The use of molecular biology techniques for the diagnosis and epidemiological study of foot-and-mouth disease virus in Thailand

The detection of foot-and-mouth disease (FMD) virus from various kinds of field samples (tissue extract and cell culture isolate) was studied using the polymerase chain reaction (PCR) technique. The gene selected for diagnosis was the polymerase gene and an amplification target product of 454 bp in length was produced using AP5/AP6 primer sets. The PCR product was further examined by NcoI endonuclease digestion. The presence of the internal restriction site was confirmed by demonstration of two small fragments of 330 bp and 124 bp in length. Forty-nine samples that gave positive and negative results by ELISA typing and were positive by the PCR test were tested by NcoI digestion to confirm the results. About 10% of PCR products could not be confirmed by the method. Furthermore the FMD RNA polymerase gene could be detected by the PCR method in samples negative in both ELISA typing and the virus isolation test. A total of 23 samples were examined and compared after each stage of the testing process. At the end of the extraction for ELISA the amplification product band at 454 bp was detected in 74% of the negative tissue extract samples, and in 48% at the end of the virus isolation procedure. The PCR technique was shown to rapidly and sensitively detect FMD viral genome, when compared with virus titration by tissue culture infectious dose 50% (TCID{sub 50}) method. The PCR was about 10 times more sensitive than the virus titration technique in detection of virus. Therefore, the PCR technique can be used in conjunction with current procedures for FMD diagnosis, to support the routine standard ELISA typing and virus isolation test on clinical samples. The first step of the nucleotide sequencing technique was introduced with a view to study genomic differentiation of FMD outbreak viruses. The appropriate primer sets for each of the three endemic sero-types were optimized and used to detect the PCR products from field isolate viruses. The PCR products of FMDV type O, A and Asia I showed a clear band at 720 bp, 814 bp and 914 bp respectively. This work showed that the technique could be introduced to perform the nucleotide sequencing to support epidemiological investigations. (author)