应用免疫磁珠分离法及荧光定量PCR技术快速检测肠出血性大肠埃希菌O157:H7  

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

作　　者：王涛[1] 刘凯[2] 王艳[2] 綦廷娜[1] 叶长芸[2] 熊衍文[2] 

机构地区：[1]贵阳医学院微生物学教研室,贵阳550004 [2]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《中国人兽共患病学报》2011年第4期291-293,315,共4页Chinese Journal of Zoonoses

基　　金：国家"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(2009ZX10004-101)资助

摘　　要：目的建立免疫磁珠分离法(immunomagnetic separation,IMS)联合荧光定量PCR(real-time PCR)技术快速检测肠出血性大肠埃希菌O157∶H7的方法。方法根据肠出血性大肠埃希菌O157∶H7抗原特异性基因rfbEO157设计引物和荧光探针,建立real-time PCR检测体系,并检测其特异性及敏感性;用O157免疫磁珠特异性捕获标本菌液中的O157∶H7大肠埃希菌,结合real-time PCR对磁珠捕获菌细胞的DNA进行检测分析。结果 Real-time PCR检测结果显示,O157∶H7大肠埃希菌可产生荧光信号,而其他常见致病菌均未见明显荧光信号;采用IMS-real-time PCR方法检测标本,菌液含菌量为3.3×102CFU/mL时即可被检出,检测全过程需时约4h。结论 IMS-real-time PCR检测方法具有特异性强、敏感度高、快速易操作等特点,该检测方法能提高O157∶H7大肠埃希菌的检出率和准确性,可用于标本的快速诊断、疾病监测和暴发疫情的病原学调查。To establish rapid detection method of enterohemorrhagic E.coli O157∶H7 by using immunomagnetic separation（IMS） and real-time PCR,a pair of primers and TaqMan probe were designed based on the rfbEO157 gene of E.coli O157∶H7.E.coli O157∶H7 was captured by the O157 immunomagnetic beads from bacterial suspensions,and then assayed by the real-time PCR.The results of detection were positive for the samples containing E.coli O157∶H7,but negative for samples containing other bacteria.The lowest detection limit of immunomagnetic separation and real-time PCR was 3.3×102 CFU per milliliter.It just took 4 hours to get the results.It is concluded that the immunomagnetic separation and real-time PCR for detected E.coli O157∶H7 in specimen shows high specificity,sensitivity,simplicity and celerity.Therefore,the method could be used to detect E.coli O157∶H7 quickly and identify the causes of outbreak.

关 键 词：免疫磁珠分离法 荧光定量PCR 大肠埃希菌O157∶H7 

分 类 号：R378.2[医药卫生—病原生物学]