Influence of PCR parameters on amplifications of HIV-1 DNA: establishment of limiting sensitivity. NLM AIDSLINE Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.

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Influence of PCR parameters on amplifications of HIV-1 DNA: establishment of limiting sensitivity.

Biotechniques. 1991 Sep;11(3):384-6, 388-91. Unique Identifier : AIDSLINE MED/92030127
Varas F; Medrano L; Ballester S; Najera R; Subdireccion General de Investigacion, Instituto de Salud; Carlos III, Madrid, Spain.

Abstract: We have investigated the optimal reaction conditions and the limiting sensitivity for detection of HIV-1 DNA by PCR. The amplification systems studied were gag (SK38/SK39); pol (P3/P4); and two other systems described here for the first time, LTR (LTR1/LTR2) and nef (Nef1/Nef2), which amplify fragments of 115 bp, 308 bp, 632 bp and 643 bp, respectively. Two PCR profiles were assayed, and the requirements for deoxynucleoside triphosphate and MgCl2 concentrations for each amplification reaction were determined. Optimal reaction conditions were oriented toward selecting maximal amplification of the expected size fragment. Limiting sensitivity was estimated by testing the decreasing copy number of a plasmid containing HIV-1 genome and obtaining a positive amplification signal with at least 5, 5, 10 and 5 copies for LTR, gag, pol and nef, respectively. We conclude that the establishment of the detection sensitivity on a PCR is an important parameter to be considered for the interpretation of results on HIV-1 infection.
Keywords: Base Sequence DNA, Viral/*ANALYSIS Genes, Viral HIV Long Terminal Repeat/GENETICS HIV-1/*GENETICS Magnesium Chloride Molecular Sequence Data Nucleotides Polymerase Chain Reaction/*METHODS Sensitivity and Specificity Support, Non-U.S. Gov't JOURNAL ARTICLEKWDbasesequencedna,viral/KWDanalysisgenes,viralhivlongterminalrepeat/geneticshiv-1/KWDgeneticsmagnesiumchloridemolecularsequencedatanucleotidespolymerasechainreaction/KWDmethodssensitivityandspecificitysupport,non-uKWDsKWDgov'tjournalarticle
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