Rapid detection of a highly virulent Chinese-type isolate of Porcine Reproductive and Respiratory Syndrome Virus by real-time reverse transcriptase PCR.

ABSTRACT

An outbreak of highly virulent Chinese-type of Porcine Reproductive and Respiratory Syndrome Virus (H-PRRSV) in most areas of China recently has led to huge economic losses and drawn great attention to its diagnosis and disease control. To facilitate rapid identification of H-PRRSV, a fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase PCR for H-PRRSV has been developed. Primers and probe specificity were evaluated with RNA extracted from 5 strains of H-PRRSV and 24 strains of other viruses, the results showed 100% specificity for the selected panel. The assay met the sensitivity of 1 50% tissue culture infective dose (TCID50) per ml of samples from infected pigs. Analysis with 10(5)-1TCID50/ml H-PRRSV samples demonstrated high reproducibility with a coefficient of variation (CV) of 0.5-2.5%. More than two hundred samples from lung, spleen, blood serum specimens obtained from 22 outbreaks of suspected H-PRRS from March to June in 2007 were verified using this assay. The results showed that 68.5% (146 out of 213) of these samples were positive which is 100% consistent with that of the sequencing method. The assay can be performed in less than 3h and thus provide a rapid method for the diagnosis of H-PRRSV as well as for elucidation of the epidemiology of H-PRRSV infections.