A multiplex RT-PCR for rapid and simultaneous detection of porcine teschovirus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex RT-PCR (mRT-PCR) assay was developed and evaluated for its ability to detect multiple viral infections of swine simultaneously. One pair of primers was selected carefully for each of the following three RNA viruses: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine teschovirus (PTV). Each target produced a specific amplicon with a size of 451 bp (PRRSV), 343 bp (CSFV), or 163 bp (PTV). The sensitivity of the mRT-PCR using purified plasmid constructs containing the specific viral target fragments was 2.02 × 102, 2.90 × 103, and 6.16 × 103 copies for PRRSV, CSFV, and PTV, respectively. Among 69 clinical samples from Heilongjiang, Jilin, and Henan provinces, co-infection by PRRSV and CSFV was 4.4%, co-infection by PRRSV and PTV was 11.6%, co-infection by PTV and CSFV was 13.0%, and co-infection by the three viruses was 8.7%. In conclusion, the mRT-PCR should be useful for routine molecular diagnosis and epidemiology.