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Expression of GM content in mass fraction from digital PCR data
Nowadays the quantification of the content of genetically modified (GM) constituents in food or feed products is performed by using either quantitative real-time PCR (qPCR) or digital PCR (dPCR). The latter is increasingly used. Therefore, experimental protocols for the quantification of 52 GM event...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8756621/ https://www.ncbi.nlm.nih.gov/pubmed/35241875 http://dx.doi.org/10.1016/j.foodcont.2021.108626 |
Sumario: | Nowadays the quantification of the content of genetically modified (GM) constituents in food or feed products is performed by using either quantitative real-time PCR (qPCR) or digital PCR (dPCR). The latter is increasingly used. Therefore, experimental protocols for the quantification of 52 GM events authorised in the EU have been converted into a digital format and minimum performance characteristics for dPCR methods are detailed. Because of the need to harmonise the transformation of PCR results between two different measurement scales, 50 conversion factors for Certified Reference Materials (CF(CRM)) have been experimentally determined by three and sometimes four independent expert laboratories. The uncertainty of each CF(CRM) has been estimated to express dPCR results in mass fraction with a consistent uncertainty contribution. In 38 out of 58 cases, the validated qPCR methods (for 52 event-specific and 6 taxon-specific measurements) could easily be transferred into dPCR methods by using the same oligo sequences, final oligo concentration or annealing temperatures for the dPCR procedure. Laboratories have nevertheless used different strategies to improve the resolution or to reduce the so-called rain in their dPCR outcome. Those modifications were needed for PCR procedures that could not be converted without changes into a digital format. Therefore, exclusion/quality criteria such as the maximum rate of partitions with intermediate fluorescence “rain”, the minimum resolution and repeatability are suggested for dPCR methods. The CF(CRM) determined in this study were generally in agreement with the declared zygosity of the GM parental donor for hemizygous maize events. In a limited number of GM events the CF(CRM) values were significantly different when measured with different maize-specific (ZmAdh1 or hmgA) genes. |
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