중합 효소 연쇄 반응을 이용한 탄저의 진단법 개발

Development of a new diagnostic method for anthrax using polymerase chain reaction

Anthrax is one of the most important zoonotic diseases in the worldwide. To control and prevent the disease effectively, a fast, specific and sensitive method has been required for diagnosis of the anthrax in both animals and human. Diagnosis of the disease had been carried out by several methods including identification of an etiological agent, serological methods. However, traditional methods had shown several problems in the confirmation of the disease, especially identification of Bacillus anthracis which is a causative agent of anthrax. Therefore, with polymerase chain reaction (PCR), this study had been carried out to develop a fast, specific and sensitive methods for identification of Bacillus anthracis by detection of genes encoding virulence factors of the bacteria. Therefore, we designed and synthesized primers on the genes encoding tripartite toxins (protective antigen, edema factor, and lethal factor) and capsule based on information from Genebank. Specificity of the methods was confirmed by comparison of the PCR results using B. anthracis reference strains, field isolates, and other Bacillus spp. The PCR method was very sensitive to detect up to 100 fg of genomic DNA as template. Identity of the PCR products was confirmed by comparison of patterns of restriction endonuclease analysis with HindⅢ, EcoRI, PstI, Sau3Al. To detect and identify B. anthracis effectively and simultaneously, Multiplex PCR was also established with primers on genes encoding lethal factor, protective antigen and capsule. Based on the results from this study, the PCR method could be used as a fast and specific diagnostic tool for anthrax.