Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder.

Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/μL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs.