title: Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.
abstract: 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.
authors.affiliation: Research Center for Food and Development, Culiacán, Sinaloa México.|National Metrology Center, El Marqués, Querétaro México.
authors.forename: Dalmira
authors.initials: D
authors.lastname: Félix-Urquídez
authors.affiliation: National Metrology Center, El Marqués, Querétaro México.
authors.forename: Melina
authors.initials: M
authors.lastname: Pérez-Urquiza
authors.affiliation: Research Center for Food and Development, Culiacán, Sinaloa México.
authors.forename: José-Benigno
authors.initials: JB
authors.lastname: Valdez Torres
authors.affiliation: Research Center for Food and Development, Culiacán, Sinaloa México.
authors.forename: Josefina
authors.initials: J
authors.lastname: León-Félix
authors.affiliation: Research Center for Food and Development, Culiacán, Sinaloa México.
authors.forename: Raymundo
authors.initials: R
authors.lastname: García-Estrada
authors.affiliation: Reference National Center for Detection of Genetically Modified Organisms, Tecámac, Estado de México México.
authors.forename: Abraham
authors.initials: A
authors.lastname: Acatzi-Silva
mesh_terms: D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics*; D010316:Particle Size; D030821:Plants, Genetically Modified / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D013499:Surface Properties; D013696:Temperature; D003313:Zea mays / Q000235:genetics*
chemical_list: D018744:DNA, Plant