QUANTITATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 DNA BY 2 PCR PROCEDURES COUPLED WITH ENZYME-LINKED OLIGOSORBENT ASSAY

Two quantitative PCR methods with our nonisotopic enzyme-linked oligos orbent assay (ELOSA) in microtiter plate format were developed for qua ntitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild -type nef region with a mimic competitive nef gene template carrying m utations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy nu mbers in the range of 20 to 2,000 copies per mu g of DNA. Internally c ontrolled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to kn own amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 cop y numbers in the range of 10 to 2,000 copies per mu g of DNA. QC- and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC- and IC-PCR-ELOSA were identi cal for 77% of patient samples. The copy level ranged between 443 +/- 156 and 21,453 +/- 13,511 copies per 10(5) CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC- and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.