埃博拉病毒莱斯顿亚型实时荧光定量PCR检测方法的建立

发布时间：2018-01-05 21:41

  本文关键词：埃博拉病毒莱斯顿亚型实时荧光定量PCR检测方法的建立　出处：《病毒学报》2015年03期 　论文类型：期刊论文

  更多相关文章： 埃博拉病毒莱斯顿亚型 荧光定量PCR 病毒载量

【摘要】：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。
[Abstract]:The aim of this study was to establish a sensitive and rapid method. Quantitative detection of Ebola virus Leston subtype nucleic acid real-time fluorescence quantitative PCR detection method and kit. First select the conserved gene NP gene of Ebola virus Leston subtype as the target detection target. The conservative sequence was selected and a pair of specific primers were designed and synthesized. Then the recombinant plasmid with NP full-length gene was used as the quantitative standard, and the 10-fold diluted recombinant plasmid was amplified by fluorescence quantitative PCR. The standard curve was drawn, and the repeatability, sensitivity and specificity were detected. The results showed that the sensitivity of the established fluorescent quantitative PCR method for detection of Ebola virus Leston subtype could reach 102 copies / 渭 L. The linear relationship between different gradients was 0.997, the slope was -0.3101, and the amplification efficiency was 110.145%. All the standard samples showed sharp and narrow specific melting peak at 79.94 鈩，

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