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本研究的目的为建立一种灵敏、快速、定量检测埃博拉病毒莱斯顿亚型核酸的实时荧光定量PCR检测方法及试剂盒。首先选择埃博拉病毒莱斯顿亚型的保守基因NP基因作为检测靶目标,筛选出保守序列并设计合成一对特异性引物。然后将连有NP全长基因的重组质粒作为定量标准品,将10倍系列稀释的重组质粒进行荧光定量PCR扩增,绘制标准曲线,并进行重复性、灵敏度及特异性检测。结果显示建立的埃博拉病毒莱斯顿亚型荧光定量PCR检测方法,其灵敏度可达102拷贝/μL,不同梯度标准品间线性关系(R2)达0.997,斜率为-0.3101,扩增效率为110.145%,所有标准品均在79.94℃出现尖且窄的特异性熔解峰。利用该检测系统可以快速定量检测埃博拉病毒莱斯顿亚型核酸,灵敏度高、重复性好,可用于基础及临床实验室对埃博拉病毒莱斯顿亚型感染的快速诊断和临床效果的监测,具有实际的应用价值。
The purpose of this study is to establish a sensitive, rapid and quantitative detection of Ebola Reston subtype nucleic acid real-time fluorescence quantitative PCR detection kit and kit. Firstly, we selected the conservative gene NP gene of Ebola Reston subtypes as the target of detection, screened the conserved sequences and designed and synthesized a pair of specific primers. Then, the recombinant plasmid with NP full length gene was used as a quantitative standard. The 10-fold serial dilutions of the recombinant plasmids were amplified by fluorescence quantitative PCR and the standard curve was drawn. The repeatability, sensitivity and specificity of the recombinant plasmids were also tested. The results showed that the sensitivity of the established Reeseton subtype fluorescence quantitative PCR assay was 102 copies / μL. The linearity (R2) between different gradient standards reached 0.997 with a slope of -0.3101. The amplification efficiency was 110.145%. All standards showed sharp and narrow melting peaks at 79.94 ℃. The detection system can be used to rapidly and quantitatively detect Ebola Reston subtype nucleic acid with high sensitivity and repeatability. It can be used in the rapid diagnosis and clinical effect of Ebola Reston subtype infection in basic and clinical laboratories Monitoring, with practical value.