通过环介导恒温扩增技术检测空肠弯曲杆菌

Detection of Campylobacter jejuni by loop-mediated isothermal amplification method

目的建立基于DNA环介导恒温核酸扩增法(LAMP)快速检测空肠弯曲杆菌。方法以空肠弯曲杆菌马尿酸酶基因(hip O基因)为靶序列,设计6套特异性引物进行LAMP反应,通过比较扩增效率筛选出最佳引物用以快速检测空肠弯曲杆菌。对检测方法的特异性进行验证,敏感性与PCR法比较。结果实时浊度仪监测反应结果表明,LAMP反应在62℃恒温条件60 min内完成;如果在反应前添加钙黄绿素与氯化锰混合液(FD),黄绿色的阳性结果明显区别于橙色阴性结果。LAMP法最低检出限为6.97×10~2拷贝/μl,敏感性为PCR法(6.97×10~3拷贝/μl)的10倍。结论 LAMP法用于快速检测空肠弯曲杆菌具有检测过程简单、实验装置简便、反应结果判读方便、敏感性高、特异性强等特点,特别适于现场和基层检疫及医疗机构快速诊断。

Objective To establish a loop-mediated isothermal amplification method for detection of Campylobacter jejuni.Methods Six sets of primers were designed to recognize Campylobacter jejuni specific gene hipO.One was selected as the optimal primer and its specificity and sensitivity to Campylobacter jejuni were evaluated by LAMP reaction in 60 minutes at 62℃.Results The results recorded by the turbidity meter showed that the sensitivity of LAMP with a detection limit of 6.97＆#215;102 copies/μl was ten times that of PCR.Conclusion LAMP is a potential and valuable method of detection of Campylobacter jejuni due to its rapidity,simplicity,low cost and accuracy.It is especially suitable for grass-roots medical units.