Real-Time Quantitative Polymerase Chain Reaction for the Demonstration of Lyssavirus Nucleic Acid
Real-time PCRs have become a de facto standard for the molecular diagnosis of many infectious pathogens. As closed-tube systems, they reduce the possibility of amplicon or cross-contamination that may lead to false positive results. Besides the relatively higher analytical sensitivity in comparison to conventional reverse transcription-polymerase chain reaction (RT-PCR) protocols, the real-time PCR allows for a quantitation of the template RNA. Also, a generally smaller fragment size causes shorter times for the PCR, and without the need for a confirmation as in gel-based assays, the real-time PCR is faster in the turnaround time and allows for a higher throughput. Various assays have been described, and here we present a cascade-like approach whereby initially two independent pan-lyssa RT-PCRs using intercalating dye are applied. The confirmation and further specification can be made using hydrolysis probe-based assays that are specific to the respective lyssavirus species.