Study On Oligonucleotide Chip For Detecting FMDV And Preliminary Development Of 3B-ELISA For Differentiating Infection From Vaccination Against FMD

Foot-and-mouth disease virus (FMDV) is the most economically important veterinary pathogen in the world. It ranks first in the A list animal of infectious diseases accroding to Office International des Epizooties (OIE) .The crucial steps to control and prevent FMD are to develop a sensitive diagnostic tests and an effective vaccine. Traditional methods include virus isolation, infection of animal model. DNA chip is a new technology that has been used in microbiological research. The more accurate detection results could be achieved in less time than tradition methods, allowing its application in FMDV detection. Moreover, to differ the vaccinated animals from the naturally infected ones becomes a trend in the clinical diagnose as it will facilitate the control of FMD. Therefore, we carried out the following innovative explorations.1. Development of an oligonucleotide chip for detection of FMDVWe have developed a low-density DNA array for detecting and genotyping of foot-and-mouth disease virus (FMDV). Six species-specific probes and twelve topotype probes of FMDV type O were designed according to the published sequences of FMDV using DNAman5.2.2 and Primer Premier5.0 software. Oligonucleotides containing 12 T as spacers were synthesized and were spotted on to specially treated glass slides. The extracted RNA from FMDV infected cells were reverse transcripted, followed by PCR amplification and labeling with fluorescent dyes Cy3. The labeled PCR products were subjected to specific hybridization and subsequently photoscanned using optimal light intensity. The results of hybridization were approximately coincident with those predicted using DNAman5.2.2 software. These data showed that the oligonucleotide chip-based detection of FMDV was developed.2. Expression of the non-structural protein 3B gene of FMDV anddevelopment of 3B-based ELISAcDNA of 3B gene was synthesized using template RNA extracted from cells infected with FMDV. A size of 230bp of FMDV 3B gene was amplified and subcloned into the expression vector, named as pGEX-KG-3B, followed by transformation of E.coli and identification. The 3B protein expression was induced with IPTG at 27℃ and was analyzed by SDS-PAGE and protein dot blotting. The results showed the successful expression of 3B gene in E.coli and possessed biological activity. The 3B-ELISA was developed using the purified fusion protein and was optimized. The cut-off value was determined by testing 36 FMDV antibody free sera. The 3B-ELISA is of good stabilityand specificity. Comparison between 3B-ELISA with UBI? FMDV NS—ELISA Kit (United Biomedical Inc. UBI) demonstrated a good agreement. The clinical diagnostic potential was evaluated as well.In brief, the thesis describes two new methods for etiological and serological detection of FMDV which have good potential of clinical application, paving the way for prevention and control of FMD, a highly contagious disease in the globe.