[1]王欢,黄庆,史大川,等.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[J].陆军军医大学学报(原第三军医大学学报),2012,34(22):2264-2268.
 Wang Huan,Huang Qing,Shi Dachuan,et al.Rapid detection of Pseudomonas aeruginosa by color loop-mediated isothermal amplification[J].J Amry Med Univ (J Third Mil Med Univ),2012,34(22):2264-2268.
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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(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
34卷
期数:
2012年第22期
页码:
2264-2268
栏目:
论著
出版日期:
2012-11-30

文章信息/Info

Title:
Rapid detection of Pseudomonas aeruginosa by color loop-mediated isothermal amplification
作者:
王欢黄庆史大川揣征然府伟灵
第三军医大学西南医院检验科
Author(s):
Wang Huan Huang Qing Shi Dachuan Chuai Zhenran Fu Weiling
Department of Medical Laboratory, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China
关键词:
铜绿假单胞菌 环介导恒温扩增OprL基因快速检验
Keywords:
loop-mediated isothermal amplification Pseudomonas aeruginosa OprL gene rapid detection
分类号:
R378.991;R446.5
文献标志码:
A
摘要:
目的      建立了一种基于颜色判定的,能够快速准确地检测铜绿假单胞菌的LAMP方法。      方法      针对铜绿假单胞菌OprL基因设计3对LAMP引物, 通过引物特异性识别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羟基萘酚蓝(HNB)或钙黄绿素(Calcein),其结果可以通过肉眼观察反应前后颜色变化来判定并经琼脂糖凝胶电泳验证。对该技术进行特异性、灵敏度分析,利用LAMP和PCR同时检测临床标本。      结果      设计出针对铜绿假单胞菌OprL基因的恒温扩增检测方法。本恒温扩增检测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。其最低检测限为17.414 pg/μl, PCR 方法最低检出限为174.414 pg/ μl, LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍, 灵敏度高。LAMP法对临床阳性标本检出率与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,适合用于现场和基层检疫及医疗单位的快速诊断。
Abstract:
Objective      To establish a rapid and sensitive method of color loop-mediated isothermal amplification (LAMP) for detecting Pseudomonas aeruginosa (P. aeruginosa).       Methods      LAMP method with hydroxy naphthol blue (HNB) and calcein was designed to amplify and identify the OprL gene of P. aeruginosa. Three pairs of LAMP primers (inner, outer and ring primers) were designed according to the eight zones of the OprL gene. A positive reaction was indicated by a color change before and after the reaction, and was verified by agarose gel electrophoresis. The reaction conditions and system were optimized. The sensitivity and specificity of the detection method were evaluated, and were compared with those of conventional PCR. Both LAMP and PCR were applied to detect clinical specimens.       Results      A LAMP method for detecting P. aeruginosa was successfully established. The LAMP method showed 100% specificity for P. aeruginosa without other bacteria amplification, and the sensitivity (with the minimum detection limit of 17.414 pg/μl) for the detection of P. aeruginosa was 10-fold higher than that of PCR (with the minimum detection limit of 174.14 pg/μl). The detection rates of LAMP and PCR in clinical specimens were the same (27/27).       Conclusion      LAMP method, in which the amplification can be completed within 60 min, is faster than PCR. The established LAMP method in this study enables rapid, sensitive and specific detection of P. aeruginosa, and can be applied for grass roots and small-scale laboratories as well as field surveillance.

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更新日期/Last Update: 2012-11-20