中国畜牧兽医 ›› 2014, Vol. 41 ›› Issue (9): 35-40.

• 生物技术 • 上一篇    下一篇

马疱疹病毒1型LAMP检测方法的建立

杨夷平, 刘建华, 冉多良, 许全圆, 李娜, 加尔肯   

  1. 新疆农业大学动物医学学院, 新疆乌鲁木齐 830052
  • 收稿日期:2014-01-15 出版日期:2014-09-20 发布日期:2014-09-24
  • 通讯作者: 冉多良 E-mail:xjrdl7@163.com
  • 作者简介:杨夷平(1987- ),男,新疆人,硕士生,研究方向:动物传染病诊断与防治。
  • 基金资助:

    国家科技支撑计划课题“马匹疫病诊断及综合防控技术研究与示范”(2012BAD46B00);新疆维吾尔自治区重大专项“马(运动、肉用、乳用)生产关键技术研究与示范”(201130101)。

Establishment of Loop-mediated Isothermal Amplification Assay Detection Method of Equine Herpesviruses Type 1

YANG Yi-ping, LIU Jian-hua, RAN Duo-liang, XU Quan-yuan, LI Na, Jiaerken   

  1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
  • Received:2014-01-15 Online:2014-09-20 Published:2014-09-24

摘要: 本试验旨在建立一种检测马疱疹病毒1型(EHV-1)的快速、灵敏、特异的环介导等温扩增技术(LAMP),同时评价该方法的可靠性。根据马鼻肺炎糖蛋白B(gB)基因特异保守序列设计多对LAMP引物,利用LAMP Real Time Turbidimeter LA-320仪监测反应进程,进行引物筛选和反应条件的优化,建立能特异性扩增EHV-1 DNA的LAMP检测方法,并加入SYBR GreenⅠ通过肉眼判断结果。该方法在65 ℃恒温下作用50 min,使EHV-1 DNA获得了高效率的特异性扩增,与其他马易感病毒如马疱疹病毒4型(EHV-4)等无交叉反应;且具有极高的灵敏性,可检测到10-4稀释的目标病毒,比普通PCR的灵敏度高10倍;反应结束后加入SYBR GreenⅠ肉眼观察的结果与LAMP Real Time Turbidimeter LA-320仪监测结果一致。通过将4份临床样品的LAMP检测结果与已得到验证的PCR结果进行比对,结果显示符合率为100%。本研究建立的LAMP检测方法具有快速、特异、灵敏、简单易操作且设备要求低等特点,具有实地检测EHV-1的前景。

关键词: 马疱疹病毒1型; 环介导等温扩增技术; 快速检测

Abstract: The objective of this study was to establish a rapid detection method of loop-mediated isothermal amplification assay (LAMP) for equine herpesvirus-1 (EHV-1) and evaluate the reliability of the method. Designing several pairs of LAMP primers targeting to the conserved sequence of gBgene of EHV-1, using the LAMP Real Time Turbidimeter LA-320 meter to monitor the reaction so as to screen the primers and optimize the conditions, we established a LAMP method which could amplify specific DNA of EHV-1, then added SYBR Green Ⅰ to judge the result by eye. Specific DNA of EHV-1 was amplified efficient under 65 ℃ in 50 min. There was no cross reaction to other similar viruses such as EHV-4, and the reaction system also had very high sensitivity, 10-4 dilution multiple target could be detected, the established LAMP was 10 times higher than ordinary PCR. After the reaction, add SYBR Green Ⅰ to observe results, the results were consistent with LAMP Real Time Turbidimeter LA-320 meter. Compared LAMP method result with PCR method result of four clinical samples, coincidence rate was 100%. The LAMP method in this test was rapid, specific, sensitive, easy operation and low equipment requirement, and had application prospects.

Key words: equine herpesvirus-1 (EHV-1); loop-mediated isothermal amplification assay (LAMP); rapid detection

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