Rapid and sensitive polymerase chain reaction based detection and typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses

Abstract

Reverse transcription followed by the polymerase chain reaction method (PCR) allowed the detection of foot-and-mouth disease virus (FMDV), regardless of the serotype. A primer set corresponding to highly conserved regions of the 2B sequence was selected. By combining in a single reaction tube specific primer pairs for FMDV, swine vesicular disease virus, (SVDV), encephalomyocarditis virus (EMCV) and bovine viral diarrhea virus (BVDV), all four viruses could be identified and differentiated in one amplification reaction, based on the different lengths of the respective amplified segments. In a similar way, FMDV types O, A, C, SAT 2 and Asia 1 could be identified and differentiated, using primers selected from the ID (VP1) genome region. All results were confirmed by direct sequencing of the PCR product. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within three hours after samples (cell culture isolates as well as clinical material) arrived, which is of great importance in case of an FMDV suspicion. Furthermore, a very high sensitivity was achieved (less than one PFU). Therefore, our powerful detection assay by means of PCR for FMDV as well as for SVDV, EMCV and BVDV, has advantages compared to the presently used procedures.