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[1]吕 恒,张锦海,顾海涛,等.肠出血性大肠杆菌O157:H7的快速可视化检测[J].医学研究与战创伤救治(原医学研究生学报),2013,15(04):321-324.[doi:10.3969/j.issn.1672-271X.2013.04.001]
　LV Heng,ZHANG Jin-hai,GU Hai-tao,et al.A quicker and visual method for detecting Enterohemorrhagic Escherichia coli O157:H7[J].JOURNAL OF MEDICALRESEARCH —COMBAT TRAUMA CARE,2013,15(04):321-324.[doi:10.3969/j.issn.1672-271X.2013.04.001]
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肠出血性大肠杆菌O157:H7的快速可视化检测()
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《医学研究与战创伤救治》（原医学研究生学报）[ISSN:1672-271X/CN:32-1713/R]

卷:
第15卷
期数:
2013年04期
页码:
321-324
栏目:
出版日期:
2013-08-05

文章信息/Info

Title:
A quicker and visual method for detecting Enterohemorrhagic Escherichia coli O157:H7
作者:
吕 恒1; 2; 张锦海2; 顾海涛2; 王 平2; 王长军2; 王玉邦1
1.210029 江苏南京,南京医科大学公共卫生学院; 2.210002 江苏南京,南京军区疾病预防控制中心
Author(s):
LV Heng1; 2; ZHANG Jin-hai2; GU Hai-tao2; WANG Ping2; WANG Chang-jun2; WANG Yu-bang1
1.School of Public Health,Nanjing Medical University,Nanjing,Jiangsu 210029,China; 2.Center for Disease Control and Prevention of Nanjing Military Command,Nanjing,Jiangsu 210002,China
关键词:
肠出血性大肠杆菌O157:H7;  可视化检测;  环介导等温扩增;  羟基萘酚蓝
Keywords:
enterohemorrhagic Escherichia coli O157:H7;  visual detection;  loop-mediated isothermal amplification(LAMP);  hydroxynaphthol blue(HNB)
分类号:
R378.2
DOI:
10.3969/j.issn.1672-271X.2013.04.001
文献标志码:
A
摘要:
目的 应用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术和羟基萘酚蓝(Hydroxynaphthol blue,HNB)指示剂建立肠出血性大肠杆菌(EHEC)O157:H7快速可视化检测方法。方法 针对EHEC O157:H7脂多糖编码基因rfbE保守区设计LAMP引物及环引物,反应体系加入染料HNB作为LAMP扩增的指示剂,结合LAMP浊度仪优化扩增条件,根据HNB的颜色变化进行结果判定,并评价检测方法的特异性和灵敏度。结果 本方法最低检出限约为100 拷贝/反应管,高于普通PCR; 检测特异性高,仅EHEC O157:H7反应管HNB颜色由紫罗兰变成天蓝色,而肠产毒性大肠杆菌(ETEC)、肠致病性大肠杆菌(EPEC)、肠侵袭性大肠杆菌(EIEC)、肠聚集性大肠杆菌(EAEC)、宋内志贺菌、福氏志贺菌均不发生变色反应; 体系的扩增效率高于普通PCR,40 min内出结果。结论 建立的基于颜色判定的EHEC O157:H7 LAMP检测方法具有特异、灵敏、设备要求简单等特点,适用于EHEC O157:H7现场快速检测。
Abstract:
Objective A loop-mediated isothermal amplification(LAMP)assay was developed for rapid visual detection of Enterohemorrhagic Escherichia coli O157:H7.Methods A set of six specific primers specific to eight regions of rfbE gene were designed.The reaction was performed in a single tube at 63 ℃with the addition of hydroxynaphthol blue(HNB)dye prior to amplification.Use Loopamp Realtime Turbidimeter to grope for amplification conditions.The result was determined by the color change of HNB,and the sensitivity and specificty of LAMP method were evaluated.Results The results showed the detection limit of the LAMP assay was 100 copies per tube which was higher than conventional PCR method.The specificity of the LAMP assay was high.Only Enterohemorrhagic Escherichia coli O157:H7 tube changed color.Conclusion These results suggested that the LAMP assay with HNB dye provided a useful tool for the rapid detection of Enterohemorrhagic Escherichia coli O157:H7.

参考文献/References:

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相似文献/References:

[1]孙茂华,孟祥升,夏兴霞,等.大肠杆菌O157:H7实验感染动物排菌动态检测[J].医学研究与战创伤救治(原医学研究生学报),2012,14(01):5.
　SUN Mao-hua,MENG Xiang-sheng,XIA Xing-xia,et al.Detection of the faeces of animals inoculated with E.coli O157:H7[J].JOURNAL OF MEDICALRESEARCH —COMBAT TRAUMA CARE,2012,14(04):5.

备注/Memo

备注/Memo:
基金项目: 江苏省科技支撑计划(社会发展)项目(BE2012609); 国家重大传染病专项课题(2013ZX10004-103、2013ZX10004-218); 南京军区重点课题(10Z038、10Z040); 全军后勤科研“十二五”重大项目(AWS11C001、AWS11C009); 南京军区重点课题(11Z040)
更新日期/Last Update: 2013-08-05