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 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.