环介导恒温扩增技术快速检测幽门螺杆菌方法的建立及评价

Establishment and evaluation of loop-mediated isothermal amplification method for rapid detection of Helicobacter pylori

目的建立基于DNA环介导恒温核酸扩增技术(loop-mediated isothermal amplification method,LAMP)的快速检测幽门螺杆菌的方法,并对该方法的灵敏度及特异度加以验证。方法以幽门螺杆菌尿素酶B基因(Ure B基因)为靶序列设计5套特异性引物,通过比较各引物的扩增效率筛选出最佳引物组合用以快速检测幽门螺杆菌;收集临床标本,以13C呼气试验为金标准验证LAMP法检测胃液中幽门螺杆菌的灵敏度及特异度。结果幽门螺杆菌反应最佳引物组合为HPUB3,反应均在60 min内完成;应用LAMP法对80例胃液样本中幽门螺杆菌进行检测,其准确率为98.75%,灵敏度为97.06%,特异度为100%,与13C呼气试验结果无统计学差异(P>0.05)。结论 LAMP法检测幽门螺杆菌用时少且灵敏度、特异度均较高。本研究为实现胃镜下同步、无创、快速检测幽门螺杆菌奠定了基础。

Objective To establish a Loop-mediated isothermal amplification method （LAMP） for detection of Helicobacter plyori through turbidity meter and colorimetric method and verify the specificity and sensitivity of this method. Methods Five sets of primers were designed to recognize the specific gene, UreB gene of H. pylori. The optimal set of primers was determined by amplification efficiency. Eighty gastric juice samples were collected to compare the sensitivity and specificity of LAMP with 13C urea breath test. Results The optimal primer for LAMP of H. pylori was HPUB3. All reactions were finished at a constant temperature in 60 minutes. The accuracy, sensitivity and specificity of LAMP in detection of H. pylori was 98.75%, 97.06% and 100%, which was not significantly different with those of 13C urea breath test （P 〉 0.05）. Conclusion LAMP is a rapid method for detecting H. pylori with high sensitivity and specificity. This study lays a foundation for real-time, noninvasive, rapid detection of H. pylori during endoscopy examination.