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 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.
Gérard
B
B
Service de Biochimie Génétique, Hôpital Robert Debré, Paris, France.
Peponnet
C
C
Brunie
G
G
Cavé
H
H
Denamur
E
E
d'Auriol
L
L
Monplaisir
N
N
Simon
F
F
Elion
J
J
Grandchamp
B
B
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis* / Q000821:virology; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D005188:False Negative Reactions; D005260:Female; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D007288:Inosine; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D007288:Inosine