Abstract: A highly sensitive and specific duplex Real-time TaqMan MGB-fluorescent quantitative RT-PCR (FQ-PCR) assay had been developed to identify and quantitate the highly and lowly pathogenic porcine reproductive and respiratory syndrome virus (HP/LP-PRRSV) infection using PRRSV nonstructural 2 (Nsp2) gene-derived primers and TaqMan fluorescent probe. The sensitivity, specificity and repetition assays of duplex FQ-PCR were tested, and 25 clinic suspicious PRRSV infected samples were detected by the FQ-PCR in contrast to the routine duplex RT-PCR method. The results indicated FQ-PCR assay as well as the quantitative standard curves for HP/LP-PRRSV with good linearities (0.998 and 0.997,respectively) were successfully established. The developed FQ-PCR assay was able to detect as little as 10¹ copies/μL of recombinant plasmid DNA. The specificity assay exhibited that positive signals could be obtained from the HP/LP-PRRSV positive control, but not from the genomic DNA of the other 5 kinds of pathogenic microorganism as the control. The repetition test indicated that the FQ-PCR was reproducible by repeatedly amplifying 3 times of different concentrations of HP/LP-PRRSV recombinant plasmids. 92% positive results from 25 clinic suspicious PRRSV infected samples were obtained, which showed better sensitivity than the detection results of the same 25 suspected samples by duplex RT-PCR.

Key words: highly/lowly pathogenic porcine reproductive and respiratory syndrome virus; duplex Real-time fluorescent quantitative RT-PCR; TaqMan MGB fluorescence probe; detection; application