CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

Abstract

Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials".The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium).The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation).The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand).Good agreement was observed between the reported results of eleven participants.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 (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 ).…”

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

“…A series of four key comparisons enabled NMIs and DIs to demonstrate and document their capabilities in measurements of the number of copies of specified intact sequence fragments extracted from different matrices and to determine their relative quantity. 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].…”

Improvement of digital PCR conditions for direct detection of KRAS mutations

“…Chemical analysis methods based on isotope‐dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) can be accurately calibrated with solutions of DNA 17,18 . Moreover, an international comparison study was conducted between metrology institutes using the dPCR method 19 . More recently, the droplet digital PCR (ddPCR) method was developed as a powerful analytical technique for clinical applications 20,21 .…”

“…17,18 Moreover, an international comparison study was conducted between metrology institutes using the dPCR method. 19 More recently, the droplet digital PCR (ddPCR) method was developed as a powerful analytical technique for clinical applications. 20,21 For example, ddPCR can be used to detect somatic mutations, amplifications, and deletions of specific genes.…”