马疱疹病毒1型LAMP检测方法的建立

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

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。

The objective of this study was to establish a rapid detection method of loop-mediated isothermal amplification as- say （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 gB gene 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 am- plify specific DNA of EHV-1, then added SYBR Green I to judge the result by eye. Specific DNA of EHV-1 was amplified ef- ficient 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 ordi- nary PCR. After the reaction, add SYBR Green I to observe results, the results were consistent with LAMP Real Time Tur- bidimeter 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.