基于颜色判定的环介导恒温扩增技术快速检测铜绿假单胞菌的研究王.DOC

基于颜色判定的环介导恒温扩增技术快速检测铜绿假单胞菌的研究 王欢 黄庆 史大川 揣征然 府伟灵* (解放军第三军医大西南医院检验科，重庆，400038) 　摘　要：目的　建立了一种基于颜色判定的简单、快速和灵敏的检测方法，即利用环介导恒温扩增技术，设计快速检测铜绿假单胞菌的方法，以便对铜绿假单胞菌感染进行快速检测。方法 （１）利用DNA环介导恒温核酸扩增法(LAMP)针对铜绿假单胞菌OprL基因设计三对引物, 通过引物特异性识别上OprL的8个独立区域来快速检测铜绿假单胞菌。如果在反应前添加荧光染料HNB(羟基萘酚蓝)或Calcein（钙黄绿素）作为反应指示，其结果可以通过肉眼观察反应前后颜色变化来判定标准并经琼脂糖凝胶电泳验证。（２）对铜绿假单胞菌恒温扩增法进行特异性试验。（３）对铜绿假单胞菌恒温扩增法进行灵敏度试验。(4)LAMP和PCR法同时检测经传统方法已确诊的阳性标本27例，阴性标本12例。结果　（１）设计出针对铜绿假单胞菌的恒温扩增检测方法。（２）本恒温扩增检测方法只扩增铜绿假单胞菌ＤＮＡ，对其他菌不扩增，显示出良好的特异性。（３）本恒温扩增检测方法的最低检测限为LAMP方法的最低检出限为17.414 pg/uL, PCR 方法最低检出限为174.414 pg/uL, LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍, 灵敏度高。（4）两种染料LAMP法对临床标本检出率与PCR法相当（27/27）。 结论 LAMP方法用于快速检测铜绿假单胞菌具有检测过程简单、实验装置简便、反应结果肉眼可辨别、灵敏度高和特异性强的特点, 所以LAMP方法检测铜绿假单胞菌特别适合用于现场和基层检疫及医疗单位的快速诊断。　 关键词：铜绿假单胞菌；　环介导恒温扩增；OprL基因；快速检验 Rapid detection of Pseudomonas aeruginosa by colorimetric loop-mediated isothermal ampli?cation with hydroxynaphthol blue dye and Calcein WANG Huan，HUANG Qing，Shi Dachuan，Chuai Zhenran ， FU Weiling* (Southwest Hospital, the Third Military Medical University, Chongqing 400038,China) Abstract :　Objective　To establish a rapid, sensitive detection approach for Pseudomonas aeruginosa based on the colorimetric loop-mediated isothermal amplification (LAMP) technique1. Methods Two colorimetric loop-mediated isothermal ampli?cation (LAMP) assay with hydroxy naphthol blue and calcein were designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa.six LAMP primers ( two inner, two outerand two ring prime ) were designed according to the eight zones of the OprL gene of Pseudomonas aeruginosa. A positive reaction is indicated by the the color change afer adding an intercalating dye to the reaction solution.The reaction conditions and system were optimized. The sensitivity and specificity of the detection approach were evaluated and compared to conventional PCR methods. LAMP and PCR both detects the Clinical specimens . Results　The LAMP assay showed 100% speci?city for the serogroup and other ba