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.

•A one-tube EvaGreen-based multiplex real-time PCR by using melting curve analysis was developed to simultaneously distinguish between six swine viruses.•The assay is specific, sensitive and highly reproducible.•The system revealed higher accuracy than conventional PCR.•This method is an alternative rapid, reliable and cost–effective method for routine surveillance of viral infections in pigs.