多重PCR方法快速检测粪样中产气荚膜梭菌

Multiplex PCR Assay for the Rapid Detection of Clostridium Perfringens in Feces of Pigs

建立了多重聚合酶链反应 (multiplexPCR)检测产气荚膜梭菌的α_毒素和肠毒素 (enterotoxin)基因。该方法可以检测猪粪便样品或小肠内容物中肠毒性产气荚膜梭菌。首先猪粪便样品经前处理后 ,接种选择性培养基 ,再挑取单个菌落溶于超纯水中 ,煮沸提抽 ,制备样品DNA ,进行PCR检测。从实验中我们成功的扩增出 32 4bp的α毒素基因片段和 2 33bp的肠毒素基因片段。以人工污染样品 10倍系例稀释后进行检测 ,该方法的检测灵敏度达到 1.0× 10 4 CFU/g并对 5 8份猪粪便样品检测 ,获得 5 3株产气荚膜梭菌 ,分离率为 91%,其中 4株为带有肠毒素的产气荚膜杆菌 ,占整个分离菌株的 7.5 %。因此多重PCR方法在检测粪样中的产气荚膜梭菌是一种非常有用的方法并可以快速检出肠毒素 ,从而避免了使用动物作定型试验。

A multiplex polymerase chain reaction(PCR)assay,developed to detect the alpha toxin and enterotoxin genes of Clostridium perfringens,was used to idientify enterotoxigenicisolates of this organism from feces and intestinal contents of pigs from pig farm.The organism was firstly grown on the Tsc_Agar.DNA was extracted from single colony on TSC_Agar by boiling and the PCR assay was Carried out using reagents from a commercial kit.The 324bp PCR product of CI.Pertringens a_toxin and the 233bp product of CI.Perfringens enterotoxin were achieved in this assay.The average sensitivity of assay,determined on artificially contaminated feces,was 1.0×10 4CFU/g. 53 CI.Perfringens isolates were obtained from 58 Samples of feces and intestinal contents,only 4 CI.Perfringens isolates were Contained enterotoxin,and about 7% of total isolates,We concluded that multiplex PCR is a useful assay for rapid detection of CI.Pertingens in feces and for confirmation of the identity of isolates presumed to be CI.Perfringens.