Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction.
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 TCID50/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 (R2 = 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.
Shin
J
J
Department of Clinical and Population Sciences, University of Minnesota, St. Paul 55108, USA.
Bautista
E M
EM
Kang
Y B
YB
Molitor
T W
TW
D000818:Animals; D002138:Calibration; D002460:Cell Line; D018628:Gene Dosage; D006146:Guanidines; D016133:Polymerase Chain Reaction / Q000379:methods*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*; D012661:Semen / Q000821:virology*; D012822:Silicon Dioxide; D013552:Swine; D013861:Thiocyanates; D014158:Transcription, Genetic; D014771:Virion
D006146:Guanidines; D012367:RNA, Viral; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012822:Silicon Dioxide