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目的建立基于分子生物学技术结合传统分离方法检测5种致泻性大肠埃希菌(DEC),并应用于浦东新区腹泻人群的DEC监测,从而构建DEC感染病原谱,以了解浦东新区感染性腹泻患者DEC的感染情况。方法从GenBank基因数据库中筛选5种DEC毒力基因序列,合成引物探针,优化设计TaqMan实时荧光PCR法结合传统分离方法,最终获得单克隆菌株。应用该方法对2014年的11家腹泻监测点1 846份粪便拭子进行DEC检测。结果 1 846件粪便拭子中DEC总阳性率为14.90%(275/1 846),其中检出致病性大肠埃希菌102株(5.53%),产毒性大肠埃希菌102株(5.53%),侵袭性大肠埃希菌3株(0.16%),聚集性大肠埃希菌68株(3.68%)。结论 Taq Man实时荧光聚合酶链反应(PCR)结合传统分离方法分离DEC体现出非常高的准确性和检测效率,同时避免了志贺菌的漏检;为期1年的监测数据显示,DEC已成为细菌性感染性腹泻病原谱中首位病原,其组成结构呈多样性及显著的季节性特点。
OBJECTIVE: To establish a method to detect five kinds of diarrhea-causing Escherichia coli (DEC) based on molecular biology techniques and traditional separation methods and apply it to the monitoring of diarrhea population in Pudong New Area so as to construct the infectious disease spectrum of DEC and understand the infectious diarrhea in Pudong New Area Patients with DEC infection. Methods Five strains of DEC virulence genes were screened from GenBank database and primer pairs were synthesized. The TaqMan real-time PCR method and the traditional separation method were optimized to obtain the monoclonal strain. This method was used for DEC testing of 1 846 fecal swabs at 11 diarrhea monitoring sites in 2014. Results The total positive rate of DEC in 1 846 fecal swabs was 14.90% (275/1 846), of which 102 strains (5.53%) were pathogenic Escherichia coli, 102 strains (5.53% ), 3 strains of Escherichia coli (0.16%), and 68 strains of Escherichia coli (3.68%). Conclusion The isolation of DEC by Taq Man real-time PCR combined with traditional separation method shows very high accuracy and detection efficiency, and avoids the missed detection of Shigella. One-year monitoring data show that DEC has become Bacterial infectious diarrhea pathogenic spectrum of the first pathogen, the composition of the structure showed a diversity and significant seasonal features.