Evaluation of RT-PCR procedures for diagnosis of clinical samples of foot-and-mouth disease virus (serotypes O, A, C and Asia 1) under the European Union Concerted Action Group Project Pl 98-4032

A collaborative study initiated from the first annual European Union (EU) Concerted Action Group meeting (project PL 98-4032, 5-7 May 1999, Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany) was organised between the Veterinary and Agrochemical Research Centre in Brussels, the Federal Research Centre in Tübingen and the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright. Each laboratory was supplied with 40 epithelium samples of FMD virus positive clinical material (ten each of the serotypes O, A, C and Asia 1) and the supernatant fluids resulting from their passage in cell culture to evaluate the sensitivity and specificity of locally employed RT-PCR procedures. The 40 samples were supplied unlabelled with respect to FMD virus serotype. In the WRL, FMD virus was detected (but not serotyped) in 36 of the 40 epithelial suspensions and in 39 of the 40 cell culture supernatant fluids by a diagnostic RT-PCR protocol using a universal O/A/C/Asia 1 primer set (1F/1R). These primers also detected 36 of the 40 epithelial suspensions in a prototype antigen capture (immunocapture) RT-PCR but variable results were achieved in a diagnostic RTPCR using serotype-specific primers. The latter primers were sensitive on the FMD virus type Asia 1 samples but less sensitive on virus samples of the other serotypes. The sensitivities of the universal O/A/C/Asia 1 primer set and the serotype-specific primers in the diagnostic RT-PCR mirrored results previously achieved on other virus samples while the antigen capture format was suitable for diagnosis of FMD virus and could be further developed for serotype-specific detection. FMD virus in all of the cell culture supernatants was detected by a universal primer/probe set for FMD using a quantitative PCR machine. The results will be compared with those from the other laboratories to evaluate and improve existing PCR protocols designed for FMD virus diagnosis.