Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 4 2 2012 06 15 47 54 S Mohammadi-Yeganeh Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. AND Molecular Biology and Genetic Engineering Department, Stem Cell Technology Research Center, Tehran, Iran. M Paryan Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. AND Molecular Biology and Genetic Engineering Department, Stem Cell Technology Research Center, Tehran, Iran. S Mirab Samiee Day General Hospital Laboratory, Tehran, Iran. AND Food and Drug Laboratory Research Center, Ministry of Health and Medical Education, Tehran, Iran. V Kia Department of Medical Biotechnology, Tarbiat Modares University, Tehran, Iran. H Rezvan Day General Hospital Laboratory, Tehran, Iran 2015 10 16 Background and Objectives: Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. Materials and Methods: A well-conserved region in the HIV-1 pol gene and 5’-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. Results: The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was < 1000 copies/ml for HIV-1 and < 500copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. Conclusion: This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients. https://ijm.tums.ac.ir/index.php/ijm/article/view/712 https://ijm.tums.ac.ir/index.php/ijm/article/download/712/479