Real-Time PCR을 이용한 유럽 부저병(European foulbrood) 원인균 Melissococcus plutonious의 신속 검출

European foul brood (EFB) is a disease of larval honeybees caused by the bacterium Melissococcus plutonious. It is particularly difficult to isolate this bacterium due to its stringent anaerobic growth requirement and competition with other bacteria. EFB was related to Streptococcus apis, Paenibacillus alvei and Bacterium eurydice that were known as secondary invader bacteria. Bacillus subtilis may be easily contaminated in the infected or dead larvae. For the rapid identification of this pathogen, PCR primers were designed on the basis of the 16S rRNA gene of M. plutonious. The unique 564 bp fragment was amplified by PCR using specific primer-pairs (EFB-NSF/NSR). For the specificity of this PCR based detection, we tested with the artificial mixture of chromosomal DNAs of other bacteria, such as P. larvae, P. alvei, B. pulvifaciens, B. subtilis, and E. coil. These are phylogenetically closed to M. plutonious and commonly found in honey bee larvae. Optimal PCR was established by using gradient PCR and Real Time PCR.