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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 To establish a sensitive, specific and rapid SYBR GreenⅠqRT-PCR method for detecting Dengue virus type 2 (DENV-2). Methods Based on the nucleotide sequence of DENV-2 strain published in GenBank, Primer Express 3.0 software was used to design specific primers in conserved regions. SYBR GreenⅠ fluorescence quantitative PCR was optimized. The specificity and sensitivity of this method were verified by Sanger sequencing. Results The method did not cross-react with other serotypes of dengue virus, influenza virus and so on, and detected a minimum of 20 copies of virus RNA per μl. Fourteen known positive and negative sera were tested and the results were as expected. Conclusion The DENV-2 SYBR GreenⅠqRT-PCR established in this study is highly specific and sensitive and can be used to diagnose early DENV-2 infection.