A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses

Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.