魏氏梭菌α-毒素基因探针的制备及应用

Preparation and Application of α- Toxin Gene Probe of Clostridium welchii

以A型魏氏梭菌贵州分离株为材料,提取染色体DNA,经PCR扩增和酚:氯仿抽提回收得到242bp的α 毒素基因部分片段,用地高辛标记制备了α 毒素基因探针。该探针不与埃希氏大肠杆菌、都柏林沙门氏菌、金黄色葡萄球菌、猪丹毒杆菌、蜡样芽胞杆菌及多杀性巴氏杆菌等细菌的DNA样品发生反应,只与魏氏梭菌DNA样品呈阳性反应,能检出300pg的DNA样品;应用探针对40株魏氏梭菌进行了Dot Blotting检测,结果与生化试验鉴定的符合率达到90%,表明该探针具有较高的特异性和灵敏度,可应用于临床上魏氏梭菌的检测。

The chromosomal DNA of Clostridium welchii type A was extracted,which was isolated from Guizhou province.A 242 bp DNA fragment containing part of α- toxin gene was amplified by PCR and recovered.The fragment was labeled with Digoxigenin(Dig) and the α- toxin gene probe was developed.This probe reacted only with DNA samples of Clostridium welchii and 300 pg chromosomal DNA could be detected in the experiment.No reaction was observed with DNA samples of E.coli,S.dublin,S.aureus,E.rhusiopathiae,B.cereus and P.multocide.Bot- blotting has been conducted to detect 40 strains of Clostridium welchii with this probe.The results showed that the conformity rate reaction 90% between Dot- blotting and biochemical reaction identification.It was demonstrated that this probe was of high specificity and sensitivity,and can be used for diagnosis of Clostridium welchii clinically.