Optimization of multiplex real-time rt-pcr for respiratory syncytial viruses detection

Background: Multiplex real-time RT-PCR (rRT-PCR) is a fast, sensitive and specific test to detect more than one target in single PCR reaction. In this study we developed multiplex rRT-PCR for RSV-A and RSV-B since those viruses are the most common pathogen found in respiratory tract. However, in order to gain optimal reaction for RSV-A and RSV-B detection, the optimization of primers and probes specific for RSV-A and RSV B are needed. Method: The primers and probes of multiplex rRT-PCR for RSV-A and RSV-B were selected and optimized utilizing PerlPrimer software and BLAST to analyze the secondary structures and specificity, respectively. Further testing of selected primers and probes for rRT-PCR was done using annealing temperature based on in silico analysis as mentioned above. This includes sensitivity testing with the utilization of synthesized DNA of RSV-A and RSV-B and specificity testing targeting the common viruses found in respiratory tract. Results: The primer set and probes selected for RSV-A and RSV-B detection were specific only for RSV-A and RSV-B and showed no secondary structure. Based on primer and probe criteria for rRT-PCR such as annealing temperature, no secondary structure formed, % GC content and limit of detection, the multiplex rRT-PCR test using selected primers and probes was able to detect synthesized DNA of RSV-A and RSV-B. Conclusion: Multiplex rRT-PCR that employing primer sets and probes targeted N gene of RSV-A and RSV-B in this study were able to be detect RSV-A and RSV-B in single PCR reaction