牛病毒性腹泻病毒LAMP检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2015.12.001

›› 2015, Vol. 42 ›› Issue (12): 3111-3118.doi: 10.16431/j.cnki.1671-7236.2015.12.001

• 生物技术 • 下一篇

牛病毒性腹泻病毒LAMP检测方法的建立与应用

李家伟^1,2, 郭利¹, 杨勇¹, 王建科¹, 张淑琴¹, 张加力², 程世鹏¹

1. 中国农业科学院特产研究所, 特种动物分子生物学重点实验室, 长春 130112;
2. 吉林农业大学研究生院, 长春 130118

收稿日期:2015-01-05 出版日期:2015-12-20 发布日期:2015-12-30
通讯作者: 郭利, 程世鹏 E-mail:piaogl110@163.com;tcscsp@126.com
作者简介:李家伟(1991-),男,吉林安图人,硕士生,研究方向:临床兽医学,E-mail:lijiawei1205@163.com
基金资助:
牛呼吸道综合征临床快速诊断试剂盒的研制(20130206024NY);中国农业科学院"创新工程"项目

Establishment and Application of LAMP Detection Method of Bovine Viral Diarrhea Virus

LI Jia-wei^1,2, GUO Li¹, YANG Yong¹, WANG Jian-ke¹, ZHANG Shu-qin¹, ZHANG Jia-li², CHENG Shi-peng¹

1. Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China;
2. Graduate School, Jilin Agricultural University, Changchun 130118, China

Received:2015-01-05 Online:2015-12-20 Published:2015-12-30

摘要/Abstract

摘要： 针对牛病毒性腹泻病毒(BVDV) 5'UTR基因(GenBank登录号:AY278459.1)序列设计4条特异性环介导等温扩增 (LAMP) 引物,特异性识别靶基因序列上4个独立区域,采用LAMP技术,利用实时浊度仪实时检测LAMP 反应过程中所产生的焦磷酸镁白色沉淀,实时监测反应液浊度来判断反应结果,实现对扩增反应全过程的监控,建立BVDV的LAMP快速检测方法。通过实时浊度仪在恒温63 ℃下50 min完成检测,对方法的特异性、灵敏度、重复性进行了评价。结果显示,经优化该方法只检测BVDV阳性,特异性强;病毒10^-6倍稀释时仍能被检测到,比PCR方法灵敏度至少高100倍;重复性良好。LAMP实时浊度法具有简单、快速、灵敏度高、特异性强的优势,为BVDV的临床检测提供了一种简单快速的试验手段。

关键词: 牛病毒性腹泻病毒; 环介导等温扩增技术; PCR

Abstract: Since the 5'UTR gene (GenBank No.:AY278459.1) had 4 isolated regions,we designed a set of 4 LAMP primers to specifically recognize target gene sequences.This study developed a loop-mediated isothermal amplification (LAMP) method for detecting BVDV,using the pyrophosphate magnesium white precipitate for Real-time detection in LAMP reaction process of turbidity instrument,Real-time monitor liquid turbidity to determine result.The whole reaction lasted only 50 minutes at a constructed temperature of 63 ℃ to evaluate specificity,sensibility and repeatability of the method.The result demonstrated that the LAMP assay could only react with BVDV,its specificity was high;It could detect at least 10^-6-fold diluted samples,which was 100 more sensitive than PCR assay;And repeatability was good.The simple,rapid,high siensitivity and specificity LAMP assay was a potential tool for the detection of BVDV in field conditions.

Key words: bovine viral diarrhea virus (BVDV); loop-mediated isothermal amplification method (LAMP); PCR

中图分类号:

李家伟, 郭利, 杨勇, 王建科, 张淑琴, 张加力, 程世鹏. 牛病毒性腹泻病毒LAMP检测方法的建立与应用[J]. , 2015, 42(12): 3111-3118.

LI Jia-wei, GUO Li, YANG Yong, WANG Jian-ke, ZHANG Shu-qin, ZHANG Jia-li, CHENG Shi-peng. Establishment and Application of LAMP Detection Method of Bovine Viral Diarrhea Virus[J]. , 2015, 42(12): 3111-3118.

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牛病毒性腹泻病毒LAMP检测方法的建立与应用

Establishment and Application of LAMP Detection Method of Bovine Viral Diarrhea Virus

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