解旋酶依赖性等温扩增快速检测O157:H7及其毒力基因的研究  

Rapid detection of Escherichia coli O157: H7 and its virulence genes by helicase-dependent isothermal DNA amplification

作　　者：雷飏[1] 刘爱平[1] 谢群[1] 陈柏塘[1] 郑文[1] 李征莉[1] 朱韩武[1] 谭徽[1] 

机构地区：[1]郴州市疾病预防控制中心,湖南郴州423000

出　　处：《实用预防医学》2014年第3期288-290,共3页Practical Preventive Medicine

基　　金：湖南省科学技术厅项目(2012FJ3064)

摘　　要：目的建立一种快速、特异的检测大肠杆菌O157:H7及其毒力基因的解旋酶依赖性等温扩增法(HDA)。方法以大肠杆菌O157:H7的溶血素基因(hly)、粘附抹平因子(eae)和O抗原编码基因(rfb)为扩增对象,设计3对特异性HDA引物,通过优化条件后进行灵敏度和特异度试验,并与普通PCR法比较。结果 HDA法从标准菌株和实验室分离株中均扩增出hlyA、eaeA和rfb基因片段,产物大小分别为100、93和109 bp,而其他非大肠杆菌O157:H7的扩增结果均为阴性。通过对各梯度大肠杆菌O157:H7菌悬液的检测表明HDA法可以检测的最低菌液浓度为3.0×101cfu/ml,该法与普通PCR法灵敏度相当。结论本HDA法具有较高的灵敏度和特异度,可快速、高效地检出大肠杆菌O157:H7菌和其毒力基因,而且对试验仪器要求低,特别适合应急处置现场和基层检验机构作为临床大肠杆菌O157:H7感染的辅助诊断。Objective To develop a quick and sensitive helicase-dependent isothermal DNA amplification （HDA） assay for the detection of Escherichia coli O157 ∶ H7 and its virulence genes.Methods Three pairs of primers were designed in the segments of the hemolysin gene （hlyA）,the adhesion gene （eaeA）,and the O157 gene （rfb）.After establishing and optimizing the HDA method,specific and sensitivity were tested,and then compared with PCR method.Results The hl.yA,eaeA and rfb genes were detected positive in the standard O157∶H7 strains and the isolated strains,with the product sizes of 100bp,93bp and 109bp,respectively.And no cross-reactions were found in the background bacteria.The sensitivity of HDA assay was 3.0 × 101 cfu/ml which was similar to the result of PCR method.Conclusions The HDA assay is highly specific and sensitive in detecting Escherichia coli O157∶H7 and has lower instrumental requirements.It is particularly suitable for auxiliary diagnosis in emergency disposal fields and grassroots medical institutions.

关 键 词：解旋酶依赖性等温扩增 大肠杆菌O157∶H7 hlyA基因 eaeA基因 rfb基因 

分 类 号：R378.2[医药卫生—病原生物学]