methods to measure specifically proviral HIV DNA are required for investigating the mechanisms of HIV integration, for screening novel integrase inhibitors in cell culture and for monitoring levels of persistent integrated viral DNA in patients. In this report, the linker primer polymerase chain reaction (LP-PCR) and Alu-PCR methods for the quantitation of integrated HIV-1 DNA have been modified and evaluated. Each of the two modified assays allowed the quantitative detection of 4 copies of integrated HIV DNA in presence of 2 x 10(5) cell-equivalents of human chromosomal DNA. The results show that proper DNA isolation procedures and the inclusion of appropriate controls in these assays are important for the accurate quantitation of integrated HIV DNA. With further improvements, it should be possible to use these methods as diagnostic tools to monitor closely the efficacy of antiretroviral therapy.
Kumar
Raman
R
National Centre for HIV Virology Research, Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, Australia.
Vandegraaff
Nick
N
Mundy
Linda
L
Burrell
Christopher J
CJ
Li
Peng
P
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured; D016662:Virus Integration; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral