Development of NASBA-ELISA technique for detection of HIV-1 RNA

Background and ObjectivesViral RNA is the first blood marker which appears in HIV-1 infection. RT-PCR and NASBA are the commonly used techniques for amplification of RNA. NASBA technique provides more advantages over RT-PCR. In this study, an NASBA assay combined with an easy, sensitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of HIV-1 RNA.Materials and Methods11-dig-UTP was added to an NASBA reaction mixture and the RNA amplicons were labeled. Then, the dig-labeled NASBA products hybridized to a biotinylated specific probe in hybridization solution buffer and the hybrids were transferred to a streptoavidin-coated plate. After several washing processes and emission of nonspecific products, the final detection of the captured RNA was done by addition of anti-dig antibody-enzyme conjugate and the substrate. ResultsThe results demonstrated that, NASBA is an efficient method for amplification of the target genome. Detection of NASBA products by agarose gel electrophoresis showed a unique 176 bp band corresponding to the specific target. For the detection of NASBA products, an ELISA assay was performed; using 0.01 µM probe, the OD of 1.049 ± 0.11 was obtained Conclusions In this study, by using suitable primers and probe a highly sensitive and specific NASBA-ELISA method for detection of HIV-1 developed.