基于毛细管电泳的环介导恒温扩增技术检测方法研究

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。

Detection of Loop-meditated Isothermal Amplification Products by Capillary Electrophoresis

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.