土拉弗朗西斯氏菌LAMP快速检测方法的应用

Application of a Loop-mediated Isothermal Amplification Method for Rapid Diagnosis of Francisella tularensis

旨在应用环介导恒温扩增技术(LAMP)检测土拉弗朗西斯氏菌。针对土拉弗朗西斯氏的FopA基因设计LAMP引物,优化LAMP反应条件,并对其特异性、灵敏度进行评价。结果显示,(1)对12种细菌和蜱的DNA(含大量假单胞菌属细菌)进行LAMP扩增,仅土拉弗朗西斯氏菌发生了扩增反应;(2)土拉弗朗西斯氏菌纯培养物检出限为4.9×102 pg/mL;克隆株质粒检出限为10 copies/μL,低于普通PCR的最低检出值1×103 copies/μL;(3)土壤模拟样本检出限为44 CFU/g;(4)和进口荧光定量PCR试剂对比检测24份(8个稀释度)土拉弗朗西斯氏菌污染土壤样本,在灵敏度范围内,均能准确检测。LAMP技术检测土拉弗朗西斯氏菌特异性强、灵敏度高、高效,对土拉热的病原学诊断具有应用价值。

This study aims to apply the loop-mediated isothermal amplification(LAMP)to detect Francisella tularensis.LAMP primers were designed for amplifying FopA gene from F.tularensis,reaction conditions were optimized,and the specificity and sensitivity of the method were evaluated.Results showed that:(1)12 bacteria and tick’s DNA(Containing a large number of Pseudomonas bacteria)were amplified with LAMP reaction;however,only F.tularensis demonstrated positive reaction;(2)the detection limit of pure F.tularensis culture was 4.9×10^2 pg/mL,the minimum threshold for cloned plasmid was 10 copies/μL,which were below those of conventional PCR,1×10^3copies/μL;(3)the minimum threshold for simulated soil samples was 44 CFU/g;(4)24 soil samples(8 dilutions)contaminated by F.tularensis were compared with the imported fluorescence quantitative PCR reagent,and all of them were accurately detected in the sensitivity range.In conclusion,applying LAMP technology in the detection of F.tularensis shows superiority of efficiency,high specificity and sensitivity,thus this has practical value for etiological diagnosis of tularemia.