Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Biotechnology Department, National Reference Laboratory for Genetically Modified Organisms (GMO) Analysis, Rome, Italy.
J Agric Food Chem. 2009 Dec 9;57(23):11086-91. doi: 10.1021/jf902560x.
The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.
报告了针对玉米（Zea mays）内源序列的五种实时聚合酶链反应（PCR）方法的比较。PCR 的目标是三种方法的醇脱氢酶（adh）基因和另外两种方法的高迁移率族（hmg）基因。在几种经过基因改造（GM）玉米认证参考物质（CRM）的混合 DNA 模板和单个 CRM DNA 提取物上，在重复条件下，对这五种实时 PCR 方法在几个稀释水平下进行了检查。对同一方法内以及所有五种方法之间获得的采用回归模型的所有曲线的斜率和 R(2)系数进行了比较，并对每个 PCR 系统的检测限和定量限进行了分析。此外，基于在每个浓度水平估计目标单倍体基因组拷贝数的能力，评估了方法等效性。结果表明，在所测试的五种方法中，一种 hmg 靶向 PCR 系统可以被认为与其他方法等效，但在稀释范围内显示出最佳的回归参数和更高的可重复性。因此，建议将其作为一种有效的模块与不同事件特异性的实时 PCR 相结合，用于玉米转基因生物（GMO）定量。讨论了对 GMO 分析控制的实际改进。
