A RT-PCR ASSAY FOR THE DIFFERENTIAL-DIAGNOSIS OF VESICULAR VIRAL DISEASES OF SWINE

A RT-PCR assay based on specific amplification of RNA sequences from e ach of the etiological agents of three important vesicular diseases th at affect swine, foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), was develo ped. Genotype-specific primers that amplified DNA fragments of differe ntial size from SVDV 3D gene or VSV L gene were selected with the aid of a computer program. Experimental testing of the primers predicted a s SVDV-specific identified a primer pair, SA2/SS4, that rendered a spe cific product from SVDV RNAs, but did not amplify RNA from either FMDV or coxsackie B5 virus (CV-BS), a highly related picornavirus. Primers SA2/SS4 were used in combination with primers 3D2/3D1, which amplify a product of different size on FMDV 3D gene (Rodriguez et al., 1992). This combined RT-PCR reaction allowed a sensitive and specific differe ntial detection of FMDV and SVDV RNAs in a single tube, by means of th e analysis of the amplified products in agarose gels. The results obta ined were similar when RNA extracted from viral stocks or plastic well s coated with either viral supernatants or extracts from lesions of in fected animals, were used as starting material in the reactions. Using a similar approach, VSV serotype-specific primers IA/IS and NA/NS wer e selected for the specific amplification of VSV-Indiana and VSV-New J ersey RNAs, respectively. The combined use of SVDV, FMDV and VSV speci fic primers in a single reaction resulted in a genotype-specific ampli fication of each of the viral RNAs. Thus, differential diagnosis of FM DV from SVDV and/or VSV can be carried out in a single RT-PCR reaction , using a rapid and simplified methodology. (C) 1998 Elsevier Science B.V. All rights reserved.