聚合酶链式反应快速检测E型肉毒神经毒素基因

Rapid detection of type E botulinal neurotoxin gene by polymerase chain reaction

目的Ｅ型肉毒中毒是人类肉毒中毒的主要型别之一，本试验旨在建立用于Ｅ型肉毒梭菌鉴定的ＰＣＲ方法。方法用人工合成的寡核苷酸引物扩增Ｅ型肉毒神经毒素基因的一段３６８ｂｐ的ＤＮＡ片段，快速检测Ｅ型肉毒神经毒素基因，对梭状芽胞杆菌属的４０株保藏菌株及３３份土壤标本进行了鉴定，用Ｅ型肉毒梭菌ＣＭＣＣ（Ｂ）６４５０１对其灵敏度进行了检查。结果从所有Ｅ型神经毒素原性菌株或标本均能扩增出目的片段，且能用特定的限制性内切酶切成相应的片段。其它菌株均未能扩增出目的片段。灵敏度试验可从３０个菌体扩增出目的片段。结论此方法用于Ｅ型神经毒素原性梭菌的鉴定具有较高的灵敏度及特异性。

Objective Type E botulism is one of main types of botulism in human beings.A polymerase chain reaction (PCR) method was established for rapid detection of type E botulinal neurotoxin gene and identification of Clostridium botulinum type E. Methods The PCR was developed by using a pair of oligonucleotide primers which were designed to amplify a fragment of 368bp on the heavy chain of type E neurotoxin gene.Forty strains belonging to 10 clostridial species and thirty three soil samples were detected using the PCR.The sensitivity of the PCR was determined by using Clostridium botulinum strain CMCC(B)64501. Results All of type E neurotoxigenic strains and samples whose cultures containing type E neurotoxin were positive but the others were negative.The restriction endonuclease digestion patterns of amplified products were in conformity with the sizes estimated from the sequence data.In test of sensitivity,target DNA fragment could be amplified from 30 CFU by the PCR. Conclusion The PCR is specific and sensitive for detection and identification of type E neurotoxigenic clostridial species.