交叉引物等温扩增技术在肠出血性大肠杆菌O157:H7快速检测中的应用  

Rapid detection of enterohemorrhagic E. coli O157: H7 by using cross priming amplification technology

作　　者：祁军[1] 詹曦菁[1] 李智慧[1] 左锋[1] 王馨[1] 柴宏森[1] 刘振宇[1] 

机构地区：[1]天津国际旅行卫生保健中心,天津300456

出　　处：《口岸卫生控制》2016年第1期11-16,共6页Port Health Control

基　　金：国家质检总局科技计划项目(2010IK221)

摘　　要：目的建立一种快速、敏感、特异的肠出血性大肠杆菌(EHEC)O157:H7的交叉引物等温扩增(Cross Priming Amplification,CPA)检测方法。方法根据EHEC O157:H7 stx1基因特征性保守序列(Gen Bank83460),设计交叉引物、剥离引物和检测探针,建立了EHEC O157:H7的CPA检测技术。用该方法检测19株细菌常见的食源性细菌,以验证其特异性;并用菌液浓度梯度稀释的方法对该方法的检测灵敏度进行评价。并通过60份食品模拟样品验证其在实际应用中的可性行,该检测结果与经典的细菌分离培养鉴定方法及常用的荧光定量PCR法进行比较。结果在检测的19株细菌中,只有EHEC O157:H7菌株的检测结果为阳性。其它菌株检测结果均为阴性,证明其特异性良好;通过浓度梯度稀释证明该方法检测的灵敏度可达l02cfu/m L;与分离培养鉴定法及荧光定量PCR法比较,其检测的灵敏度均为96.77%和96.67%、特异度分别为93.10%和90%、准确性分别为95%和93.33%。结论用于EHEC O157:H7的CPA检测体系具有简便、快速、灵敏、特异的优点,可用于进出境口岸现场及食品EHEC O157:H7的快速检测。Objective This article aim to develop a kind of cross priming amplification（ CPA） assay for the detection of EHEC O157： H7. Methods Based on the characteristic conserved sequence of stx1 gene（ Gen Bank83460） of EHEC O157： H7,the CPA method is developed by designing crossing primers,bumper and detection probe separately. Specificity and sensitivity of the CPA are verified,and the consistency between CPA and other methods such as isolation- culture method and real- time quantitative PCR are also determined. Results The analysis of 19 bacterial strains demonstrates that CPA method can detect the EHEC O157： H7 specifically,all non EHEC O157： H7 strains can not be detected by this method. The detecting sensitivity of CPA is 102 CFU / ml. Compared with isolation- culture method and real- time quantitative PCR,the specificity,sensitivity and accuracy of CPA are 96. 77% and 96. 67% 、93. 10% and 90% 、95% and 93. 33% respectively. Conclusion The CPA assay for dectction of EHEC O157： H7 is a simple,rapid,sensitive and specific method suitable for detection of EHEC O157： H7 in simulated food samples.

关 键 词：交叉引物等温扩增 CPA 肠出血性大肠杆菌O157:H7 EHEC O157:H7 

分 类 号：R378.2[医药卫生—病原生物学]