Comparison of competitive and positive control-based PCR quantitative procedures coupled with end point detection.
Two PCR methods using internal standards, coupled with our sandwich nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format, were developed for quantitation of human immunodeficiency virus type 1 (HIV-1) provirus. We present an overview of both methodologies focusing on two major features, i.e., the conditions of equivalency of replication efficiency and the definition of criteria of acceptance validating a result. Quantitative competitive PCR (QC-PCR) was based on the coamplification of the HIV-1 nef gene with different amounts of a pNEFmut plasmid that contains the nef gene with different amounts of a pNEFmut plasmid that contains the nef region but with mutations in the capture probe recognition region. The NEF wild-type (NEF) and the NEF mimic (NEFmut) amplification products were differentiated in ELOSA. NEFmut OD to NEF OD ratios were plotted against the number of mimic copies, and the deduced linear curve permitted quantitation of HIV-1 copy number. Internally controlled PCR (IC-PCR) was based on coamplification of the HIV-1 nef gene with an internal endogenous standard, the ras gene, as a positive control of amplification. HIV-1 copy number was determined using external standard of known amounts of HIV-1 DNA. We address the advantages as well as the limitations of individuals protocols and discuss future improvements of quantitative amplification process.
Mallet
F
F
UMR 21742 CNRS-bio Mérieux, Ecole Normale Supéricure de Lyon, France. fmallet@ens-bma.cnrs.fr
D004279:DNA, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015754:Genes, nef / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014779:Virus Replication
D004279:DNA, Viral