脉冲场凝胶电泳分子分型技术在一起肉毒中毒诊断中的应用

Application of pulsed field gel electrophoresis molecular typing technique in the diagnosis of a botulism event

目的 实验室检测2例疑似肉毒毒素中毒病例的3份相关样本,探究中毒事件发生原因,为临床治疗提供参考。方法 参照GB 4789.12-2016《食品安全国家标准食品微生物学检验肉毒梭菌及肉毒毒素检验》对1份自制腐乳和2份病例的粪便样本进行前处理,应用实时荧光定量PCR法检测样本中肉毒毒素基因,以血琼脂平板和营养琼脂平板分离培养单菌落,将分离出的3株肉毒梭菌进行脉冲场凝胶电泳(pulsed-field gel electrophoresis),通过BioNumerics(BN)软件对图谱进行聚类分析。结果 1份自制腐乳和2份病例粪便样本的肉毒毒素基因检测结果,均为A型肉毒梭菌核酸阳性,3份样本均分离出A型肉毒梭菌。3株肉毒梭菌通过脉冲场凝胶电泳分子分型检测,得到指纹图谱,经BN软件聚类分析同源性为100%。结论 本次事件是一起因食用受到A型肉毒梭菌污染的自制腐乳而引起的肉毒中毒,分离出的3株肉毒梭菌通过PFGE分子分型确定为同一来源。今后应加强大众预防肉毒中毒的宣传教育,减少此类中毒事件的发生。

Objective To test 3 samples collected from 2 cases with suspected botulinum in the laboratory, explore the reason of the poisoning event, and provide reference for clinical treatment. Methods According to the National Food Safety Standard, Food Microbiological Examination: Clostridium Botulinum and Botulinum Toxin Examination(GB 4789.12-2016), the pre-treat was carried out in 1 sample of the homemade fermented bean curd and 2 stool samples from the cases for botulinum toxin genes examination with the real-time PCR method. The blood agar plates and nutrient agar plates were used for isolation and culture of single colony. After the pulsed field gel electrophoresis(PFGE) of 3 isolated strains, the BioNumerics(BN) software was used for the clustering analysis to the chromatogram. Results The examination for botulinum toxin gene showed that the nucleic acid of type A clostridium botulinum was positive in 1 sample of the homemade fermented bean curd and 2 stool samples, and the strains of type A clostridium botulinum were isolated from all 3 samples.The print-finger chromatogram of 3 strains of clostridium botulinum were obtained by the PFGE, and the homology was100% after the clustering analysis with the BioNumerics(BN) software. Conclusions The event is a botulism caused by eating the homemade fermented bean curd which was contaminated with type A clostridium botulinum. The 3 isolated strains are from one source with PFGE genetic typing method. The publicity and education of botulism prevention should be strengthened for reducing the occurrence of botulism event in the future.