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[1]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**.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):1-10.[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):1-10.[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:
1-10
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12陈刚毅1李美12王嘉钰12陈蓉3邹家尉12杜凤1董娟1黄鑫1崔欣1唐卓1**
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 Zhuo 1**
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
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的检测铜绿假单胞菌、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。由此,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 参15)
Abstract:
The detection of pathogens is an integral part for the environmental surveillance, food safety and clinical diagnosis. Loop-mediated isothermal amplification (LAMP) is the emerging technology used in detecting pathogen. However, the suitable reporting way is still a challenge for fast on-site detection and the currently used reporting systems always require uncapping operation. This study aimed to construct an isothermal and one-step visual detection method for pathogens based on LAMP. The fluorescent reporting dye was used to detect eight common pathogens in order to determine the specificity of the method. Then the pH indicator was introduced to realize the colorimetric detection and the sensitivity of the method was investigated using gradient dilution sample of Escherichia coli. The multiplex detection was also evaluated. Based on our results, the pH indicator could realize the single-step colorimetric detection and 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 specifically. 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 reported here a novel method in which the one-step colorimetric detection of pathogens could be achieved by combining LAMP with pH indicator. The method is cost-effective, user-friendly and shows a great potential in on-site detection.

相似文献/References:

[1]王嘉钰 董娟** 唐卓**.基于环介导等温扩增法可视化检测玉米转基因成分*[J].应用与环境生物学报,2019,25(06):1.[doi:10.19675/j.cnki.1006-687x.2019.04020]
 WANG Jiayu,DONG Juan** & TANG Zhuo**.Colorimetric detection of genetically modified organisms (GMO s) based on loop-mediated isoth ermal amplification (LAMP)*[J].Chinese Journal of Applied & Environmental Biology,2019,25(05):1.[doi:10.19675/j.cnki.1006-687x.2019.04020]

备注/Memo

备注/Memo:

收稿日期 Received: 2019-01-24 接受日期 Accepted: 2018-03-11
*国家自然科学基金项目(21708037)和中国博士后科学基金(2017M612998)资助 Supported by the National Natural Science Foundation of China (21708037), and the China Postdoctoral Science Foundation (2017M612998)
**通讯作者 Corresponding author (E-mail: tangzhuo@cib.ac.cn)
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更新日期/Last Update: 2019-03-13