集成式肠道致病菌血清型分子鉴定系统的研发及应用

Cholera, typhoid, dysentery and other infectious diarrhea are still main diseases of human. At present, serological detection is the gold standard of clinical identification and surveillance of enteropathogens. As we known, serological detection is based on slide agglutination test. Because of disadvantages of complexity operation, time consuming, poor repeatability and expensive expense, a new technology based on molecular identification system for serotyping which will replace the conventional one is needed urgently. With the occurrence of new technologies, several new methods have been developed in recent years. But those methods still had unacceptable defects, as listed below:(1)low detective channels; (2) contaminated PCR outcomes and complicated operation process of microarray and microspheres technology; (3) expensive equipment. Our research group has invented a novel technology named MeltPleX, which is based on several new technologies (Probe melting, multiplex real-time PCR). By using MeltPleX, 42-48 target genes can be detected in one tube on real-time PCR machine. MeltPleX would be applied to detect and classify the serotypes of 4 common enteropathogens (Cholerae,Shigella, V.P, Diarrhea-causing Escherichia coli)by using an integrated molecular identification system (extraction, amplification and detection). The whole detection of MeltPleX costs 4 hours and the result can be analyzed by software automatically. The developed method could be used in surveillance and detection of intestinal infectious diseases to ensure food safety and human health.

霍乱、伤寒、痢疾及其他感染性腹泻仍是影响人类健康的重要疾病。目前血清型鉴定是肠道致病菌临床检测和疾病监测的金标准,主要依赖玻片凝集实验。该经典检测手段存在操作繁琐、耗时长、重现性差、价格贵、易受主观因素干扰等诸多缺陷。基于分子生物学的血清型分子鉴定成为最有前途的替代方案。近年国内外报道了多种血清型分子鉴定方法,但实际应用仍有限，原因在于：通量低，覆盖的血清型种类少；芯片或微球技术操作繁琐且易产物污染，且需昂贵的专用设备。本课题组提出的基于荧光探针熔解曲线的多重检测技术（MeltPleX），可在荧光PCR仪上单管一步检测42-48个靶基因。本项目拟用该技术研发针对4种肠道致病菌（霍乱弧菌、志贺菌、副溶血弧菌、致泻性大肠杆菌）的一套集成核酸提取、扩增和检测的一体式快速血清型分子鉴定系统，鉴定结果4小时内，软件自动判读结果。旨在为基层防控重要肠道传染病提供适宜的技术手段，保障食品安全和人民健康。

目的：霍乱弧菌、副溶血弧菌、志贺菌、致泻性大肠杆菌以及其他感染性腹泻病原菌仍然是威胁人类健康的重要因素，研究快速简便的重要病原细菌的血清型分子鉴定技术，为基层防控肠道传染病提供适宜的技术手段，提高基层防控肠道传染病的监测能力和应急处置能力，服务我国传染病防控的整体需求，具有重大意义。基于分子生物学的血清型分子鉴定成为传统的玻片凝集血清分型方法的最有前途的替代方案。.方法：本课题将深圳市疾病预防控制中心444株副溶血弧菌菌株、179株大肠杆菌菌株、262株志贺菌菌株和5株霍乱弧菌菌株进行测序、拼接组装和注释，并从美国国立生物信息中心（NCBI）genbank数据库下载相关菌株基因组数据，最终建立这四种肠道致病菌基因组数据库，用于肠道致病菌比较基因组分析、血清型抗原基因靶标序列筛选、进化分析和SNP变异分析。应用荧光探针熔解曲线的多重检测技术（MeltPleX），在荧光PCR仪上单管一步检测42-48个靶基因。.结果：建立了基于MeltPleX 的4套肠道致病菌血清型分子鉴定系统，鉴定结果4小时内，与传统血清型鉴定方法相比：灵敏度＞95%，特异度为100%，CV<5%，kappa值＞0.75。在此基础上，制定了4个肠道致病菌血清型分子鉴定系统标准化技术方案，在深圳市10个区CDC和15家哨点医院推广应用。.结论：本集成式肠道致病菌血清型分子鉴定系统，具有灵敏度高、特异性强、操作简便、自动化程度高、价格低廉和省时省力等优点，适宜临床和疾控机构的大规模检测，为基层CDC和医院提供适宜的检测手段，满足突发公共卫生事件的应急处置和疾病监测需求，具有广阔的应用前景。