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当前位置: 首页 > 文章 > 马疱疹病毒1型LAMP检测方法的建立 中国畜牧兽医 2014,41 (9) 35-40
Position: Home > Articles > Establishment of Loop-mediated Isothermal Amplification Assay Detection Method of Equine Herpesviruses Type 1 China Animal Husbandry & Veterinary Medicine 2014,41 (9) 35-40

马疱疹病毒1型LAMP检测方法的建立

作  者：
杨夷平;刘建华;冉多良;许全圆;李娜;加尔肯
单  位：
新疆农业大学动物医学学院
关键词：
马疱疹病毒1型;环介导等温扩增技术;快速检测
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-1DNA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50min,使EHV-1DNA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。
译  名：
Establishment of Loop-mediated Isothermal Amplification Assay Detection Method of Equine Herpesviruses Type 1
作  者：
YANG Yi-ping;LIU Jian-hua;RAN Duo-liang;XU Quan-yuan;LI Na;Jiaerken;College of Veterinary Medicine,Xinjiang Agricultural University;
关键词：
equine herpesvirus-1(EHV-1);;loop-mediated isothermal amplification assay(LAMP);;rapid detection
摘  要：
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 GreenⅠ to judge the result by eye.Specific DNA of EHV-1was 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 GreenⅠ 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.

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