Final report of CCQM-K86.c. Relative quantification of genomic DNA fragments extracted from a biological tissue

Abstract

Key comparison CCQM-K86.c was performed to demonstrate the capacity of National Metrology Institutes (NMIs) and Designated Institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a canola powder. The study provides direct support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length up to 150 nucleotides following extraction from an unprocessed, high fat/oil ground seed matrix, with a copy number ratio from 0.001 to 1". The study was carried out under the auspices of the Nucleic Acids Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) and was jointly coordinated by the National Research Council of Canada (NRC) and the EU Joint Research Centre, Geel (JRC). The following laboratories (in alphabetical order) submitted measurement results in this key comparison study: Centro Nacional de Metrología, Mexico ("CENAM"); D.I. Mendeleyev Institute of Metrology, Russia ("VNIIM"); EU Joint Research Centre, Geel (JRC); Hong Kong Government Laboratory ("GLHK"); Instituto Nacional de Metrología de Colombia ("INM"); LGC (United Kingdom); National Institute of Biology, Slovenia ("NIB"); National Institute of Metrology, P.R. of China ("NIM China"/"NIMC" [figures]); National Institute of Metrology, Thailand (NIMT); National Measurement Institute, Australia ("NMIA"); National Metrology Institute of Japan, AIST, Japan ("NMIJ"); National Metrology Institute of Turkey ("TÜBITAK"). Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Expression of GM content in mass fraction from digital PCR data

“…Moreover, the parental origin of the MON810 trait could be verified by dPCR. The equivalence of measurement results obtained by dPCR and qPCR using an appropriate plasmidic calibrant has also been demonstrated by other studies ( Burns et al, 2006 ; Caprioara-Buda et al, 2012 ) including interlaboratory studies on different plant matrices ( Corbisier et al, 2012 ; Mester et al, 2020 ). The applicability of dPCR for the assessment of detection limits in GMO analysis has been reviewed ( Burns, Burrell, & Foy, 2010 ) and dPCR methods were successfully applied on food and feed samples in simplex ( Morisset, Štebih, Milavec, Gruden, & Žel, 2013 ), duplex ( Félix-Urquídez et al, 2016 ) and multiplex formats ( Dobnik, Spilsberg, Bogožalec, Holst-Jensen, & Žel, 2015 ; Dobnik, Štebih, Blejec, Morisset, & Žel, 2016 ).…”

Consistency plots: a simple graphical tool for investigating agreement in key comparisons

“…Figure 7a) shows results and uncertainties for one of two materials examined in key comparison CCQM-K86c, a comparison on determination of a specified gene target ratio in Brassica napus (canola) [22]; for the data, see table S3 in supplementary information. The CCQM-K86 range of studies [22][23][24] supports the quantification of proportion of genetically modified (GMO) material present in otherwise wild-type materials. For this material, the nucleic acid target was a fragment introduced in modification event RT73, conferring glyphosate resistance.…”

Metrological framework to support accurate, reliable, and reproducible nucleic acid measurements

“…In the first two studies, matrices were rich in polymeric carbohydrate (amylose and amylopectin) and poor in fat: maize (Zea maize L.) seed powder in the CCQM-K86 study [84] and rice (Oryza sativa L.) seed powder in the CCQM-K86.b study [85]. In the third study, CCQM-K86.c, high-fat/oil matrix was selected, represented by rapeseed/canola (Brassica napus L.) seed powder [86]. Currently, a study on measuring DNA in high-protein matrix (meat) is in progress.…”