TaqMan探针实时荧光定量RT-PCR法检测SIV/SHIV病毒RNA拷贝数方法的建立

Quantitation of Simian Immunodeficiency Virus (SIV) and SHIV Virus RNA Load Using Real-Time Quantitative RT-PCR with TaqMan Probe

目的建立TaqMan探针实时荧光定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)及SHIV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,通过对本室SIV/SHIV病毒RNA定量外标准品RS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4.60×101copies/μL,特异性及重复性良好,对同一样品进行16次重复检测,其循环阈值的平均标准偏差为0.066。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,可用于定量测定猴免疫缺陷病毒(SIV)及SHIV病毒RNA载量。

Objective To develop a real-time quantitative RT-PCR method with TaqMan Probe to assess the RNA load of SIV and SHIV. Methods The primers and TaqMan probe targeted at signature and conserved sequence of SIVmac239-gag gene were designed and applied to real-time RT-PCR assays. The reaction system, was optimized, and the sensitivity, specificity and reproducibility were evaluated. The gag gene was also amplified by conventional RT-PCR, then sensitivity of the two methods was compared. Results The sensitivity of this assay was 4.60 × 10^1 copies/μL. Further, the detection results showed high specificity and reproducibility. The standard deviation of sixteen times detection of one sample was 0.066. Conclusions The method showed high sensitivity, specificity and stability and will be used to quantify the SIV and SHIV RNA.