Laboratory Detection of Respiratory Viruses by Automated Techniques

Pérez-Ruiz, Mercedes; Pedrosa-Corral, Irene; Sanbonmatsu-Gámez, Sara; Navarro-Marí, José-María

Real-time PCR using dual labelled probes such as Taqman ® probes is widely used by virologists in their "in house" protocols, since it is easy to optimize, and despite the higher costs compared to conventional PCR protocols, the reduction in hands-on-time and the high efficiency compensates favourably the use of this approach. Common PCR parameters of 45 cycles of denaturation at 95ºC for 30 s and annealing and extension at 60ºC for 60 s, can be applied to any real-time assay [36] . This protocol is carried out in our laboratory for any RNA virus, including RV such as Flu, RSV, HRV, hMPV, hBoV and HCoV, by using the same instrument and the same protocol (personal communication). The technical disadvantage of real-time PCR is the limited number of fluorophores that can be included in a single assay, thus, limiting the multiplexing capabilities. In the best case, 3 targets can be detected in a single tube. A published protocol is able to detect 12 RV by a multiplex real-time PCR, including 4 tubes per sample and 3 viral targets per tube [37] .