Scientific Journal of

  Abstract

 Background and Objectives

 Viral 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 Methods

 11-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.

 Results

 The 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.

  Abstract

 Background and Objectives

 Hepatitis C is the most common viral disease among intravenous drug users transmitted mainly through blood transfusion. In this study, we used an isothermal nucleic acid sequence-based amplification (NASBA) in combination with a molecular beacon probe-based real-time assay for detection of HCV.

 Materials and Methods

 In this experimental study, the conserved 5’NCR region with the length of 241 bp was used for probe and primers design. In comparison with the standard, RNA virus detection sensitivity of the method was 100 percent for up to 500 copies/ ml.

 Results

 No serological positive sample was detected with this assay the clinical sensitivity of reaction was 96.6%. NCBI Nucleotide Blast showed 100% analytical specificity and no cross was observed with viruses or human genome. Investigation of 10 serological negative samples showed the clinical specificity to be 100%.

 Conclusions

 In summary, due to its isothermal nature, its speed, and its use for RNA specific amplification, NASBA real-time assay will have broad applications for the rapid detection of HCV in plasma samples.