变形杆菌环介导等温扩增检测法的可视化方法的比较  

Comparison of the visualization methods of loop-mediated isothermal amplification for detecting Proteus

作　　者：申培立 王羽[1] 李志辉[2] 郭梦冉[1] 马晓燕[1] 张伟 马雯[1] 李英军[1] 檀建新[1] 

机构地区：[1]河北农业大学食品科技学院/河北省农产品加工工程技术中心,河北保定071001 [2]河北农业大学理工学院,河北黄骅061100

出　　处：《河北农业大学学报》2016年第3期73-77,82,共6页Journal of Hebei Agricultural University

基　　金：国家自然科学基金资助项目(31371772和31071494);河北省科技计划项目(152776120D)

摘　　要：通过比较现有几种可视化检测环介导等温扩增反应产物的方法,建立一种可视化检测变形杆菌的方法。利用已有的LAMP检测变形杆菌的方法作为出发检测体系,比较了焦磷酸镁反应沉淀观察法和分别单独添加核酸染料SYBR Green I、孔雀绿(Malachite Green)、钙黄绿素-氯化锰(Calcein-MnCl2)的可视化检测方法,结果显示,焦磷酸镁沉淀的指示效果较差,SYBR Green I法会造成实验场所的交叉污染,孔雀绿法指示反应不够灵敏,钙黄绿素-氯化锰法综合考虑较优;并证明该方法用于可视化LAMP检测变形杆菌,具有良好的特异性,灵敏度达到4.8CFU/mL。试验证明钙黄绿素-氯化锰法是一种较优的LAMP可视化检测法。A visual loop-mediated isothermal amplification（LAMP）assay method for detection of bacteriumProteus was developed by using Calcein-MnCl2 as the better visualizing indicator after comparing four visualization indicators usually employed in LAMP assay.Based on the basic LAMP assay system we established before,four LAMP visualizing methods were compared which included（a）checking the presence of a white precipitate of magnesium pyrophosphate in the final reaction mixture of LAM;and checking the color changes after applying（b）Malachite Green,（c）SYBR Green I and（d）Calcein-MnCl2 in the LAMP reaction mixture,re-spectively.Results showed that magnesium pyrophosphate precipitate method was not sensitive enough;SYBR Green I method might cause cross contamination in the test room;Malachite Green method was not sensitive enough if the DNA amplification is not efficient.Thus,Calcein-MnCl2 method is considered as the better one for indicating the LAMP results.By employing Calcein-MnCl2 as the indicator,the LAMP assay method showed a high specificity for detection of Proteus,and the sensitivity reached 4.8CFU/mL.These results indicated that Calcein-MnCl2 is the better indicator of the visual LAMP assay method for detecting Proteus.

关 键 词：环介导等温扩增 可视化检测 变形杆菌 

分 类 号：R378.2[医药卫生—病原生物学]