登革Ⅱ型病毒SYBRGreenⅠqRT-PCR方法的建立

Establishment of SYBR GreenⅠqRT-PCR for identification of dengue-2 virus

目的建立敏感、特异、快速的检测登革Ⅱ型病毒(DENV-2)SYBR GreenⅠqRT-PCR方法。方法根据GenBank上发表的DENV-2株核苷酸序列,应用Primer Express 3.0软件在保守区设计特异性引物,优化SYBR GreenⅠ荧光定量PCR反应条件,利用Sanger测序验证该方法的特异性和敏感性。结果该方法与其他血清型登革病毒、流感病毒等均无交叉反应,最低检出20个病毒拷贝/μl RNA。检测14份已知阳性和阴性血清,结果与预期一致。结论本研究建立的DENV-2 SYBR GreenⅠqRT-PCR特异性强、敏感性高,可用于输入性DENV-2早期感染诊断。

Objective We developed a method of identifying dengue-2 virus （DENV-2） using SYBR Green ⅠqRT-PCR. Methods All sequences in GenBank representing the Dengue-2 variants were downloaded. The DENV-2 specific primers were designed in the conserved region using Primer Express 3.0 software. SYBR Green Ⅰ qRT-PCR was optimized. Specificity and sensitivity were obtained and the corresponding amplicons were confirmed by Sanger sequencing. Results The established assay had no cross reaction with three other serotypes of Dengue, Influenza, Lassa, and yellow fever virus. The detection sensitivity of the optimized assay was 20 copies/μl of extracted RNA. Fourteen clinical samples confirmed beforehand were detected with this assay. The detection results were consistent with earlier results. Conclusion This SYBR Green Ⅰ based qRT-PCR assay was highly specific and sensitive. It has the potential for use in the rapid diagnosis of acute primary dengue infection in imported cases.