SYBR Green I实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

Quantification of Simian Immunodeficiency Virus(SIV) Viral Load Using Real-Time Quantitative RT-PCR with SYBR Green I

目的建立SYBR Green I荧光染料实时定量RT-PCR方法,测定猴免疫缺陷病毒(SIV)RNA拷贝数。方法巢式RT-PCR扩增SIV病毒RNAgag基因上1360-1837之间的长度为477 bp的片段,将该片段克隆到pGEMT载体上,构建pGEM-SIVgag477质粒。该质粒经限制性内切酶NotⅠ酶切后,进行体外转录,转录出的RNA产物(RS)纯化后10倍系列稀释,作出标准曲线,作为SIV病毒RNA荧光定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCR Kit,该标准品可精确定量到100 copies/μL。结论制备的RS外标准品纯度高,SYBR Green I荧光染料实时定量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)RNA拷贝数。

Objective To develop a real-time quantitative RT-PCR method with SYBR Green Ⅰ to assess the viral load of SIN. Methods A 477 bp specific fragment was amplified with nested RT-PCR and ligated into a pGEM T vector. The recombinant pGEM-SINgag477 was transformed into E. coli DH5α competent ceils. Large amount of ceils were collected and purified after cultured in LB medium. Plasmids DNA were digested by restriction enzyme Not Ⅰ to hnearize DNA after purified from the cells. The linearized plasmids DNA were transcripted into RNA using riboprobe in vitro transcription systems （Promega, P1420）. 10-fold serial dilutions of the RNA standards were quantified and the actual copy numbers were assessed using real-time quantitative RT-PCR with SYBR Green 1 by Roche LightCycler. Results From 1 ×10^8 copies/μL to 1×10^2 copies/μL of 10-fold serial diluted RNA could be quantified with real-time quantitative RT-PCR. The standard curve showed that they had good hnear correlation and could be served for the quantification of other samples. The specification of amplified products is checked by melting curve analysis. Conclusions The RNA standards could be used as an external standards of the real-time quantitative RT- PCR method with SYBR Green Ⅰ. The method has high sensitivity, specificity and stability so that it could be used for the quantification of SIN RNA load.