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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
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
