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目的建立一种基于实时荧光定量PCR技术的寨卡病毒检测方法。方法根据代表性亚洲型、非洲型寨卡病毒株NS5基因序列,设计特异性引物ZK-1F/R、ZK-2F/R;利用PCR、Real-time PCR方法对引物特异性、灵敏度、可重复性进行评价和优化;利用寨卡病毒感染细胞对本方法进行验证。结果 PCR结果表明两对引物的特异性良好,其Real-time PCR检测灵敏度分别为1.0×103copies/μL和1.0×101copies/μL,扩增效率分别为0.68和0.90,说明引物ZK-2F/R的综合效果优于ZK-1F/R,通过优化实验条件建立基于引物ZK-2F/R的实时荧光定量PCR检测方法,对含有寨卡病毒PRVABC59株和MR766株的样本进行检测,结果分别为9.0×104copies/μL、8.5×104copies/μL。结论本研究建立了一种检测寨卡病毒的方法,可用于临床患者ZIKV感染的检测和预防。
Objective To establish a method for detecting Zika virus based on real-time fluorescence quantitative PCR. Methods Specific primers ZK-1F / R and ZK-2F / R were designed according to the sequences of representative Asian and Zika virus NS5 genes. The specificity, sensitivity and repeatability of primers were determined by PCR and real-time PCR Sexual evaluation and optimization; the use of Zika virus-infected cells to verify the method. Results The PCR results showed that the specificity of the two primers was good. The detection sensitivity of Real-time PCR was 1.0 × 103 copies / μL and 1.0 × 101 copies / μL, respectively. The amplification efficiencies were 0.68 and 0.90, which indicated that the primers ZK-2F / R ZK-1F / R was better than ZK-1F / R. The ZK-2F / R-based real-time PCR method was established and the samples containing Zika virus (PRVABC59) and MR766 were tested. The results were 9.0 × 104 copies / μL, 8.5 × 104 copies / μL. Conclusion This study established a method for the detection of Zika virus, which can be used for the detection and prevention of ZIKV infection in clinical patients.