马疱疹病毒1型LAMP检测方法的建立

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。

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

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.