A Triplex Propidium Monoazide (PMA) qPCR Assay Enables Rapid Discrimination of Live Porcine Reproductive and Respiratory Syndrome Viruses

The devastating swine disease, porcine reproductive and respiratory syndrome (PRRS), can only be caused by live PRRS virus (PRRSV) infection. However, the most commonly used detection methods cannot discriminate PRRSV infectivity. Here we developed a triplex propidium monoazide (PMA) qPCR assay for differential detection of infectious PRRSV isolates (NADC34-like PRRSV-2, NADC30-like PRRSV-2, and HP-PRRSV-2) prevalent in China. First, the PRRSV inactivation strategy was selected by comparing distinct inactivation methods. Subsequently, we optimized PMA pretreatment parameters and concentrations of primers and probes. The triplex PMA-qPCR assay displayed favorable specificity, sensitivity, and reproducibility. Moreover, 452 clinical samples (environmental feces, lungs, lymph nodes (LNs), and sera) were submitted to differential detection by triplex qPCR and triplex PMA-qPCR assays. A total of 83 PRRSV-positive samples were detected by the triplex qPCR assay, including 25 NADC34-like, 48 NADC30-like, and 15HP-PRRSV-2-positive samples (two samples were coinfected by NADC34-like and NADC30-like PRRSV-2, while three samples were coinfected by NADC30-like and HP-PRRSV-2). Meanwhile, 65 samples were identified by the PMA-qPCR method, including 21 NADC34-like, 36 NADC30-like, and 9HP-PRRSV-2 positive samples (one sample was coinfected by NADC34-like and NADC30-like PRRSV-2). No PRRSV could be isolated from the 18 qPCR-positive but PMA-qPCR-negative samples. Overall, this study provides the first triplex PMA-qPCR assay for rapid discrimination of live PRRSV isolates in clinical samples, particularly in environmental feces.