猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2017.02.006

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (2): 344-349.doi: 10.16431/j.cnki.1671-7236.2017.02.006

猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用

罗亚坤^1,2, 梁琳^1,2, 王静^1,2, 刘存^1,2, 刘琪^1,2, 刘畅^1,2, 蔺文成³, 崔尚金^1,2

1. 中国农业科学院北京畜牧兽医研究所, 北京 100193;
2. 农业部兽用药物与兽医生物技术北京科学观测实验站, 北京 100193;
3. 华南农业大学动物科学学院, 广州 510642

收稿日期:2016-08-16 出版日期:2017-02-20 发布日期:2017-02-25
通讯作者: 崔尚金 E-mail:cuishangjin@caas.cn
作者简介:罗亚坤(1991-), 男, 黑龙江哈尔滨人, 硕士生, 研究方向:动物传染病的诊断与防治, E-mail:lykzxz2016@163.com
基金资助:
中国农业科学院创新工程项目（ASTIP-IAS16）

Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus

LUO Ya-kun^1,2, LIANG Lin^1,2, WANG Jing^1,2, LIU Cun^1,2, LIU Qi^1,2, LIU Chang^1,2, LIN Wen-cheng³, CUI Shang-jin^1,2

1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Beijing Observation Station for Veterinary Drug and Veterinary Biotechnology, Ministry of Agriculture, Beijing 100193, China;
3. College of Animal Science, South China Agricultural University, Guangzhou 510642, China

Received:2016-08-16 Online:2017-02-20 Published:2017-02-25

摘要/Abstract

摘要：

试验旨在建立一种快速检测猪流行性腹泻病毒（PEDV）的环介导等温扩增方法（LAMP），为诊断PEDV提供简便、敏感、准确可靠的工具。参考GenBank中PEDV基因序列（登录号：KT799997），针对PEDVN基因设计了6条引物，对所建立的LAMP反应体系、反应温度进行优化，建立可特异性扩增PEDV的LAMP方法。结果显示，本试验成功建立了PEDV LAMP检测方法，在60℃恒温下反应60 min，能特异性地检测PEDV，检测限量为91拷贝/μL，比常规PCR方法的敏感性高100倍。对比75份临床样本的LAMP和常规RT-PCR法检测结果，显示两种方法符合率为97.3%。综上所述，本试验建立的LAMP方法具有特异性强、敏感性高，操作简单，设备要求低的特点，适用于PEDV临床样本的快速检测。

关键词: 猪流行性腹泻病毒; 环介导等温扩增技术; 检测

Abstract:

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of porcine epidemic diarrhea virus (PEDV), and provide a simple, sensitive, accurate and reliable tool for diagnosis of PEDV.The conservative PEDV N gene (GenBank accession number: KT799997) of PEDV was selected as a target to design six specific primers.The reaction system and temperature of LAMP were optimized, and the LAMP method for specific amplification of PEDV was established. Results showed that the PEDV LAMP detection method was established successfully, and it could detect PEDV specifically at 60℃ for 60 min,and the detection limit was 91 copies/μL, which was one hundred-fold higher than conventional RT-PCR method.75 clinical samples were detected by LAMP and PCR, respectively, the coincidence of LAMP and PCR was about 97.3%. All the data suggested that the LAMP assay had strong specificity, high sensitivity, simple operation, low equipment requirement, and was suitable for rapid detection of PEDV clinical samples.

Key words: porcine epidemic diarrhea virus (PEDV); loop-mediated isothermal amplification (LAMP); detection

中图分类号:

S823.9⁺1

罗亚坤, 梁琳, 王静, 刘存, 刘琪, 刘畅, 蔺文成, 崔尚金. 猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用[J]. 《中国畜牧兽医》, 2017, 44(2): 344-349.

LUO Ya-kun, LIANG Lin, WANG Jing, LIU Cun, LIU Qi, LIU Chang, LIN Wen-cheng, CUI Shang-jin. Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus[J]. , 2017, 44(2): 344-349.

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猪流行性腹泻病毒环介导等温扩增检测方法的建立及应用

Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus

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