B. Gerard et al., FLUOROMETRIC DETECTION OF HIV-1 GENOME THROUGH USE OF AN INTERNAL CONTROL, INOSINE-SUBSTITUTED PRIMERS, AND MICROTITER PLATE FORMAT, Clinical chemistry, 42(5), 1996, pp. 696-703
We describe a PCR-based fluorometric assay for the detection of the HI
V-1 genome, This technique consists of a reverse hybridization with ol
igonucleotide probes covalently coated onto a microtiter plate as a so
lid support, Several improvements to the PCR amplification and detecti
on steps gave greater sensitivity and specificity for HIV-1 screening
and resulted in a convenient and rapid technique. False-positive resul
ts were avoided by using uracyl DNA glycosylase. False-negative result
s 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 a
nd by incorporating inosine at the primer positions corresponding to v
iral polymorphic nucleotides. Furthermore, the hybridization buffer an
d enzymatic reaction were optimized to increase the assay's sensitivit
y. The sensitivity and specificity of the fluorometric detection were
similar to those of radioisotopic oligonucleotide solution hybridizati
on; however, hands-on time was reduced, and the use of radioactivity w
as eliminated, We have used this technique routinely on 115 samples an
d obtained 100% specificity and high sensitivity (only one false-negat
ive result) according to viral culture and (or) serological status of
the patients.