免疫磁珠分离联合荧光定量聚合酶链式反应快速检测阪崎肠杆菌的试验研究  

Establishment of Real time PCR Assay for Detection of Enterobacter Sakazakii Based on Immunomagnetic Separation

作　　者：姚骅珊[1] 陈悦科 YAO Huashan;CHEN Yueke(Suzhou Chien-shiung Institute of Technology,Taicang 215411,China;Suzhou Bacme Biotech CO.,LTD,Taicang 215411,China)

机构地区：[1]苏州健雄职业技术学院医药科技学院,太仓215411 [2]苏州华益美生物科技有限公司,太仓215411

出　　处：《江苏工程职业技术学院学报》2018年第2期1-6,共6页Journal of Jiangsu College of Engineering and Technology

基　　金：太仓市应用基础研究计划(编号TC2013YY03);江苏省自然科学基金(编号SBK201340091)

摘　　要：建立免疫磁珠分离(Immunomagnetic Separation,简称"IMS")联合荧光定量聚合酶链式反应PCR(Polymerase Chain Reaction,简称"PCR")快速检测阪崎肠杆菌的方法。制备以生物素介导偶联的链霉亲和素免疫磁珠,优化偶联和捕获条件。合成阪崎肠杆菌ITS(Internal Transcribed Spacer简称"ITS")靶基因序列的引物和探针,同时构建内参质粒(Internal Amplification Control,简称"IAC"),建立能够实时监控反应过程的荧光定量PCR反应体系。在1 mL体系中添加粒径为80 nm的链霉亲和素免疫磁珠0.3 mg,孵育30 min,可获得80.5%以上的捕获效率。建立的基于内参的荧光PCR体系针对质粒检测时,Ct值(Cycle threshold,简称"Ct值")与模板拷贝数有着良好的线性关系(R^2=0.998),IMS-PCR检测体系对阪崎肠杆菌菌液的检测灵敏度为33.3 CFU/mL,可在3 h内完成。针对4株阪崎肠杆菌标准菌株和5株非阪崎肠杆菌菌株的检测也显示出较好的特异性和稳定性,人工模拟样品的检测结果与采用国标法检测的结果完全一致,可用于快速检测样品中的阪崎肠杆菌和疾病预防。Objective:To establish a rapid method for detecting enterobacter sakazakii by immunomagnetic separation(IMS)combined with real time quantitative PCR(real time PCR).Methods:Biotin mediated coupling of streptavidin immunomagnetic beads were prepared to optimize the coupling and capture conditions.Primers and probes for the target gene ITS of enterobacter sakazakii were synthesized and the internal reference plasmid was constructed to establish a fluorescence quantitative PCR reaction system that could monitor the reaction process in real time.Results:The efficiency of capture could reach more than 80.5%by incubating 30min with streptavidin immunomagnetic beads at a room temperature of 0.3 mg/mL.When the real time PCR system based on the internal reference system was established for plasmid detection,there was a good linear relationship between the Ct value and copy number of the template(R 2=0.998).The sensitivity of the IMS-PCR detection system of enterobacter sakazakii was 33.3 CFU/mL,and could be completed in the 3 h.The detection of 4 strains of enterobacter sakazakii and 5 strains of non-enterobacter sakazakii showed good specificity and stability.The results of the simulated samples were in good agreement with the national standard method,which could be used for the rapid detection of enterobacter sakazakii and prevention of disease.

关 键 词：免疫磁珠 荧光定量PCR 内参 阪崎肠杆菌 快速检测 

分 类 号：R378.2[医药卫生—病原生物学]