Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system.
We have developed a quantitative competitive PCR (QC-PCR) assay in a microplate format for quantifying human immunodeficiency virus Type 1 (HIV-1) DNA or RNA in a broad range of source materials. Our QC-PCR assay is a modification of technique originally described by Piatak et al. (1993), which is based on the presence of a competitive internal standard containing an internal 80-bp deletion of HIV-1 gag target sequence. For improved detection and quantification of the wild-type and internal-standard PCR products in a microplate format, we introduced a non-HIV, 31-bp insert into the internal standard as a probe hybridization site that does not cross-hybridize with wild-type HIV-1 products. By using a primer pair in which one primer is biotinylated, QC-PCRs can be bound to a streptavidin-coated microplate, denatured and probed with a digoxigenin (Dig)-labeled, wild-type or internal-standard probe. The hybridized Dig-labeled probes are detected with an anti-Dig antibody conjugated to detector molecules for luminometry (aequorin) or optical densitometry (peroxidase), yielding results that are quantifiable over the range of 100-10,000 copies of HIV gag. Tested source materials for HIV-1 DNA or RNA quantification include plasma, vaginal lavage and cultured cells. The application of the QC-PCR assay using the microplate format affords a convenient and cost-effective method for quantifying HIV-1 proviral and viral loads from a variety of body fluids, cells and tissues.
Guenthner
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Centers for Disease Control and Prevention, Atlanta, GA, USA.
Hart
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D000331:Aequorin; D004279:DNA, Viral / Q000032:analysis / Q000097:blood; D004076:Digoxigenin; D005260:Female; D015658:HIV Infections / Q000097:blood / Q000821:virology; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D015345:Oligonucleotide Probes; D009195:Peroxidase; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D012367:RNA, Viral / Q000032:analysis / Q000097:blood; D007507:Therapeutic Irrigation; D014621:Vagina / Q000737:chemistry
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D000331:Aequorin; D009195:Peroxidase; D004076:Digoxigenin