猴免疫缺陷病毒(SIV)实时荧光定量PCR检测方法的建立

Quantitation of simian immunodeficiency virus(SIV) proviral DNA load using real-time quantitative PCR with TaqMan probe

目的建立快速、敏感、特异的猴免疫缺陷病毒(SIV)TaqMan探针实时荧光定量PCR检测方法,对SIV病毒核酸进行定量检测。方法 RT-PCR扩增SIVmac251保守gag基因序列796 bp片段,进行TA克隆,构建标准品质粒pMD-SIVgag。通过对SIV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PCR方法的灵敏度、特异性和重复性。结果所建立的SIV QPCR检测方法,质粒DNA模板在107～102拷贝之间表现较好线性和相关性,标准曲线所得斜率为-3.26,相关系数为0.999。检测灵敏度达到200拷贝,方法重复性测试,检测25份临床样品CV%均小于1%。结论建立的SIV QPCR检测方法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)核酸拷贝量。

Objective To develop a rapid, sensitive and specific assay based on taqman probe real-time PCR to quantitate the proviral load of simian immunodeficiency virus （SIV）. Method A 796bp specific fragment was amplified with RT-PCR, then cloned into pMD19T vector to construct a recombinant plasmid pMD S1Vgag, which was used as SIV proviral DNA standards of this QPCR method. The QPCR system was optimized by using serial dilution of standards, and the sensitivity, specificity, repeatability and quantitation range of the method were also evaluated. Result The quantitative standard curve from 107 copies/well to 102 copies/well serial diluted plasmid DNAs showed that they had good linear correlation, the lowest limit of the standard curve reached 2 ~ 102 copies/well, the correlation （ r 〉 0. 999）. And coefficient of variability from 25 samples was below 1% （ CV 〈 1% ）. Conclusion The method showed high sensitivity, specificity and stability and will be used to quantify the SIV proviral DNA.