[Detection of HIV-1 in plasma samples using microplate hybridization after RT-PCR]

We have developed a microplate hybridization(MH) technique, which utilizes the non-isotopic method of enzyme-linked assay for detection of HIV in the amplified product after PCR. HIV RNA extracted from plasma was amplified by RT-Nested-PCR using biotinylated-inner primers of gag, pol and env regions, respectively. The PCR product was visualized by 5% polyacrylamide gel electrophoresis and ethidium bromide staining. The heat-denatured PCR product was hybridized with HIVcDNA of each region which was immobilized to a microplate. The hybridized microplate was reacted with streptavidin-conjugate peroxidase and then the optical density(O.D.) was read at 490nm. The cut off value was determined at O.D. 0.25. The results of the electrophoresis of gag, pol and env regions were all positive in 53 HIV-1 seropositive samples from Japan and the USA, and all negative in 55 HIV-1 seronegative samples. Using the MH technique, USA samples showed a higher O.D. than the Japan samples, particularly in the pol region. The results of MH technique in gag and pol regions coincided with that of electrophoresis. But, one of 27 Japanese HIV-1 seropositive samples showed O.D. of less than 0.2 in only env region. This particular sample was classified by V3 peptide-based enzyme immunoassay as subtype E, which differs from the typical subtype B of Japan and USA samples. This suggests the presence of several genotypes in HIV-1 seropositive individuals in Japan. Based on this data, the MH technique using gag, pol and env region is a simple, sensitive, safe and specific assay for detection of HIV-1 RNA in plasma, and would be useful in clinical testing.