产气荚膜梭菌肠毒素基因地高辛探针制备与检测方法建立的初步研究

Study on Digoxigenin-labeled DNA Probe and In Situ Hybridization Analysis For Detection of CP Enterofoxin Gene

产气荚膜梭菌（Ｃｌｏｓｔｒｉｄｉｕｍ ｐｅｒｆｒｉｎｇｅｎｓ，ＣＰ）是人类和家畜重要的厌氧性病原菌，产生肠毒素的ＣＰ是人类食物中毒病原菌。本研究用地高辛标记肠毒素基因制备探针，并建立了原位杂交检测方法，直接检测肠毒素基因（ｃｐｅ）。这是继ＰＣＲ检测方法后又一项特异性基因诊断技术。由于利用了光学显微镜观察结果，形态学上比较直观，增加了实验的准确性。与ＰＣＲ、乳胶凝集相比，原位杂交检测方法具有较高的敏感性和特异性，使用方便，在没有ＰＣＲ仪等设备的条件下，可以直接检测ｃｐｅ。

Clostridium perfringens CP with toxigenic pttential has been implicated as a causative agent of foodborne poisoning, gas gangrene, infectious diarrhea, and several veterinary diseases. In this study we used non-radioactive digoxigenin-labeled DNA probe specific for the enterotoxin gene and developed an in situ hybridization analysis for the detection of the gene.Compared with other methods, i. e.PCR for enterotoxin gene and Reverse Passive Latex Agglutination Test (PET-RPLA) for enterotoxin, it has shown high sensitivity and specificity for detection and also directly differ entiated enterotoxigenic strains of this bacterium and made the diagnosis at gene level.All the results could be identified under light microscope within a short time.