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Journal of Clinical Microbiology, Dec 1995, 3201-3208, Vol 33, No. 12
Copyright © 1995 by the American Society for Microbiology. All rights reserved.

Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay

F Mallet, C Hebrard, JM Livrozet, O Lees, F Tron, JL Touraine and B Mandrand
Unite Mixte de Recherche 103 Centre National de la Recherche Scientifique-bioMerieux, Ecole Normale Superieure de Lyon, France.

Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation 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 mutations in the capture region. Correlation of wild-type HIV- 1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per micrograms of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC- PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per microgram 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 identical 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.

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