NASBA方法制备HIV/SHIV RNA定量测定标准品及其应用

Preparation of RNA Standards for Quantification of Simian Immunodeficiency Virus(SIV) Load Using NASBA Technique

目的应用NASBA方法制备SIV／SHIV RNA定量测定标准品。方法应用NASBA方法直接扩增SIVmac251病毒gag基因上1476～1685之间的片段，扩增的RNA产物（RS-NASBA）纯化后10倍系列稀释，测定定量曲线、标准曲线，测定该标准品的稳定性和重复性。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCRKit，该标准品可精确定量到2．033×10 copies／μL。结论外标准品RS．NASBA纯度高，稳定性好，可用于定量测定SIV／SHIV RNA拷贝数。

Objective To prepare standards for quantifying the viral load of SIV/SHIV using NASBA technique. Methods A specific fragroent（ 1476 - 1685 ） located at gag gene of SIVmac251 was amplified exponentially in vitro under isothermal conditions by using three enzymes essential for retroviral replication： reverse transcriptase, RNase H, and a DNAdependent RNA polymerase. Ten million-fold amplifieatin occurred after 1.5 hours incubation, lO-fold serial dilutions of the RNA products （RS-NASBA） were quantified the actual copy numbers using real-time quantitative RT-PCR with SYBR green I （Roche LightCycler） . The specification of amplified products was checked by melting curve analysis. Results From 2.033 ×107 copies/μ L to 2.033 ×10 copies/μL of RS-NASBA in lO-fold serial dilutions could be quantified by real-time quantitative RT-PCR. The standard curves showed that they had good linear correlation and can be used to serve the quantification of other samples. Conclusions The RS-NASBA could be used as an external standards for quantitative measurement of the SIV/SHIV RNA copy number.