【摘要】 目的建立非洲型寨卡病毒(ZIKV)实时荧光定量PCR检测技术。方法人工合成非洲型ZIKV E基因上2 074～2 210 bp的保守片段,克隆到pUC57载体作为非洲型ZIKV质粒标准品;以108～102 copies/μl共7组质粒标准品制作标准曲线;设计的上游引物为5’-TCAAAGATGATGTTGGAGCT-3’、下游引物5’-CCTCTCACAGTGGCYTCA-3’、探针5’FAM-CCACCATTTGGGGATTCTTACATTGTC-BQ1-3’,采用实时荧光定量PCR检测其特异性、灵敏度及重复性。结果非洲型ZIKV质粒标准品浓度为76.61 ng/μl;以非洲型ZIKV质粒标准品制作标准曲线,在2.47×(108～102)copies/μl之间有较好线性关系,Rsq为1;能特异检测出非洲型ZIKV质粒标准品,与登革病毒1～4、亚洲型ZIKV质粒标准品、HCV-1b病人血清RNA无交叉反应,检测限为2.47×101copies/μl。结论本研究建立了可用于非洲型ZIKV实验室检测的实时荧光定量PCR技术。更多还原

【Abstract】 Objective To establish a method to detect African genotype Zika virus based on real-time fluorescence-based quantitative PCR. Methods The 2074-2210 bp conserved fragment of E gene was artificially synthesized and sequenced. The gene fragment was cloned into pUC57 recombinant plasmid to construct a standard recombinant plasmid. Specific primers and probes were designed based on this gene fragment: the upstream primer 5’-TCAAAGATGATGTTGGAGCT-3’, the downstream primer 5’-CCTCTCACAGTGGCYTCA-3’, and the probe 5’FAM-CCACCATTTGGGGATTCTTACATTGTC-BQ1-3’. The specificity, sensitivity and repeatability of this method were evaluated by real-time fluorescence-based quantitative PCR. Results The standard curve of the standard recombinant plasmid has a good linear relationship between2.47×108～102 copies/μl. This method can specifically detect the standard plasmid without cross reactions with Dengue type I～IV, Asian genotype Zika virus and HCV-1 b RNA. The detection limit of this method was 2.47×101 copies/μl. Conclusion The study established a high specificity, sensitivity and repeatability African genotype Zika virus detection method, which can provide support for prevention and control of African genotype Zika virus.更多还原