环介导等温扩增技术检测粪便幽门螺杆菌方法的建立及应用  

Development and clinical application of loop-mediated isothermal amplification method on feces Helicobacter pylori detection

作　　者：韩慧[1] 胡子有[1] 姚芳[1] 曾勇[1] 吴炳义[1] 熊婧[2] 

机构地区：[1]南方医科大学南方医院临床医学实验研究中心,广州市510515 [2]南方医科大学南方医院消化科,广州市510515

出　　处：《实用医学杂志》2011年第19期3485-3487,共3页The Journal of Practical Medicine

基　　金：广东省"211工程"三期重点学科建设项目

摘　　要：目的:建立一种快速、简单的检测粪便幽门螺杆菌的环介导等温扩增方法(loop-medi atedisotherma lamplification,LAMP)。方法:针对幽门螺杆菌的16SrRNA基因设计4条特异性引物(2条内引物和2条外引物),优化反应条件后,对方法的敏感性、特异性进行评估,并评估检测粪便模拟标本的敏感性。结果:使用LAMP方法可以在2h内得到检测结果,加入染料后阳性结果为绿色,电泳后有明显的阶梯状条带。幽门螺杆菌的基因组DNA检测敏感性为12pg,是普通PCR的10倍,对模拟粪便标本进行检测,检测限为102CFU/mL。金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、粪肠球菌的检测结果均为阴性。结论:本研究建立的检测幽门螺杆菌的LAMP方法敏感性高、特异性强,操作简单,不需要特殊设备,是一种很有前景的检测方法。Objective To develop a loop-mediated isothermal amplification （LAMP） method for detecting Helicobacter pylori （Hp） of feces rapidly and simply.Methods Four specific LAMP primers （two inner primers and two outer primers） were designed by targeting 16S rRNA gene of Hp.After optimizing reaction condition,the sensitivity and specificity of the method were evaluated by simulated feces samples detection.Results The method could be finished within 2 h,and positive results were green and had a ladder-like pattern on the gel.The sensitivity of Hp genome DNA was 12 pg,and was 10 times as much as PCR,the detection limit of feces simulated samples was 102 CFU/mL.The results of Staphylococcus aureus？Escherichia coli？ Pseudomonas aeruginosa and Enterococcus facealis were negative.Conclusion LAMP assay is high sensitive,specific and simple to operate with no need special equipments,which can be taken as a promising method for Hp detection.

关 键 词：螺杆菌 幽门 环介导等温扩增技术 16srRNA 

分 类 号：R440[医药卫生—诊断学]