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[1]丁盛,陈刚毅,李美,等.基于LAMP的病原菌一步等温可视化检测[J].应用与环境生物学报,2019,25(05):1215-1221.[doi:10.19675/j.cnki.1006-687x.2019.01041]
　DING Sheng,CHEN Gangyi,et al.One-step isothermal visual detection of pathogens based on LAMP[J].Chinese Journal of Applied & Environmental Biology,2019,25(05):1215-1221.[doi:10.19675/j.cnki.1006-687x.2019.01041]
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基于LAMP的病原菌一步等温可视化检测
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年05期
页码:
1215-1221
栏目:
研究论文
出版日期:
2019-10-31

文章信息/Info

Title:
One-step isothermal visual detection of pathogens based on LAMP
作者:
丁盛; 陈刚毅; 李美; 王嘉钰; 陈蓉; 邹家尉; 杜凤; 董娟; 黄鑫; 崔欣; 唐卓
1中国科学院成都生物研究所 成都 610041 2中国科学院大学 北京 100049 3成都中医药大学 成都 610041
Author(s):
DING Sheng1;  2;  CHEN Gangyi1;  LI Mei1;  2;  WANG Jiayu1;  2;  CHEN Rong3;  ZOU Jiawei1;  2;  DU Feng1;  DONG Juan1;  HUANG Xin1;  CUI Xin1 & TANG Zhuo1**
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China
关键词:
病原菌; 环介导等温扩增; 可视化检测; 多重检测
Keywords:
pathogens;  loop-mediated isothermal amplification;  visual detection;  multiplex detection
分类号:
Q71
DOI:
10.19675/j.cnki.1006-687x.2019.01041
摘要:
病原菌检测是环境监测、食品安全以及临床诊断中重要的研究手段，环介导等温扩增技术是病原菌等温检测的新兴技术，为实现病原菌的可视化检测，利用环介导的等温扩增技术结合灵敏的颜色指示剂对多种病原菌建立一步等温可视化检测. 结果显示基于LAMP的实时荧光分析能够特异性地检测铜绿假单胞菌、鼠伤寒沙门氏菌、副溶血性弧菌、志贺氏菌、大肠杆菌O157:H7、阪崎肠杆菌、单核细胞增生李斯特氏菌以及金黄色葡萄球菌. 该方法的检测限低至能够检测体系中少于10个拷贝的菌样. 其次，确定了以pH指示剂中性红作为可视化报告分子时的反应缓冲体系应含低于10 mmol/L Tris-HCl. 通过将多种引物混合用于考察该方法在多重检测方面的可行性，结果表明该方法能够实现病原菌的五重检测. 本研究建立了一种适合于现场检测的一步等温可视化病原菌检测体系，该方法较传统方法更加简单、价格低廉，能够通过肉眼对检测结果进行判断，对实际应用具有重要意义. （图5 表2 参16）
Abstract:
The detection of pathogens is an integral part of environmental surveillance, food safety, and clinical diagnosis. Loop-mediated isothermal amplification (LAMP) is an emerging technology used to detect pathogens. However, a suitable method of reporting is still a challenge for fast on-site detection and the currently used reporting systems always require uncapping. This study aimed to construct an isothermal and one-step visual detection method for pathogens based on LAMP. Fluorescent reporting dye was used to detect eight common pathogens and determine the specificity of the method. Then a pH indicator was introduced to realize the colorimetric detection, and the sensitivity of the method was investigated using gradient dilution of Escherichia coli. The multiplex detection was also evaluated. Based on our results, the pH indicator could realize the single-step colorimetric detection; the optimal buffer for colorimetric detection contained less than 10 mmol/L Tris-HCl. The method was capable of detecting Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio parahaemolyticus, Shigella and Escherichia coli O157: H7, Cronobacter sakazakii, Listeria monocytogenes, and Staphylococcus aureus. The sensitivity of the method could detect as few as 10 copies of Escherichia coli and the multiplex detection could be realized when mixing the primers of Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio parahaemolyticus, Shigella and Escherichia coli O157:H7 to detect these pathogens. In conclusion, we report here a novel method in which the one-step colorimetric detection of pathogens could be achieved by combining LAMP with a pH indicator. The method is cost-effective, user-friendly, and shows great potential for on-site detection.

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更新日期/Last Update: 2019-10-25