Establishment of a Quadruplex RT-qPCR Method for the Detection of All Lineages of PPRV

Peste des petits ruminants (PPR) is an infectious disease with high morbidity and mortality rates, and four distinct lineages have been discovered in different regions globally. In this study, a quadruplex RT-qPCR method capable of differentiating all four lineages of PPRV was established. By screening specific conserved regions across all viral genomes, we designed primers, as well as four TaqMan probes capable of distinguishing all lineages. The established method underwent validation of its relevant characteristics. The sensitivity of the detection method was determined by testing plasmid serial dilutions ranging from 10⁸ to 10⁰ copies/μL; results showed that the method could detect as few as 10 copies per microliter of PPRV. No cross-reactivity was observed among the four probes or with other common pathogens of goats and sheep. The coefficient of variation (CV) values for inter-assay and intra-assay repeatability of each probe were both below 2% (intra-assay: 0.11% to 0.98%; inter-assay: 0.18% to 1.95%), demonstrating excellent repeatability. Testing of 62 clinical samples also confirmed that the method could effectively detect and differentiate clinical samples of different PPR lineages. This method, for the first time, enabled the differentiation of all PPRV lineages in a single reaction, improving the detection efficiency of the PPR virus and providing robust technical support for the global PPR eradication program.