东方马脑脊髓炎病毒PCR-DHPLC检测方法的建立与初步应用

Development of PCR-DHPLC assays for detection of Eastern equine encephalitis virus

为建立检测东方马脑脊髓炎病毒(EEEV)的方法,本研究利用PCR结合变性高效液相色谱技术(DHPLC),针对EEEV NS1基因,设计特异性引物,通过优化反应条件,对核酸扩增产物采用DHPLC进行检测分析,核酸扩增产物形成DHPLC特征峰图,建立了EEEV的PCR-DHPLC快速检测方法。结果显示该方法与其他6种常见马病病毒均无交叉反应,未检测到其他马病病毒的阳性吸收峰,具有较强的特异性;对梯度稀释的标准阳性模板的检测限可达到10拷贝/μL;利用本研究建立的方法与荧光定量RT-PCR对30份临床样品进行检测,两者符合率达100%。本研究建立的PCR-DHPLC法具有特异、敏感、快速、重复性好等优点,可以用于EEE的病原学诊断及流行病学调查。

To establish a sensitive assay for the eastern equine encephalitis virus （EEEV） detection, a PCR-denaturing high performance liquid chromatography （PCR-DHPLC） assay was developed with the primers designed according to the conserved sequence of EEEV. The specificity test showed that the PCR-DHPLC assay was specific for EEEV detection with a detection limit of 10 copy/μL, but no corss-reaction was found to Western equine encephalitis, Venezuelan equine encephalomyelitis virus, West Nile virus, equine adenovirus, equine influenza virus（H3N8） and equine herpevirus-1. The method was used to detect the 30 clinical samples and the results were 100% in agreement with the real-time PCR. Therefore, the assay could be used in epidemiological investigation of EEEV.