Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.
Anal Chem
; 88(1): 812-9, 2016 Jan 05.
Article
em En
| MEDLINE
| ID: mdl-26605751
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.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Plantas Geneticamente Modificadas
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Dosagem de Genes
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Zea mays
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Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Evaluation_studies
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2016
Tipo de documento:
Article