Rapid detection and characterization of foot-and-mouth disease virus by restriction enzyme and nucleotide sequence analysis of PCR products

Abstract

Reverse transcription coupled with PCR was used for the detection of foot-and-mouth disease virus serotypes A, C, and O in organ extracts from experimentally infected cattle. Primers were selected from conserved sequences flanking the genome region coding for the major antigenic site of the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of the capsid is highly variable its coding sequence is considered to be the most appropriate for the characterization of virus isolates and, therefore, for the determination of the epidemiological relationships between viruses of the same serotype. For differentiation between serotypes and for detailed characterization of individual virus isolates restriction enzyme cleavage and nucleotide sequence analysis of the respective PCR products were carried out. In order to minimize the time required for sample preparation from clinical material, viral RNA was released from particles by heating the sample for 5 min at 90 degrees C. Finally, an air thermocycler was used, which allows performance of a PCR of 30 cycles in approximately 20 min. The results show that reverse transcription PCR followed by restriction enzyme analysis and/or nucleotide sequence analysis of the PCR products is useful for the rapid detection and differentiation of foot-and-mouth disease virus.