title: Quantitative assessment of HIV-1 DNA load by coamplification of HIV-1 gag and HLA-DQ-alpha genes.
abstract: We developed an assay for simultaneous amplification, detection and quantitation of HIV-1 gag gene and the DQ-alpha locus of the histocompatibility (HLA) region of the human genome by polymerase chain reaction (PCR). Crude cell lysates from control cell lines and peripheral blood mononuclear cells (PBMC) from HIV-1-infected and control individuals were coamplified using optimized concentrations of primers directed at both loci, followed by simultaneous hybridization with radioactively labeled HIV-1-gag and HLA-DQ-alpha probes. Simultaneous quantitation of the 242-base-pair HLA and 115-base-pair HIV products was accomplished by both end-point dilution analysis and image analysis of autoradiographs relative to standard curves derived from infected cell lines. We observed good agreement between input cell counts on fresh samples and the HLA-DQ-alpha target copy number values determined by both end-point dilution analysis and comparison of band intensities with standard curves. HIV-1 proviral load in symptomatic patients ranged from 200 to 4000 HIV-PCR-units per 1 x 10(6) PBMC (mean of 1245 copies), whereas asymptomatic patients had levels ranging from two to 1000 HIV-PCR-units per 1 x 10(6) PBMC (mean of 213 copies). This HIV/HLA coamplification approach should be particularly useful for analysis of frozen repository samples from natural history studies, and may facilitate wider application of quantitative PCR analysis.
authors.affiliation: Irwin Memorial Blood Centers, San Francisco 94118.
authors.forename: T H
authors.initials: TH
authors.lastname: Lee
authors.forename: F J
authors.initials: FJ
authors.lastname: Sunzeri
authors.forename: L H
authors.initials: LH
authors.lastname: Tobler
authors.forename: B G
authors.initials: BG
authors.lastname: Williams
authors.forename: M P
authors.initials: MP
authors.lastname: Busch
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D005802:Genes, MHC Class II; D015750:Genes, gag; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics*; D006683:HLA-DQ Antigens / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000382:microbiology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics
chemical_list: D004279:DNA, Viral; D006683:HLA-DQ Antigens