Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction.
Two polymerase chain reaction (PCR) assays are described for the detection of swine vesicular disease virus (SVDV) RNA, a reverse transcription PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR). Both the RT-PCR and RT-nPCR were able to detect representative members of each of seven phylogenetically distinct groups of SVDV and gave negative results with a range of porcine enteroviruses and of viruses responsible for vesicular conditions in pigs. When combined with a commercial kit for rapid RNA extraction, the RT-PCR was useful for the detection of SVDV in samples of epithelium and faeces from animals with clinical SVD. The addition of a second amplification step to create a nested PCR (RT-nPCR) increased the sensitivity of the technique for the detection of viral RNA (vRNA) in SVDV infected tissue culture fluid by a factor of approximately 1,000, for 100 TCID50 for the RT-PCR to 0.1 TCID50 for RT-nPCR. When combined with a more elaborate extraction procedure for RNA, the RT-nPCR was considerably more sensitive than virus isolation in tissue culture for detecting SVDV in nasal swabs, tissues, and faeces collected from pigs between 7 days and 176 days after infection with a recent European isolate of SVDV. However, stringent conditions are necessary for carrying out the RT-nPCR to minimise the possibility of contamination.
Lin
F
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World Reference Laboratory for Foot-and-Mouth Disease, Pirbright Laboratory, Woking, Surrey, UK.
Mackay
D K
DK
Knowles
N J
NJ
D000818:Animals; D004769:Enterovirus Infections / Q000235:genetics* / Q000662:veterinary; D004771:Enteroviruses, Porcine / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D013552:Swine; D013555:Swine Vesicular Disease / Q000175:diagnosis / Q000235:genetics / Q000821:virology*
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase