南方医科大学学报2011，Vol.31Issue(4)：587-593,7.
转基因番茄Zeneca B,Da,F实时荧光定量PCR检测体系的建立
Construction of quantitat ive real-time PCR detection system of transgenic tomato line Zeneca B, Da,F
摘要
Abstract
To construct the plasmid reference molecules for detection of transgenic tomato line Zeneca B,Da,F using quantitative real-time PCR(qPCR). Methods Three plasmid reference molecules pEasy-T3-APX, pEasy-T3-16A and pEasy-T3-16S were cloned based on reverse genetics, which contain the target fragments of tomato endogenous reference gene apx (ERG-apx), gene-specific sequence of pg(GS-pg) and construct-specific sequence of vectors pJR16S/ pJR16A (CS-16S/CS-16A) of Zeneca B,Da,F, respectively. Primers and Taqman probes were designed by Beacon Designer 7.5.The specificity, sensitivity, reproducibility and the limit of detection(LOD) of the qualitative and quantitative PCR system based on the plasmid reference molecules were evaluated. PicoGreen was used to measure the DNA concentration of the plasmid reference molecules. Two sets of samples containing 1% or 0.1% (w/w) pEasy-T3-16A or pEasy-T3-16S mixed with pEasy-T3- APX as background DNA were prepared for evaluating the efficacy of the qPCR system. Results The target fragments for qPCR detection were anchored, ERG-apx 108 bp, GS-pg 108 bp , CS-16S 109 bp and CS-16A 102 bp. The three plasmid reference molecules were confirmed at the expected sizes by restriction enzyme digestion. The qPCR results showed that the RSD of reproducibility were 0.2% tol.5%, LOD was 25 copies, R2 values for these standard curves were 0.994 -0.998 and amplification efficiencies were 93.3% ~102.4% .The bias between the test and true values of two sets of mixed samples ranged from -9.3% to 14.7% after adjusting by conversion factors(Cf). Conclusion The plasmid reference molecules and qPCR system for qualitative and quantitative detection of transgenic tomato line Zeneca B,Da,F have been established successfully.关键词
质粒标准分子/转基因番茄/核酸检测技术/荧光定量PCRKey words
plasmid reference molecule/ transgenic tomato/ nucleic acid testing/ Quantitative real-time PCR分类
生物科学引用本文复制引用
麦荣嘉,周晓红,陈敏芳,莫倩珍,胡良勇,黎事伦,唐敏然,顾晓敏,蔡永洪,周伦彬..转基因番茄Zeneca B,Da,F实时荧光定量PCR检测体系的建立[J].南方医科大学学报,2011,31(4):587-593,7.基金项目
国家科技支撑计划(2008BAK41B01) （2008BAK41B01）
国家自然科学基金(30872204,81071378) （30872204,81071378）
广东省自然科学基金(5004747) （5004747）
