人4型腺病毒拷贝数SYBR Green Ⅰ实时定量PCR检测方法的建立

Development of SYBR Green Ⅰ Real-Time PCR Assay for Human Adenovirus Type 4 Copy Number

目的:建立检测人4型腺病毒拷贝数的荧光定量PCR方法。方法:提取本实验室构建的4型腺病毒全基因组质粒,以梯度稀释质粒为标准模板,选取人4型腺病毒六邻体区域基因设计一对特异性引物,进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线。结果:标准曲线为y=-4.284x+53.468,由全基因组质粒所构建的标准曲线线性关系良好,扩增反应Ct值与拷贝数的对数呈线性关系(R2=0.999 609),检出敏感度可达1×102拷贝/μL,且与其他几种腺病毒无交叉反应。用该方法检测4型腺病毒感染细胞2、12和24 h后的病毒拷贝数,其病毒拷贝数随时间增加,与细胞病变(CPE)变化保持一致,且荧光定量PCR的测量结果稳定(变异系数<6%)。结论:建立了检测人4型腺病毒基因拷贝数的荧光定量RT-PCR方法,该方法灵敏度高、特异性强。

Objective： To develop a method for detecting human adenovirus type 4（HAdV-4） copy number using real time quantitative PCR（RT-PCR）. Methods： The HAdV-4 whole genome plasmid was extracted and purified to 10 fold dilution series as the standard templates, primers were designed according to the hexon gene region. Then the standard curve for detecting HAdV-4 copy number was constructed. Results： The standard curve construeted by HAdV-4 whole genome plasmid reflected amplification reaction Ct value in linear with the virus DNA copy number（linearity： R2=0.999 609）. This method was used to detect the virus DNA copy number of the ceils infected with HAdV-4 at 2, 12 and 24 hours of infection, it can be seen that the copy number increased over time, which was in consistent with cytopathic effect（CPE）, and finally the stable data were got. Conclusion： The standard curve for quantitative detection of HAdV-4 gene has been constructed by fluorescence quantitative RT- PCR. It was sensitive and specific, accurate and reliable to detect HAdV-4 DNA copy number.