Simultaneous detection and identification of HBV and HTLV-I viruses by Melting curve analysis ofmultiplex Real-time PCR

Background and Objectives:HBV and HTLV-I are life threatening infectious agents in patients who receive blood and blood products. Although serological methods have been proved to be useful, detection of these viruses has remained a challengingissue due to the many obstacles. By the advent of Nucleic Acid Testing methods, especially in multiplex format, more precise detection is possible.The objective of this study was to develop a reliable, rapid and cost- effective method tosimultaneously detect HBV and HTLV-I. Materials and Methods: We have developed a multiplex Real time-PCR assay for simultaneous detection of HBV and HTLV-I. Primer sets were designed for highly conserved regions of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex Real-time PCRs were performed. Results: Analytical sensitivity was considered to be 1000 and 100 copies/ml for HBV and HTLV-I, respectively. High concentration of one virus had no adverse effect on detection of t low concentrations of the other one. By analyzing 30 samples, clinical sensitivity of the assay was determined to be 87% and 96% for HBV and HTLV-I, respectively. Using different viral and human genome samples, the specificity of the assay was verified to be 100%. Conclusions:We have developed a reliable, rapid and cost effective method tosimultaneously detect HBV and HTLV-I.Our results indicatedthe high capability of this simple and rapid method for detecting these viruses in clinical samples.