文献类型：期刊文献

中文题名：基于毛细管电泳的环介导恒温扩增技术检测方法研究

英文题名：Detection of Loop-meditated Isothermal Amplification Products by Capillary Electrophoresis

作者：李振庆[1];吴到检[1];张大伟[1];山口佳则[2,3]

机构：[1]上海理工大学光电信息与计算机工程学院,上海200093;[2]华东理工大学理学院物理系,上海200237;[3]大阪大学应用物理系

年份：2016

卷号：35

期号：6

起止页码：758

中文期刊名：分析测试学报

外文期刊名：Journal of Instrumental Analysis

收录：CSTPCD;;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；

基金：国家自然科学基金(21205078);高等学校博士学科点专项科研基金博导类资助课题(20123120110002);上海高校青年教师培养资助计划(51-13-302-102)

语种：中文

中文关键词：环介导恒温扩增反应;毛细管电泳;假阳性

外文关键词：loop-mediated isothermal amplification; capillary electrophoresis; false-positive product

摘要：以大肠杆菌（E.coli）为对象,采用环介导恒温扩增技术（LAMP）对其扩增,在实验室自制的毛细管电泳-诱导荧光平台上建立了LAMP产物的检测新方法。引物F3,B3,FIP,BIP扩增的E.coli LAMP产物大小为240 bp。优化的毛细管电泳条件为：毛细管有效长度/总长度（10 cm/15 cm）,筛分介质溶液为0.5%羟乙基纤维素（1 300 K）,电场强度（100 V/cm）,进样条件（100 V/cm,1.0 s）。毛细管电泳时,DNA长度在100~500 bp范围内与其迁移时间呈线性关系,相关系数为0.996。在相同毛细管电泳条件下对E.coli LAMP产物进行分析,并利用这种线性关系在电泳图中对E.coli LAMP产物与假阳性产物做区分,结果表明,毛细管电泳技术不仅可在15 min内实现LAMP产物及附加产物的快速检测,而且可快速区分LAMP阳性及假阳性实验产物。采用建立的毛细管电泳快速检测LAMP产物的方法,对AB0174 E.coli基因实施了LAMP,结果表明该方法适合DNA LAMP产物的快速检测。
A novel method was proposed for the detection of loop mediated isothermal amplification（ LAMP） products of Escherichia coli（ E. coli） based on self-built capillary electrophoresis（ CE） with lamp induced fluorescence system. Four primers were designed for the target DNA,and the sizes of the LAMP products of E. coli corresponding to the primers of F3,B3,FIP and BIP were 240 bp.The best result was achieved with the sieving polymer of 0. 5% HEC（ 1 300 K）,an electric field strength of 100 V / cm and an injection time of 1. 0 s at the electric field strength of 100 V / cm. The migration time of DNA from 100 bp to 500 bp is linearly related with DNA size,and the correlation coefficient is 0. 996. The LAMP products of E. coli were resolved under the same electrophoretic conditions. The sizes of the LAMP products were calculated based on the linear relationship,and the positive products was distinguished from the false-positive products. The results demonstrated that CE could not only rapidly separate the LAMP products of E. coli within 15 min,but also distinguish the positive from false-positive products. The proposed method was successfully applied in the separation of the LAMP products of AB0174 E. coli. Therefore,it is suitable for the rapid detection of LAMP products of DNA.