【摘要】 目的建立SYBR GreenⅠ荧光染料实时定量PCR方法,测定猴免疫缺陷病毒(SIV)前病毒DNA。方法巢式RT-PCR扩增SIVmac251病毒RNA gag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEM T载体上,构建pGEM-SIVgag477质粒。该质粒经大量扩增纯化后定量,10倍系列稀释后,做出标准曲线,作为SIV前病毒DNA荧光定量检测的外标准品。结果应用Roche公司FastStart DNA Master SYBR GreenⅠKit,该标准品可精确定量到10 copies/μL。结论制备的pGEM-SIVgag477质粒外标准品纯度高,SYBR GreenⅠ荧光染料实时定量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)前病毒DNA载量。更多还原

【Abstract】 Objective To develop a real-time quantitative PCR method with SYBR Green I to assess the proviral DNA of SIV.Methods A specific fragment in size of 477bp was amplified with nested RT-PCR and ligated into a pGEM T vector.The recombinant pGEM-SIVgag477 was transformed into E.coli DH5α competent cells.Large amount of cells were collected and purified after culturing in the LB medium.10-fold serial dilutions of the plasmid DNAs were quantified the actual copy numbers using real-time quantitative PCR with SYBR Green I by Roche LightCycler.Results From 1×10A8 copies/μL to 10 copies/μL of 10-fold serial diluted plasmid DNAs could be quantified with real-time quantitative PCR.The quantitative standard curve showed that they had good linear correlation and could be served for the quantification of other samples.The specificity of amplified products was checked by melting curve analysis.Conclusions The recombinant pGEM-SIVgag477 could be used as an external standards of the real-time quantitative PCR method with SYBR Green Ⅰ.The method showed high sensitivity,specificity and stabilityand will be used to quantify the SIV proviral DNA.Therefore it can detect infected cells with active virus replication and infected cells at transcription latent period as well,such as the early or the late stage of SIV infection when the viral load in the plasma is rather low or the virus is in a latent period.It may play an important and necessary role in the use of SAIDS model.更多还原