title: Real-time PCR quantitation of subtype C HIV DNA in a Zambian discordant couple cohort.
abstract: 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.
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, Georgia 30329, USA.
authors.forename: Roy W
authors.initials: RW
authors.lastname: Johnson
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Susan
authors.initials: S
authors.lastname: Sunay
authors.forename: Renee H
authors.initials: RH
authors.lastname: Moore
authors.forename: Joseph
authors.initials: J
authors.lastname: Mulenga
authors.forename: Eric
authors.initials: E
authors.lastname: Hunter
authors.forename: Susan
authors.initials: S
authors.lastname: Allen
authors.forename: Jerry L
authors.initials: JL
authors.lastname: Blackwell
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Derdeyn
mesh_terms: D015331:Cohort Studies; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D015750:Genes, gag; D015658:HIV Infections / Q000821:virology*; D018023:HIV Seronegativity; D006679:HIV Seropositivity; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012747:Sexual Partners; D015024:Zambia / Q000453:epidemiology
chemical_list: D004279:DNA, Viral