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

Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.

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

Clin Chem. 1996 May;42(5):696-703. Unique Identifier : AIDSLINE MED/96211420
Gerard B; Peponnet C; Brunie G; Cave H; Denamur E; d'Auriol L; Monplaisir N; Simon F; Elion J; Grandchamp B; Service de Biochimie Genetique, Hopital Robert Debre, Paris,; France.

Abstract: 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.

Keywords: Acquired Immunodeficiency Syndrome/*DIAGNOSIS/VIROLOGY Base Sequence Comparative Study *DNA Primers DNA, Viral/*ANALYSIS False Negative Reactions Female Human HIV-1/*GENETICS Infant, Newborn *Inosine Molecular Sequence Data Nucleic Acid Hybridization Oligonucleotide Probes Polymerase Chain Reaction Spectrometry, Fluorescence JOURNAL ARTICLE
961030
M96A1383

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