Fluorometric detection of HIV-1 genome through use of an internal control, inosine-substituted primers, and microtiter plate format

We describe a PCR-based fluorometric assay for the detection of the HIV-1 genome. This technique consists of a reverse hybridization with oligonucleotide probes covalently coated onto a microtiter plate as a solid support. Several improvements to the PCR amplification and detection steps gave greate...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 1996-05, Vol.42 (5), p.696-703
Main Authors: Gerard, B, Peponnet, C, Brunie, G, Cave, H, Denamur, E, d'Auriol, L, Monplaisir, N, Simon, F, Elion, J, Grandchamp, B
Format: Article
Language:English
Subjects:
Acquired Immunodeficiency Syndrome - diagnosis
Acquired Immunodeficiency Syndrome - virology
AIDS/HIV
Base Sequence
DNA Primers
DNA, Viral - analysis
False Negative Reactions
Female
HIV-1 - genetics
Humans
Infant, Newborn
Inosine
Molecular Sequence Data
Nucleic Acid Hybridization
Oligonucleotide Probes
Polymerase Chain Reaction
Spectrometry, Fluorescence
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Summary:We describe a PCR-based fluorometric assay for the detection of the HIV-1 genome. This technique consists of a reverse hybridization with oligonucleotide probes covalently coated onto a microtiter plate as a solid support. Several improvements to the PCR amplification and detection steps gave greater sensitivity and specificity for HIV-1 screening and resulted in a convenient and rapid technique. False-positive results were avoided by using uracyl DNA glycosylase. False-negative results from the presence of PCR inhibitors were detected by coamplifying an internal control with the viral sequence. False-negative results from viral genome variability were limited by using two pairs of primers and by incorporating inosine at the primer positions corresponding to viral polymorphic nucleotides. Furthermore, the hybridization buffer and enzymatic reaction were optimized to increase the assay's sensitivity. The sensitivity and specificity of the fluorometric detection were similar to those of radioisotopic oligonucleotide solution hybridization; however, hands-on time was reduced, and the use of radioactivity was eliminated. We have used this technique routinely on 115 samples and obtained 100% specificity and high sensitivity (only one false-negative result) according to viral culture and (or) serological status of the patients.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/42.5.696