定量PCR技术快速分析噬菌体展示肽库的应用  

Using Quantitative PCR-based Approach for Rapid Phage Display Analysis

作　　者：邬兰[1] 杜同信[1] 俞杨[1] 焦杰[1] 王自正[1] 

机构地区：[1]核医学科实验诊断部分子诊断室,南京医科大学附属南京医院/南京市第一医院,江苏南京210006

出　　处：《标记免疫分析与临床》2016年第10期1207-1211,共5页Labeled Immunoassays and Clinical Medicine

基　　金：南京市医学科技发展资金(卫生青年人才培养项目)资助项目

摘　　要：目的建立无需转染菌株的快速定量方法,用以分析噬菌体展示肽库筛选。方法选取Ph.D.-C7C噬菌体展示肽库,进行梯度稀释并提取基因组,在其载体序列上设计一对通用引物,运用SYBR Green的方法进行荧光定量PCR扩增,通过计算该方法的特异性、精密度和线性范围来确定该检测方法的可行性。结果通过溶解曲线得知该对引物可特异性筛选噬菌体展示七肽库。通过计算每个浓度的Ct值及其偏差可发现,该体系在2×10~4pfu/m L^2×10^(10)pfu/m L的浓度范围内线性关系良好,且在2×10~5pfu/m L^2×10^(10)pfu/m L的浓度范围内批间不精密度小于15%。结论建立的荧光定量PCR体系可以避免转染菌株所带来的繁琐人工劳动,并可以特异、准确、快速的对噬菌体展示肽库进行定量分析。Objective To establish quantitative PCR-based approach for rapid phage display analysis without transfecting strains. Methods The Ph. D.^TM-C7C phage display peptide library was applied with gradient dilution and DNA was extracted. A pair of general primers were designed by the vector＇ s sequence for the SYBR Green fluorescence quantitative PCR. The specificity, accuracy and linear range were calculated to determine the feasibility of this approach. Results The melting curve showed that the pair of general primerscan specifically screen this phage display peptide library. The biases calculated by each concentration＇s Ct values suggested a good linear correlation in the range of 2 × 10^4pfu/mL -2 × 10^10 pfu/mL concentration. And in the range of 2 × 10^5pfu/mL -2 × 10^10 pfu/mL concentration the non-precision is less than 15%. Conclusion This research established a faster, less labor intensive, more accurate and specific analysis of screening phage display peptide library by using quantitative PCR approach, which avoids transfecting strains.

关 键 词：噬菌体展示 实时荧光定量聚合酶链反应 文库筛选 

分 类 号：R440[医药卫生—诊断学]