通过环介导恒温扩增技术检测空肠弯曲杆菌

发布时间：2018-08-27 18:10

【摘要】：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。
[Abstract]:Objective To establish a DNA loop-mediated isothermal nucleic acid amplification (LAMP) method for the rapid detection of Campylobacter jejuni. Methods Six sets of specific primers were designed for LAMP reaction with the hippocampal uridase gene (hip O gene) of Campylobacter jejuni. The best primers were screened for rapid detection of Campylobacter jejuni by comparing amplification efficiency. Results The results of real-time turbidimetry showed that the LAMP reaction was completed within 60 minutes under the constant temperature of 62 C. If the mixture of calcein and manganese chloride (FD) was added before the reaction, the positive result of yellow-green was obviously different from the negative result of orange. ConclusionLAMP is a simple and convenient method for rapid detection of Campylobacter jejuni, which is suitable for on-site and grass-roots quarantine and rapid diagnosis of medical institutions.
【作者单位】：解放军总医院;解放军疾病预防控制所;解放军总医院海南分院;
【基金】：国家科技支撑计划资助项目(2015BAI13B07) 解放军总医院“百病妙诀”扶植项目(MJ201403)
【分类号】：R440

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