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鉴于目前埃博拉疫情在西非流行和输入国内的潜在风险,开发快速准确易普及的检测埃博拉病毒的方法对埃博拉防控具有重大意义。本文拟建立一种基于颜色判定简单、快速和灵敏的方法,即多引物自配引发等温扩增技术(isothermal multiple self-matching initiated amplification,IMSA)应用于埃博拉病毒扎伊尔亚型的检测。该技术设计了分别对应于埃博拉病毒扎伊尔型核蛋白(nuclear protein,NP)基因的7个区段的6条特异引物,在等温(63℃)条件下进行核酸扩增反应1小时,在扩增前加入HNB染料(羟基萘酚蓝)作为反应指示剂,以HNB染料颜色变化作为结果判定的标准。文中利用这种技术与RT-qPCR分别对含有目的片段的假病毒的系列稀释物以及模拟临床样本进行灵敏度分析,同时对30例健康人血浆以及登革病毒、日本脑炎病毒样本进行特异性分析。在塞拉利昂完成了81例埃博拉疑似病例血液样本的检测。结果显示RT-IMSA方法检测灵敏度与RT-qPCR方法的灵敏度相当,30例健康人血浆样本检测均为阴性,并且与登革病毒及日本脑炎病毒无交叉反应。与获得批准的荧光定量PCR试剂盒比较,RT-IMSA检测81例临床样本的结果为53例阳性,23例阴性,5例假阴性。灵敏度和特异性分别为91.4%和100%。因此,相较于RT-qPCR的高成本和复杂操作,RT-IMSA方法具有快速、简单的检测优势。
In view of the current epidemic of Ebola in West Africa and the potential risks of its entry into the country, the development of a rapid, accurate and readily available method of testing for Ebola is of great importance for the prevention and control of Ebola. This article intends to establish a simple, rapid and sensitive method based on color determination, that is, isothermal multiple self-matching initiated amplification (IMSA) is applied to the detection of Ebola Zairian subtypes . This technique designed 6 specific primers corresponding to 7 segments of the Ebola virus Zaire nuclear protein (NP) gene, respectively, and performed a nucleic acid amplification reaction under isothermal conditions (63 ° C.) for 1 hour , HNB dye (hydroxynaphthol blue) was added as a reaction indicator before amplification, and the color change of HNB dye was taken as a result of the judgment. In this paper, the sensitivity of serial dilutions of pseudoviruses containing the target fragment and the simulated clinical samples were analyzed respectively by this technique and RT-qPCR, and the specific samples of 30 healthy human plasma, dengue virus and Japanese encephalitis virus samples were also analyzed. . 81 blood samples from suspected cases of Ebola were completed in Sierra Leone. The results showed that the sensitivity of RT-IMSA method was comparable to that of RT-qPCR method. The plasma samples of 30 healthy people were negative and had no cross-reaction with dengue virus and Japanese encephalitis virus. Compared with the approved fluorescent quantitative PCR kit, the results of 81 clinical samples detected by RT-IMSA were 53 positive, 23 negative and 5 false negative. Sensitivity and specificity were 91.4% and 100%, respectively. As a result, the RT-IMSA approach offers fast and easy detection advantages over the high cost and complexity of RT-qPCR.