登革热病毒RNA实时荧光定量RT-PCR检测方法的建立

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。

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

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.