Multiplex PCR for the simultaneous detection of porcine reproductive and respiratory syndrome virus,classical swine fever virus,and porcine circovirus in pigs

A multiplex polymerase chain reaction (PCR) was designed for the simultaneous detection of three viruses involved in reproductive and respiratory failure in pigs: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV-2). Each target produced a specific amplicon with a size of 718 bp (PRRSV), 288 bp (CSFV), or 466 bp (PCV-2). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 2.0 × 10⁴, 2.5 × 10³, and 6.0 × 10² copies for PRRSV, CSFV, and PCV-2, respectively. Non-specific reactions were not observed when other viruses, bacteria, and PK-15/Marc-145 cells were used to assess the multiplex PCR. Among 82 clinical samples from Fujian province, co-infection by PRRSV and CSFV was 12.19%, co-infection by PRRSV and PCV-2 was 21.95%, CSFV and PCV-2 was 13.41%, and co-infection by the three viruses was 3.66%. In conclusion, the multiplex PCR should be useful for routine molecular diagnosis and epidemiology. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens.