文蛤内脏产气荚膜梭菌毒素基因的PCR检测  

Isolation and characterization of Clostridium perfringens from Meretrix meretrix Linnaeus

作　　者：蔡玉梅[1] 柴同杰[1] 段长民 黄丽波[1] 蔡春梅 王慧[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]德州市肿瘤医院,山东德州253000

出　　处：《水产学报》2009年第3期403-409,共7页Journal of Fisheries of China

基　　金：国家自然科学基金专项基金项目(30571381)

摘　　要：用细菌学方法分离并初步鉴定文蛤内脏中的产气荚膜梭菌，PCR方法检测分离菌株的α、β、ε、ι、β2和肠毒素等产气荚膜梭菌毒素基因,PCR扩增片段克隆后进行核苷酸序列测定，然后与GenBank相应毒素基因的核苷酸序列进行同源性比较；分离菌株α毒素全长基因克隆、测序后与不同来源分离株进行同源性比较；检测内脏产气荚膜梭菌阳性的蛤肉组织中的α毒素基因以判定食品安全性。结果表明，文蛤内脏产气荚膜梭菌分离率为71％，69％的分离菌株为C型，31％的为A型，两种毒素型均能扩增出特异性β2条带及肠毒素条带，检出基因与GenBank相应毒素基因的同源性在98％以上；蛤肉组织α毒素基因检测结果为阴性。文蛤内脏分离菌株α毒素全长基因与不同来源分离株的同源性为98.64％～99.58％。本研究首次在文蛤内脏检出产气荚膜梭菌的毒素基因，对水产动物产气荚膜梭菌的进一步研究及人类的食品安全有一定的指导意义。To study the isolation rate and toxin type of Clostridium perfringens in fresh Meretrix meretrix Linnaeus, one hundred gut samples of Meretrix meretrix Linnaeus bought from market were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction （PCR） for genes encoding the four lethal toxins （α, β, ε and ι） for classification into toxin type and for genes encoding enterotoxin and the novel β2 toxin for further subclassification. The amplified complete α toxin gene was cloned and sequenced and compared with strains isolated from other living environment. Then, alpha toxin gene detected from Meretrix meretrix Linnaeus muscle （C. perfringens positive in gut）was performed for food safety assessing. The results indicated that C. perfringens could be isolated in 71 gut samples （71%） from Meretrix meretrix Linnaeus, 49 strains （69%） were C. perfringens toxin type C （α and β toxin positive）, 22 strains （31%） were toxin type A （α toxin positive）. In addition, genes encoding for β2 toxin and enterotoxin were found in the both toxin types. These amplified toxin gene fragments were cloned and sequenced and compared with the corresponding genes in GenBank, and the identity exceeded 98%. The homology of complete α toxin gene was 98.64%-99.58% when compared with strains isolated from other living environment. This is the first report of C. perfringens toxin genes in Meretrix meretrix Linnaeus in general, the origin of this bacterium and its importance to human food poisoning are discussed.

关 键 词：文蛤 产气荚膜梭菌 毒素基因 核苷酸序列 

分 类 号：Q523[生物学—生物化学] S917[农业科学—水产科学]