一种基于实时荧光定量PCR的寨卡病毒检测方法的建立

Establishment of a method based on real-time fluorescent quantitative PCR for detection of Zika virus

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 to detect Zika virus based on real-time PCR. Methods Specific primers were designed based on the conserved regions of Zika virus（ZIKV）NS5 gene sequence from representative Asian and African genetype and designated ZK-1F/R and ZK-2F/R. The specificity,sensitivity and repeatability of the method were evaluated and optimized using PCR and real-time PCR. The method was validated using ZIKV infected cells. Results Both of the two pairs of primers showed good specificity in PCR,while the sensitivity of the primers were 1.0×103copies/μL and1.0×101copies/μL,and the amplification efficiency was 0.68 and 0.90,respectively. These results indicated that primer ZK-2F/R was better than that of ZK-1F/R in ZIKV detection. Using the real-time PCR method based on primer ZK-2F/R,the extracts of cells infected with ZIKV Asian PRVABC59 strain and African MR766 strain were detected to contain 9.0×104copies/μL and 8.5×104copies/μL of ZIKV respectively. Conclusion The study established a ZIKV rapid detection method and can be used for ZIKV detection and prevention.