Influence of PCR parameters on amplifications of HIV-1 DNA: establishment of limiting sensitivity.
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.
Subdirección General de Investigación, Instituto de Salud Carlos III, Madrid, Spain.
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Varas
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Medrano
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Ballester
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Nájera
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D005814:Genes, Viral; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D015636:Magnesium Chloride; D008969:Molecular Sequence Data; D009711:Nucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D009711:Nucleotides; D015636:Magnesium Chloride