Félix-Urquídez Dalmira, Pérez-Urquiza Melina, Valdez Torres José-Benigno, León-Félix Josefina, García-Estrada Raymundo, Acatzi-Silva Abraham
Research Center for Food and Development, Culiacán, Sinaloa México.
National Metrology Center, El Marqués, Querétaro México.
Anal Chem. 2016 Jan 5;88(1):812-9. doi: 10.1021/acs.analchem.5b03238. Epub 2015 Dec 9.
Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%-100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio.
需要有证标准物质（CRM）来确保分析测量的可靠性。转基因生物（GMO）领域中现有的CRM通过实时聚合酶链反应（qPCR）进行表征。由于该技术依赖于校准物，其应用受到限制。本研究的目的是获得一种计量学质量更高的方法，用于表征玉米中T-nos/hmg拷贝数比含量的CRM。通过中心复合设计开发并优化了一种双重液滴数字PCR（ddPCR）检测方法。所开发的方法实现了11 cP T-nos的绝对检测限（LOD）、0.034%的相对LOD、23 cP的定量限（LOQ）（相对LOQ为0.08%）以及0.08%-100% T-nos/hmg比的动态范围。该检测方法的特异性和适用性已通过对几个玉米品种中低T-nos浓度（0.9%）的分析得以确立。通过酶切分析证实了DNA消化在多拷贝结合情况下减少测量偏差的便利性。鉴于其整体性能，该方法可用于表征T-nos/hmg比含量的CRM候选物。
