Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection

• Published in: Journal of virological methods 1999, Vol.77 (1), p.87-99
• Main Authors: Callens, M, De Clercq, K
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cells, Cultured; Digoxigenin; DNA Probes; Enterovirus - genetics; Enterovirus - growth & development; Enterovirus - isolation & purification; Enzyme-Linked Immunosorbent Assay - methods; Epithelial Cells; Faecal samples; Feces - virology; Foot-and-mouth disease virus; Fundamental and applied biological sciences. Psychology; Microbiology; Reverse Transcriptase Polymerase Chain Reaction - methods; RT-PCR-ELISA detection; Sensitivity and Specificity; SVDV diagnosis; Swine; Swine Vesicular Disease - diagnosis; Swine Vesicular Disease - virology; Swine vesicular disease virus; Techniques used in virology; Virology; Virus Cultivation
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(98)00140-2

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 TCID 50/100 μl. 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.