A Multiplex RT-PCR Assay to Detect and Discriminate Porcine Reproductive and Respiratory Syndrome Viruses in Clinical Specimens.

Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses in China. The attenuated vaccine (HP-PRRSV JXA1-R) was used to control HP-PRRSV. However, in recent years, co-infection with classical PRRSV (C-PRRSV), HP-PRRSV, and/or HP-PRRSV JXA1-R has been increasing in China, resulting in a significant impact on PRRSV diagnostics and management. To facilitate rapid discrimination of HP-PRRSV JXA1-R from HP-PRRSV and C-PRRSV, a multiplex RT-PCR assay for the visual detection of HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV was established and evaluated with reference PRRSV strains and clinical samples. Primer specificities were evaluated with RNA/DNA extracted from 10 viral strains, and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 24 copies/μL for PRRSVs. A total of 516 serum samples were identified, of which 12.21% (63/516) were HP-PRRSV-positive, 2.33% (12/516) were HP-PRRSV JXA1-R-positive, and 1.16% (6/516) were C-PRRSV-positive, respectively, which was completely consistent with the sequencing method. The high specificity, sensitivity, and reliability of the multiplex RT-PCR assay described in this study indicate that it is useful for the rapid and differential diagnosis of HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV.