摘要
目的建立1种基于环介导恒温扩增(LAMP)的快速、敏感、特异检测小肠结肠炎耶尔森氏菌方法,为诊断该菌感染及食品中污染检测提供实验室诊断依据。方法根据GenBank中foxA序列,设计多套引物,筛选最佳引物、优化反应条件,建立小肠结肠炎耶尔森氏菌DNA的LAMP检测方法,加入荧光染料钙黄绿素以肉眼判断结果。结果根据扩增曲线,从4组引物中筛选出最优引物组进行LAMP检测,63℃恒温15min开始出现扩增,最低检测限为1.46×10^(-7)g/L,高于普通PCR最低检测限1个数量级;以金黄色葡萄球菌、沙门菌、假结核耶尔森氏菌等10株病原菌为模板进行LAMP检测,均未检出非特异性扩增反应。测序结果分析显示,LAMP扩增区不同菌株序列一致性约为95%,具有很好的保守性。结论建立的方法能够满足基层实验室、应急检测等使用需求,有良好的应用价值,可在有条件的基层进行推广。
Objective To establish a rapid,sensitive and specific method to detect Yersinia enterocolitica by loop-mediated i- sothermal amplification （LAMP） ; to provide diagnosis evidence for its infection and contamination surveillance for food. Meth- ods According to sequence of foxA in Genbank, several sets of specific primers and probes were designed and screened for optimal primer/probe set, the amplification reaction was optimized to establish LAMP assay to detect DNA of Yersinia entero- colitica. Calcein was added to reaction system for result adjustment by eye. Results Optimal primer/probe set was selected for LAMP analysis based on amplification curves. The amplification could be detected after 15 min reaction at 63 ℃, the limit of detection was 1.46 × 10^-7 g/L, which was 10 times sensitive than normal PCR. No specific amplification was detected when LAMP was used to analyze Staphylococcus aureus, Salmonella and Yersinia pseudotuberculosis. Sequence analysis showed the homology of amplicons of different strains was around 95%, with good conservation. Conclusion The established assay can satisfy the requirement for local labs and emergency detection, with good applicable values. It is suitable to be promoted in e- quipped local medical facilities.
出处
《江苏预防医学》
CAS
2017年第2期133-136,共4页
Jiangsu Journal of Preventive Medicine
关键词
小肠结肠炎耶尔森氏菌
环介导恒温扩增技术
快速检测
检出限
Yersinia enterocolitica
Loop-mediated isothermal amplifications Quick diagnosis
Limit of Detection
