Development of a Multiplex RT-qPCR Method for the Identification and Lineage Typing of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen that causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion of sows and the manifestation of respiratory diseases in piglets. PRRSV strains are categorized into two distinct genotypes: PRRSV-1 and PRRSV-2. PRRSV-2 can be further classified into several lineages, including sub-lineage 1.8 (NADC30-like), sub-lineage 1.5 (NADC34-like), lineage 8 (HP-PRRSV-like), lineage 5 (VR-2332-like), and lineage 3 (QYYZ-like), all of which are prevalent in China. In order to identify PRRSV-1 and PRRSV-2, two primer-probe combinations were designed, targeting the M gene. In order to further differentiate the five lineages of PRRSV-2, another five primer-probe combinations were designed, targeting the Nsp2 gene. A TaqMan-based multiplex RT-qPCR assay was subsequently developed, integrating the aforementioned seven sets into two primer pools. Following the optimization of primer concentration and annealing temperature, a comprehensive evaluation was conducted to assess the assay's amplification efficiency, specificity, repeatability, and sensitivity. The developed multiplex RT-qPCR method exhibited excellent repeatability, with coefficients of variation (CVs) less than 2.12%. The detection limits for all seven targets were found to be less than 5 copies/μL. Ultimately, the method was utilized for the detection of a total of 1009 clinical samples, with a PRRSV-positive rate of 7.63% (77/1009). Specifically, the reference method was utilized to further confirm the status of the 77 PRRSV-positive samples and another 27 samples suspected of PRRSV infection. The sensitivity of the method was 97.40% (75/77), and the specificity was 96.30% (26/27), resulting in an overall coincidence rate of 97.12% (101/104). All the PRRSV-positive samples were typed as NADC30-like strains, and the accuracy of this typing was further confirmed by Sanger sequencing. In conclusion, A one-step multiplex RT-qPCR method was successfully constructed, evaluated, and applied to detect clinical samples. The assay provides an easy-to-operate, time-saving, and highly efficient way for the quick identification of PRRSV and simultaneous detection of five PRRSV-2 lineages prevalent in China. The method could offer guidance for PRRSV prevention and control measures.