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目的比较分析4种肉毒毒素(botulinum neurotoxins,Bo NT)基因分型方法,为四川省监测和食物中毒中肉毒梭菌(Clostridium botulinum)分型鉴定提供可靠的方法。方法使用本实验室保存的6株肉毒梭菌(包括A、B、E型)验证4种肉毒梭菌PCR基因分型鉴定方法——美国食品药品监督管理局(FDA)多重PCR分型方法、国际标准化组织(ISO)的两种多重PCR分型方法和一种实时荧光PCR分型方法,比较方法间的差异,并初步分析差异原因。结果 3种多重PCR方法均可在一个反应中同时检测A、B、E 3种型别肉毒梭菌。ISO多重PCR方法 1中A型检测虽能获得预期条带,但结果条带不清晰。其余两种多重PCR方法在分型检测肉毒梭菌时,可获得清晰的预期条带。实时荧光PCR分型方法能在多重反应体系中同时检测到不同型的肉毒梭菌,但由于荧光标记相同,要获得分型结果需要分别检测各毒素型别。结论美国FDA多重PCR方法和ISO多重PCR方法 2操作较简单易行,可在四川省肉毒梭菌监测中推荐使用。
Objective To compare and analyze the genotyping methods of four botulinum neurotoxins (Bo NT) in Sichuan province, and provide a reliable method for typing and identification of Clostridium botulinum in Sichuan Province. Methods Four strains of Clostridium botulinum were used to validate the PCR genotyping using six strains of Clostridium botulinum (including type A, B and E) preserved in our laboratory - the US Food and Drug Administration (FDA) multiplex PCR typing Method, two multiplex PCR typing methods of International Organization for Standardization (ISO) and a real-time fluorescent PCR typing method to compare the differences among the methods and to analyze the causes of the differences initially. Results All three multiplex PCR methods could simultaneously detect three types of Clostridium botulinum A, B and E in one reaction. ISO multiplex PCR method 1 type A test to obtain the expected band, but the results of the band is not clear. The remaining two multiplex PCR methods in the detection of Clostridium botulinum species, you can get a clear expected band. Real-time PCR-based genotyping can detect different types of Clostridium botulinum simultaneously in multiple reaction systems. However, due to the same fluorescent label, it is necessary to separately detect the toxin types in order to obtain the typing result. Conclusions The US FDA multiplex PCR method and ISO multiplex PCR method 2 are simple and easy to operate and can be recommended for the surveillance of Clostridium botulinum in Sichuan Province.