沙门菌快速分型方法研究

Currently, the "gold standard" method of the serotyping and genotyping of Salmonella enterica (S. enterica) has the limitations, such as the complicated operation and time consuming. The representative 1000 S. enterica strains were selected and sequenced by using the next generation sequencing platform (NGS) and the representative bioinformatics database were then developed combined with the PubMed database of S. enterica. Based on the developed database, the targeted genes related to serotyping of S. enterica, such as O antigen gene, H antigen gene etc. were selected, and the multiplex real-time PCR based the multiplex-ligation-melting-analysis (MLMA) for fast DNA serotyping assay was developed, which could identify top 150 serotypes. Furthermore, unusual serotype or new serotype could be identified by analyzing the S. enterica DNA serotyping database. Meanwhile, SNPs, Pan-genome, K-me, wgMLST etc. related to genotyping of top 5 Salmonella serotypes for the outbreak detection were screened，then NGS based second-generation molecular typing assay for the outbreak detection and evolution analysis of S. enterica was developed. The development of fast DNA serotyping and molecular typing of S. enterica will improve the testing accuracy and the efficiency of operations, which will also provide the simple and rapid technique support with the field epidemiological investigation. The developed method will hold great potential as a tool for the public health emergency response and foodborne disease surveillance.

目前，沙门菌的血清分型和分子分型 “金标准”方法都存在操作繁琐，耗时耗力的缺陷。本研究利用二代测序技术，选择1000株有代表性的沙门菌菌株进行全基因组测序，并结合已有基因组信息，构建更完整的比较基因组数据库。在此基础上，拟筛选特异的膜蛋白基因、编码O抗原和H抗原基因等，建立基于多重连接探针熔解曲线的沙门菌血清型分子鉴定方法，鉴定常见150种血清型，检测时间从传统的3-6天缩短到4个小时，特异性强，通量高；并建立本地化血清型数据库，鉴定罕见血清型或发现新血清型。同时，筛选常见5种血清型的SNP位点等，建立基于高通量测序的沙门菌二代分子分型方法，2天内完成，实现爆发疫情的快速溯源和传播途径分析。该两种分型方法的研究将提高实验室检测的准确率和工作效率，为现场流行病学调查提供一种简便快速的技术手段，提升公共卫生和食品安全管理的效能和水平。

项目背景：沙门菌（Salmonella）为重要的食源性致病菌，沙门菌型别的鉴定和溯源对于沙门菌病的预防控制、传染源追踪和危险因素分析乃至临床诊疗都具有重要的意义。本课题通过NCBI公布的12547条沙门菌全基因组序列和本研究所获得的1469株沙门菌基因组序列，比对特异性靶基因序列，建立改良探针熔解曲线鉴定常见200种沙门菌血清型的方法，4小时内完成。与传统血清分型方法相比，其灵敏度为96 %，特异度为100 %，Kappa值为0.99。所建立的基于全基因组测序的沙门菌血清型数据库，可鉴定罕见血清型或新血清型，对深圳市的147株稀有血清型进行鉴定，与传统血清学鉴定方法相比， 17.28%被鉴定为其他血清型，71.60%被鉴定为新的血清型。通过对沙门菌流行病学关联菌株和无关联菌株的全基因组信息分析，建立基于基因组测序的沙门菌溯源方法，并建立了肠炎沙门菌、鼠伤寒沙门菌等5种血清型分型方法，2天内完成，实现沙门菌感染爆发疫情的快速溯源和传播途径分析。本课题为国内首次系统建立了基于探针熔解曲线鉴定常见200种沙门菌血清型的方法，操作简便，检测成本低，适宜临床和疾控机构的大规模检测；所建立的SNP-WGS标准化分型方案能提高食源性疾病暴发识别效率，为实现深圳市乃至中国食源性疾病的实时基因组监测奠定了基础。本课题研究为现场流行病学调查和溯源提供一种简便快速的技术手段和新思路，满足突发公共卫生事件的应急处置和疾病监测需求。