摘要
目的应用纳米孔测序技术,建立一种宏基因组测序筛查病原菌的方法,发现常规检测手段难以识别的食源性致病因子。方法收集一起食源性疾病暴发事件中患者的粪便样本采样液,分别取原始样本和经硫乙醇酸盐流体培养基(fluid thioglycollate medium,FTG)增菌后的样本,提取核酸、构建文库并进行纳米孔测序。通过测序数据比对分析初步筛查可疑病原菌,并结合病原菌的分离培养和鉴定进行验证。结果序列分析显示,原始样本中检出6种病原菌,经FTG培养后检出病原菌增至15种,其中包括产气荚膜梭菌。后续分离鉴定结果表明,10份样本中均分离出产气荚膜梭菌,且其生化特征一致。基于此结果,进一步对涉事饭店的工作人员、环境和食品样品共110份样本进行了产气荚膜梭菌的检测,分别检出产气荚膜梭菌6、10和3株。结论综合序列分析及分离鉴定结果,推断本次食源性疾病事件可能由食用被产气荚膜梭菌污染的食品引起。纳米孔测序结合增菌培养可实现对病原菌快速筛选,为不明原因食源性疾病暴发事件的致病因子筛查提供了可行的新策略。
Objective To establish a metagenomic sequencing method for screening pathogenic bacteria using nanopore sequencing technology,identify foodborne pathogenic factors that are difficult to detect by conventional methods.Methods Fecal suspension samples from patients involved in a foodborne disease outbreak were collected.Both the original samples and those enriched using fluid thioglycollate medium(FTG)were subjected to nucleic acid extraction,library preparation and nanopore sequencing.Suspected pathogens were preliminarily identified through sequence alignment and further confirmed by conventional isolation,cultivation and biochemical identification.Results Sequence analysis revealed 6 kinds of pathogens in the original samples,while enrichment with FTG increased the number of detectable pathogens to 15,including Clostridium perfringens.Subsequent isolation and identification confirmed the presence of Clostridium perfringens in all 10 tested samples,with consistent biochemical characteristics.Based on these findings,110 additional samples from the implicated restaurant,including samples from staff,the kitchen environment and food were detected for Clostridium perfringens.A total of 6,10 and 3 isolates were recovered from staff,kitchen environmental and food samples,respectively.Conclusion Based on the comprehensive sequence analysis and isolation identification results,it is inferred that this foodborne disease incident may have been caused by consuming food contaminated with Clostridium perfringens.Nanopore sequencing combined with enrichment culture enables rapid pathogen screening and offers a feasible new strategy for identifying causative agents in foodborne outbreaks of unknown origin.
作者
施菊萍
王翔
王亚平
钱惠芬
徐兰
郑伟强
刘芳
吴鹏程
SHI Ju-Ping;WANG Xiang;WANG Ya-Ping;QIAN Hui-Fen;XU Lan;ZHENG Wei-Qiang;LIU Fang;WU Peng-Cheng(Zhangjiagang Center for Disease Control and Prevention,Zhangjiagang 215600,China;School of Health Science and Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China;Jiangyin Food Safety Testing Center,Jiangyin 214400,China)
出处
《食品安全质量检测学报》
2025年第22期27-33,共7页
Journal of Food Safety and Quality
基金
江苏省卫生健康委科研课题项目(Ym2023077)
苏州市预防医学科研课题项目(SZYZ202409)
苏州市“科教兴卫”青年科技项目(KJXW2023066)。
