Development of a novel hot-start multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and porcine parvovirus.

A novel hot-start multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness in simultaneously detecting multiple viral infections of swine. Specific primers for each of five virus genomes, namely classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) were used. Combined nucleic acid purification was carried out using a commercial RNA/DNA extraction kit. The mPCR consisted of a two-step procedure which included reverse transcription and PCR amplification. This mPCR and the corresponding separate assays were evaluated comparatively on serial ten-fold dilutions of each virus. Analysis of the sensitivity in comparison to the corresponding single PCR (sPCR) for the detection of each of the five targets was identical for CSFV, PCV2 and PPV, 1 log lower for PRRSV and 2 logs lower for ASFV. No spurious PCR amplification reactions among all five pathogens were noticed with various amounts of DNA and RNA mixtures. All the uninfected controls were scored negative. The relative efficiency of the mPCR developed in this study compared to performing sPCR for each virus, suggests its potential application for routine molecular diagnostic purposes.