[1]陈昌国,陈秋圆,侯兵兵,等.基于 LAMP技术针对溶藻弧菌 gyrB基因快速检测方法的建立[J].现代检验医学杂志,2019,34(06):6-9.[doi:10.3969 ／ j.issn.1671-7414.2019.06.002]
　CHEN Chang-guo,CHEN Qiu-yuan,HOU Bing-bing,et al.Establishment of the Rapid Detection Method Targeting to GyrB Gene of Vibrio Parahaemolyticus Based on LAMP Technology[J].Journal of Modern Laboratory Medicine,2019,34(06):6-9.[doi:10.3969 ／ j.issn.1671-7414.2019.06.002]
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基于 LAMP技术针对溶藻弧菌 gyrB基因快速检测方法的建立 ()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第34卷
期数:
2019年06期
页码:
6-9
栏目:
论著
出版日期:
2019-12-30

文章信息/Info

Title:
Establishment of the Rapid Detection Method Targeting to GyrB Gene of Vibrio Parahaemolyticus Based on LAMP Technology
文章编号:
1671-7414（2019）06-006-04
作者:
陈昌国; 陈秋圆; 侯兵兵; 刘新萍;  董优优
（解放军总医院第六医学中心检验科，北京　100048）
Author(s):
CHEN Chang-guo;  CHEN Qiu-yuan;  HOU Bing-bing;  LIU Xin-ping;  DONG You-you
（Department of Clinical Laboratory, the Six Medical Center of PLA General Hospital, Beijing 100048, China）
关键词:
环介导等温扩增技术; 弧菌; 溶藻弧菌; 快速检测; gyrB 基因
分类号:
R378.3；Q503
DOI:
10.3969 ／ j.issn.1671-7414.2019.06.002
文献标志码:
A
摘要:
目的　应用环介导等温扩增技术（Loop-mediated isothermal amplification, LAMP）建立针对溶藻弧菌gyrB 基因的 快速检测方法。方法　以溶藻弧菌标准株（ATCC-17749）和溶藻弧菌野生株（WT）为研究对象，通过在线生物学软件（http:// primerexplorer.jp/e/）设计针对溶藻弧菌gyrB 基因的LAMP 引物，利用恒温水浴进行等温扩增，优化反应条件，建立快 速检测方法。结果　①经2g/dl 凝胶电泳结果显示反应温度为61℃时扩增产物成像效果较60℃，62℃和63℃明显，为 适宜反应温度；②在61℃条件下反应60 min 和90 min 凝胶电泳成像效果差异不大，反应时间为60 min 能满足扩增需要； ③利用同一体系对临床6 种其它常见致病菌进行等温扩增时未出现阳性条带，提示整个体系特异度较好；④采用模板稀 释法验证该LAMP 体系检测的灵敏度为10-4mg/L。结论　建立了基于LAMP 技术针对溶藻弧菌gyrB 基因的快速检测方 法, 具有良好的特异度和敏感度，对快速检测溶藻弧菌有重要意义。
Abstract:
Objective　To establish a rapid method for the detection of Vibrio alginolyticus by loop mediated isothermal amplification （LAMP）. Methods　Taking the Vibrio alginolyticus（ATCC-17749）and Vibrio alginolyticus（WT）as the research object, designing of LAMP primers for the gyrB gene by online biology software （http://primerexplorer.jp/e/）， isothermal amplification was carried out in a constant temperature water bath, and the reaction conditions were optimized, and established a quick test response system. Results　① The results of 2g/dl gel electrophoresis showed that the imaging effect of the amplified products was obviously higher than that of 60℃ , 62℃ and 63℃ at the reaction temperature of 61℃ . ② There was no significant difference in gel electrophoresis effect at 60 min and 90 min at 61℃ , and the reaction time was 60 min, which could meet the need of amplification. ③ There was no positive band in isothermal amplification of 6 other common clinical pathogenic bacteria using the same system, suggesting that the specificity of the whole system was good. ④ Template dilution method was used to verify the sensitivity of the LAMP system for detection of 10-4 mg/L. Conlusion　A rapid detection method for gyrB gene of Vibrio alginolyticus based on LAMP technology was established, which has good specificity and sensitivity, and has important significance for rapid detection of Vibrio alginolyticus.

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备注/Memo

备注/Memo:
基金项目：军队后勤科研重点项目，编号：BHJ14J005。 作者简介：陈昌国（1979-），男，博士，副主任技师，研究方向为病毒与肿瘤发生，病原微生物分子检测方法，E-mail:1234_chen@sina.com。收稿日期：2019-07-22
更新日期/Last Update: 2019-12-25