Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection.
A highly sensitive detection of swine vesicular disease virus (SVDV) based on a single tube RT-PCR system and digoxigenin (DIG)-PCR-ELISA detection was developed. Using a one tube RT-PCR system, optimisation of the PCR conditions and optimisation of the microwell hybridisation and colourimetric detection of the amplicons resulted in a method that could detect viral RNA in infected tissue culture fluid with a titre as low as 0.1 TCID50/100 microl. The same sensitivity was obtained with SVDV-spiked faeces, if the samples were pre-treated with 1,1,2-trichlorotrifluoroethane/chloroform and subsequently concentrated using an ultrafiltration system and RNA extracted with the Purescript kit. The specificity of the test was validated on 27 SVDV strains belonging to four different groups. No cross-reactivity with genetically and symptomatically related viruses was detected using RNA of foot-and-mouth disease virus (FMDV), porcine enterovirus (PEV), vesicular stomatitis virus (VSV), Coxsackie B5 virus (CV-B5) and encephalomyocarditis virus (EMCV). The test was validated successfully on clinical samples, being slightly more sensitive and much faster than virus isolation on cell cultures. Moreover the possibility of automating the procedure will allow the processing of large numbers of clinical samples.
Veterinary and Agrochemical Research Centre, Brussels (Ukkel), Belgium.
M
M
Callens
K
K
De Clercq
D000818:Animals; D002478:Cells, Cultured; D015342:DNA Probes; D004076:Digoxigenin; D004770:Enterovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004847:Epithelial Cells; D005243:Feces / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013555:Swine Vesicular Disease / Q000175:diagnosis* / Q000821:virology; D014776:Virus Cultivation
D015342:DNA Probes; D004076:Digoxigenin