Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the <i>T‑nos</i>/<i>hmg</i> Copy Number Ratio in Genetically Modified Maize

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 <i>T-nos</i>/<i>hmg</i> 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 <i>T-nos</i>, 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% <i>T-nos</i>/<i>hmg</i> ratio. The specificity and applicability of the assay were established for the analysis of low <i>T-nos</i> 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 <i>T-nos</i>/<i>hmg</i> ratio