Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is in boar semen for extended periods of time as determined by reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. The concentration of PRRSV RNA in semen and the biological significance of the detection level, however, remain to be resolved. In order to determine the concentration of PRRSV VR-2332 (a prototypic strain of North American isolates) in semen following infection, we established a ‘standard curve’-quantitative competitive (SC-QC)-RT-nPCR assay as well as an equimolar QC-RT-nPCR assay. A deletion-type competitor RNA derived from the Lelystad virus, a European strain of PRRSV, ORF-7 gene standard which shares the nested sets of primer recognition sequences with the VR-2332 ORF-7 gene was used as an internal standard. The equimolar QC-RT-nPCR assay results revealed that the number of copies of PRRSV RNA in 1 TCID 50 /ml of virus derived from CL-2621 cell culture supernatants varied depending upon the type of samples in which virus was added; 143±24.0 and 266.5±48.5 copies in serum and semen samples spiked with PRRSV VR-2332, respectively. For the establishment of SC-QC-RT-nPCR assay, a standard curve was generated from band intensity ratios versus a series of known initial numbers of wild-type RNA copies which were quantified by the equimolar QC-RT-nPCR assay. Various initial numbers of copies of wild-type PRRSV RNA and each band intensity ratio with 1000 copies of competitor RNA were well correlated within the range of 100 to 200 000 copies ( R 2 =0.947). A ‘standard curve’ quantitation assay using competitive single-tube RT-nPCR will offer a rapid and reliable way to quantify low concentrations of PRRSV RNA in semen.