DETECTION OF SWINE VESICULAR DISEASE VIRUS-RNA BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

Two polymerase chain reaction (PCR) assays are described for the detec tion of swine vesicular disease virus (SVDV) RNA, a reverse transcript ion PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR). Bot h the RT-PCR and RT-nPCR were able to detect representative members of each of seven phylogenetically distinct groups of SVDV and gave negat ive results with a range of porcine enteroviruses and of viruses respo nsible for vesicular conditions in pigs. When combined with a commerci al kit for rapid RNA extraction, the RT-PCR was useful for the detecti on of SVDV in samples of epithelium and faeces from animals with clini cal SVD. The addition of a second amplification step to create a neste d PCR (RT-nPCR) increased the sensitivity of the technique for the det ection of viral RNA (vRNA) in SVDV infected tissue culture fluid by a factor of approximately 1000, from 100 TCID50 for the RT-PCR to 0.1 TC ID50 for RT-nPCR. When combined with a more elaborate extraction proce dure for RNA, the RT-nPCR was considerably more sensitive than virus i solation in tissue culture for detecting SVDV in nasal swabs, tissues, and faeces collected from pigs between 7 days and 176 days after infe ction with a recent European isolate of SVDV. However, stringent condi tions are necessary for carrying out the RT-nPCR to minimise the possi bility of contamination. (C) 1997 Elsevier Science B.V.