用于CMV启动子序列检测的绝对定量PCR方法的建立及验证  

Establishment and Qualification of a RT-PCR Method for CMV Promoter Nucleic Acid Sequences

作　　者：苗玉发[1] 王三龙[1] 汪巨峰[1] 李波[1] 

机构地区：[1]中国食品药品检定研究院,北京100176

出　　处：《医学研究杂志》2012年第9期46-48,共3页Journal of Medical Research

基　　金：国家"重大新药创制"科技重大专项基金资助项目(2008ZX09305-002)

摘　　要：目的建立和验证用于CMV启动子序列检测的绝对定量PCR方法。方法针对CMV启动子序列设计探针和引物,用SYBR Green I熔解曲线分析引物扩增的特异性。对反应体系进行系统优化。用含CMV启动子的基因治疗产品和质粒标准品验证建立的PCR方法。结果上游引物为5'-AGACTTGGAAATCCCCGTGAGT-3',下游引物为5'-CGTATTAGTCATCGC-TATTACCATGGT-3',探针为5'-AACCGCTATCCACGCCCATTGATG-3'。扩增的特异性较强,反应体系优化后能达到较高的灵敏度。Ct值的变异系数都小于2%。标准曲线的定量范围为1.5×102～1.5×107拷贝,灵敏度为30拷贝。质粒标准品和基因治疗产品的扩增效率相近。结论建立了检测CMV启动子序列的绝对定量PCR方法。Objective To establish and qualify a RT - PCR method for CMV promoter nucleic acid sequences detection. Methods Probe and primers were designed according to CMV promoter sequences. The amplification specificity was analyzed using SYBR Green I dissociation curve. The reaction system was optimized. The RT - PCR method was qualified using a gene therapeutic product containing CMV promoter and plasmids standard article. Results Forward primer sequences were 5′- AGACTTGGAAATCCCCGTGAGT - 3′; reverse primer sequences were 5′-CGTATTAGTCATCGCTATTACCATGGT -3′; probe sequences were 5′-AACCGCTATCCACGCCCAT- TGATG - 3′. The specificity of amplification was good,and highly sensitivity was achieved when the concentrations of primers, probe, enzyme and magnesium in the reaction system were optimized. The CV for Ct was below 2%. Quantification margin was between 1.5 × 10^2 and 1.5 × 10^7 copies, and sensitivity of the reaction reached 30copies. The amplification efficiency of plamids and geue therapeutic product was similar. Conclusion The method that could absolutely quantify the copies of CMV promoter sequences was established.

关 键 词：实时定量PCR 优化 CMV启动子 质粒 

分 类 号：R155.5[医药卫生—营养与食品卫生学]