Development of Multiplex, Real-time, One-step RTPCR Protocols for the Separate Detection of Selected Respiratory Viruses

Abstract

The contagiousness and severity of respiratory tract infections, which remain among the leading causes of morbidity and mortality in the Philippines, make their accurate and rapid diagnosis necessary. Moreover, protocols which may permit rapid surveillance of respiratory viruses in the Philippines are desirable for epidemiological research. Molecular methods for detection of respiratory viruses, such as traditional and real-time PCR, have a short turnaround time and are reported to have excellent specificity and sensitivity. It was the aim of this study to develop a multiplex, real-time, one-step RT-PCR protocol for the detection of Influenza Viruses A and B (IVA/B Multiplex), Respiratory Syncytial Viruses A and B (RSVA/B Multiplex), Parainfluenza Viruses 1 to 4 (PIV1/2 and PIV3/4 Multiplexes), as well as Adenovirus and SARS Coronavirus (AV/SARS-CoV Multiplex). Development involved the separate optimization of primer, probe and manganese acetate concentration for each of the reactions, and the validation of each of the multiplex assays. Viral samples for testing were not available for RSVB, PIV2, and PIV4; therefore, their monoplex reactions were not optimized. Consequently, for the RSVA/B Multiplex, the multiplex reaction was only optimized with the RSVA template. The PIV1/2 Multiplex and PIV3/4 Multiplex were designed to distinguish PIV1 and 2 group, and PIV3 and 4 group respectively without subtyping. The results showed that the multiplex reaction for the 1VAZB, RSVA/B and AV/SARS-CoV Multiplexes were valid. The PIV1 monoplex reaction was optimized but PIV1/2 Multiplex reaction yielded negative results. The PIV3 monoplex reaction yielded a negative result as well. The study showed that multiplex, real-time, one-step RT-PCR protocols for the detection of IVA and B, RSVA and B, and AV and SARS-CoV were feasible. It is recommended that further development of real-time PCR detection assays be undertaken with samples of RSVB and PIV2 to 4.