Real-time PCR quantitation of subtype C HIV DNA in a Zambian discordant couple cohort

• Published in: AIDS research and human retroviruses Vol. 22; no. 5; p. 438
• Main Authors: Johnson, Roy W, Li, Bing, Sunay, Susan, Moore, Renee H, Mulenga, Joseph, Hunter, Eric, Allen, Susan, Blackwell, Jerry L, Derdeyn, Cynthia A
• Format: Journal Article
• Language: English
• Published: United States 01.05.2006
• Subjects: Cohort Studies; DNA, Viral - analysis; Female; Genes, gag; HIV Infections - virology; HIV Seronegativity; HIV Seropositivity; HIV-1 - classification; HIV-1 - genetics; Humans; Male; Polymerase Chain Reaction - methods; Reference Standards; Reproducibility of Results; Sexual Partners; Zambia - epidemiology; Zambia
• ISSN: 0889-2229
• DOI: 10.1089/aid.2006.22.438

To study the viral sequence diversity that is characteristic of HIV infection, PCR amplification and sequencing of viral genes is an essential step. However, a limitation of traditional PCR methods is that one viral target may be preferentially amplified over another when multiple sequences are present. This presents a particular problem when conclusions about diversity are made from one or only a few PCRs. One way to avoid resampling is to perform a large number of PCR amplifications on a single template; however, this requires that extensive dilution series be carried out on each patient sample to identify the appropriate concentration of input DNA. Here we describe the development and implementation of a quantitative real-time PCR (qPCR) method that detects a short sequence in gag and is optimized to detect subtype C HIV sequences. The standard curve was externally validated using two chronically infected cell lines carrying a known number of HIV copies per genome, and this assay yielded reproducible and accurate measurements on patient DNA samples over a wide range of input targets. The qPCR assay results were consistent with those obtained by the traditional limiting dilution method yet entailed only a fraction of the time and reagents required for the latter. This robust and quantitative real-time assay can be used to ensure that each viral sequence obtained through PCR represents a single template for studies in which the diversity of the entire population must be accurately portrayed, and can readily be applied to other research settings and viral subtypes.