Establishment and Application of Duplex RT-PCR Assay for Detection of Highly and Lowly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

A duplex RT-PCR for detection highly and lowly pathogenic porcine reproductive and respiratory syndrome virus(HP/LP-PRRSV) was developed using the primers designed basing on the Nsp2 gene coding the non-structure protein 2 of HP-PRRSV and LP-PRRSV,respectively.The total RNA of standard HP-PRRSV and LP-PRRSV strains was used as the positive control to establish the duplex RT-PCR assay.The sensitivity,specificity and repetition assay of duplex RT-PCR were tested,and samples taken from clinic suspicious PRRSV infected pigs had been testified by the established duplex RT-PCR assay.The results indicated that the duplex RT-PCR assay was successfully established.The specificity and sensitivity of the duplex RT-PCR assay revealed that the threshold of duplex RT-PCR was 100 copies/μL of HP-PRRSV or LP-PRRSV,and no products were amplified from the nucleic acid of Marc-145 cell or the other 8 kinds of pathogenic viral or bacterial microorganism acting as the negative control.The repetition test indicated that the duplex PCR was repeatible.21 were PRRSV positive detected from 25 clinic suspicious PRRSV infected pigs,while 20 and 4 were HP-PRRSV and LP-PRRSV positive,respectively.The study suggested that the established duplex RT-PCR method was highly specific and sensitive,and suitable for clinic rapid identified diagnosing of HP-PRRSV and LP-PRRSV.