Development of real-time fluorescent quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus variant strains

To identify highly pathogenic porcine reproductive and respiratory syndrome virus(PRRSV) variants rapidly using real-time PCR technique,primers and TaqMan probe for specific PCR amplification were designed based on sequence differences between variant strains and classical strains.The fluorescent quantitative RT-PCR assay for detection of PRRSV variants was established.Its specificity,sensitivity and stability were examined.This assay had differentiate PRRSV variants.1 TCID50 of template RNA could be detected.The sensitivity of this assay was 100 times higher than the conventional RT-PCR.Content could be absolutely quantified of virus.The variation coefficients were less than 10% based on 4 replications of each sample.13 lung samples were examined by the developed fluorescent quantitative RT-PCR and the conventional PCR,respectively,and 11 were detected to be infected with PRRSV by the real-time PCR,but only 9 were confirmed to be infected with PRRSV by the conventional PCR.These results showed that the developed fluorescent quantitative RT-PCR assay was rapid,specific,sensitive and simple for the detection of PRRSV variants and the identification of highly pathogenic PRRSV.