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 m icroplate format for quantifying human immunodeficiency virus Type 1 ( HIV-1) DNA or RNA in a broad range of source materials. Our QC-PCR ass ay is a modification of technique originally described by Piatak et al . (1993), which is based on the presence of a competitive internal sta ndard containing an internal 80-bp deletion of HIV-1 gag target sequen ce. For improved detection and quantification of the wild-type and int ernal-standard PCR products in a microplate format, we introduced a no n-HIV, 31-bp insert into the internal standard as a probe hybridizatio n 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 wit h a digoxigenin (Dig)-labeled, wild-type or internal-standard probe. T he hybridized Dig-labeled probes ar detected with an anti-Dig antibody conjugated to detector molecules for luminometry (aequorin) or optica l densitometry (Peroxidase), yielding results that are quantifiable ov er the rang of 100-10 000 copies of HIV gag. Tested source materials f or HIV-1 DNA or RNA quantification include plasma, vaginal lavage and cultured cells. The application of the QC-PCR assay using the micropla te format affords a convenient and cost-effective method for quantifyi ng HIV-1 proviral and viral loads from a variety of body fluids, cells and tissues.