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 transcrip tion-nested polymerase chain reaction (RT-nPCR) assay. The concentrati on of PRRSV RNA in semen and the biological significance of the detect ion level, however, remain to be resolved. In order to determine the c oncentration of PRRSV VR-2332 (a prototypic strain of North American i solates) in semen following infection, we established a 'standard curv e'-quantitative competitive (SC-QC)-RT-nPCR assay as well as an equimo lar 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-233 2 ORF-7 gene was used as an internal standard. The equimolar QC-RT-nPC R assay results revealed that the number of copies of PRRSV RNA in 1 T CID50/ml of virus derived from CL-2621 cell culture supernatants varie d 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 a ssay, 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 10 00 copies of competitor RNA were well correlated within the range of 1 00 to 200000 copies (R-2 = 0.947). A 'standard curve' quantitation ass ay using competitive single-tube RT-nPCR will offer a rapid and reliab le way to quantify low concentrations of PRRSV RNA in semen. (C) 1998 Published by Elsevier Science B,V. All rights reserved.