Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay.
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.
Mallet
F
F
Unité Mixte de Recherche 103 Centre National de la Recherche Scientifique-bioMérieux, Ecole Normale Supérieure de Lyon, France.
Hebrard
C
C
Livrozet
J M
JM
Lees
O
O
Tron
F
F
Touraine
J L
JL
Mandrand
B
B
D000163:Acquired Immunodeficiency Syndrome / Q000821:virology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D015754:Genes, nef; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011786:Quality Control; D015203:Reproducibility of Results
D017931:DNA Primers; D004279:DNA, Viral; D007202:Indicators and Reagents