登革热病毒RNA实时荧光定量RT-PCR检测方法的建立  

Development of Real- Time Quantitative RT- PCR for the Detection of Dengue Virus

作　　者：朱玉兰[1] 王佃鹏[1,2] 甄胜西[1] 黄宗炎[1] 刘胜牙[1] 高朝贤[1] 

机构地区：[1]深圳出入境检验检疫局国际旅行卫生保健中心,深圳518033 [2]内蒙古察右后旗医院,乌兰察布012400

出　　处：《热带医学杂志》2009年第11期1244-1246,1261,共4页Journal of Tropical Medicine

基　　金：国家质检总局课题(No.2002-ik102);深圳出入境检验检疫局科研计划项目(No.SZ2008011)

摘　　要：目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒(DV)及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。Objective To develop a real-time quantitative RT-PCR method with TaqMan Probe to assess the RNA copy number of Dengue virus. Methods Primers and TaqMan probe targeting the signature and the conserved sequence of four the Dengue virus subtypes were designed. The reaction protocol was optimized. The sensitivity, specificity and reproducibility were then evaluated. Conventional RT- PCR was also used to compare for the sensitivity of this method. Results The sensitivity of this assay was 1 × 10^3 copies/mL. The detection method was found to be highly specific and reproducible. The standard deviation from five separate measurements of the same sample was 0.792. Conclusion The developed method can be used to quantitatively measure the copy number of Dengue virus RNA.

关 键 词：登革热 病毒载量 实时定量RT-PCR TAQMAN探针 

分 类 号：R373.33[医药卫生—病原生物学]