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Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

On 26 November 2021, the World Health Organization (WHO) classified variant B.1.1.529, reported earlier by the South African authorities as “Variant of Concern” with the name Omicron. The decision was taken because Omicron is bearing a number of mutations with a potential impact on its transmissibility, severity of disease following infection, as well as on the effectiveness of immune protection resulting from both vaccines and natural infection. In this paper, we present an in silico developed RT-PCR based detection method (named OmMet) that was designed to be highly specific for the detection of the Omicron variant. Bioinformatic prediction tests demonstrate that the sequences in the primer sets are highly accurate, and do not match with genetic sequences of other viruses, including other coronaviruses or SARS-CoV-2 variants. The methodology presented does not rely on S-gene target failure (SGTF) of existing RT-PCR assays, widely used currently, but allows the direct specific identification of the variant B.1.1.529 (Omicron).

The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format., JRC.F.5-Food and Feed Compliance

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).

The recent ruling of the European Court of Justice has confirmed that organisms obtained by mutagenesis techniques are genetically modified organisms (GMOs). However, in contrast to organisms originating from conventional mutagenesis techniques, those obtained by new mutagenesis techniques are not exempted from the obligations of the GMO EU regulatory framework. This ruling raises questions about the detectability of the corresponding GM food and feed products. A case study is used in this document to explain and discuss possibilities and limitations for the detection and quantification of (known and unknown) genetic modifications in plant products derived from new mutagenesis techniques. Many of the mutations induced by new mutagenesis techniques cannot be unequivocally distinguished from natural mutations because such genome editing technologies are able to create very precise and limited genome changes that mimic the result of potential naturally occurring mutations. Moreover, mutations obtained by genome editing technologies could also not be differentiated from those introduced by conventional mutagenesis techniques which have been incorporated in traditional breeding programs and are often not thoroughly documented. Products of genome editing could only be detected and identified in imports of commodity products by enforcement laboratories when prior knowledge on the altered genome sequence, a validated detection method with appropriate selectivity and certified reference materials are available, similarly as required for the authorisation of current transgenic GMOs. However, when the modification involves only a SNP or few nucleotide changes, it would not be possible to identify whether the mutation originated spontaneously or was induced by conventional or new (genome editing) mutagenesis techniques. Moreover, it is unlikely that methods for the quantification of GMO products with small genome modifications in complex food or feed materials provide the level of selectivity needed for the enforcement of legislation, such as the one on labelling. In the absence of prior knowledge on the genome-edited changes, it is likely that non-authorised genetically modified food and feed products obtained by genome editing would enter the EU market undetected. The EU control system for GMOs and corresponding food and feed products may not function as efficiently for unauthorised genome-edited products compared to transgenic GMOs. In particular, the principle of zero tolerance for unauthorised GMO on the EU market is more difficult to maintain., JRC.F.5-Food and Feed Compliance

This report describes the production of a set of Certified Reference Materials (CRMs), ERM 440a, b, c, d and e, which are certified for their DAS-81910-7 cotton (unique identifier DAS-8191Ø-7) event mass fractions. These materials were produced following ISO Guide 34:2009 [1] and are certified in accordance with ISO Guide 35:2006 [2]. Genetically modified (GM) cotton seeds of the DAS-81910-7 event and seeds from a non GM cotton variety were milled to obtain GM and non-GM seed powders with a similar particle size distribution. Mixtures of non-GM and GM cotton seed powder were prepared gravimetrically. The certified values were obtained from the gravimetric preparations, taking into account the water mass fractions of the two powder materials and the genetic purity with respect to the DAS-81910-7 cotton. The certified values were confirmed by event-specific real-time PCR as an independent verification method (measurements were within the scope of accreditation to ISO/IEC 17025:2005 [3]). The uncertainties of the certified values were estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) [4] and include uncertainties relating to possible inhomogeneity (Section 4), instability (Section 5) and characterisation (Section 6). The materials are intended for the calibration or quality control of real-time PCR measurements to identify DAS-81910-7 cotton and quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs are available in glass bottles containing at least 1 g of dried cotton seed powder, sealed under an atmosphere of argon. The minimum amount of sample to be used for extraction of the DNA is 200 mg., JRC.F.6-Reference Materials

This report describes the production of a set of Certified Reference Materials (CRMs), ERM BF410ap, bp, cp, dp and ep, which are certified for their GTS 40-3-2 soya bean (unique identifier MON-Ø4Ø32-6) event mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. The materials are intended for the calibration or quality control of real-time PCR measurements to identify GTS 40-3-2 soya bean and quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials panel., JRC.F.6-Reference Materials

This report describes the production of a set of Certified Reference Materials (CRMs), ERM-BF412ak, bk, ck, dk and ek, which are certified for their Bt11 maize (unique identifier SYN-BTØ11-1) event mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. Genetically modified (GM) maize seeds of the Bt11 event and seeds from a non GM maize variety were milled to obtain GM and non-GM seed powders with a similar particle size distribution. Mixtures of non-GM and GM maize seed powder were prepared gravimetrically. The certified values were obtained from the gravimetric preparations, taking into account the water mass fractions of the two powder materials and the genetic purity with respect to the Bt11 maize. The certified values were confirmed by event-specific real-time PCR as an independent verification method (measurements were within the scope of accreditation to ISO/IEC 17025:2005). The uncertainties of the certified values were estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible inhomogeneity (Section 4), instability (Section 5) and characterisation (Section 6). The materials are intended for the calibration or quality control of real-time PCR measurements to identify Bt11 maize and quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs are available in glass bottles containing at least 1 g of dried maize seed powder, sealed under an atmosphere of argon. The minimum amount of sample to be used for extraction of the DNA is 200 mg., JRC.F.6-Reference Materials

It is important to guarantee that results expressing the GM content are reliable, comparable and fulfil the requirements of existing EU legislation. The use of different measurement units to express a GM content, the appearance of new analytical methods that do not require a calibrant and the composite EU legislation on GMOs have triggered the need for a document to clarify how to obtain reliable and comparable results. For this guidance document, past and current EU legislations have been reviewed with a special emphasis on what is meant by 'GM percentage' in the different legal texts. The metrological traceability of measurement results and the currently available guidance are explained and summarised. The particular case of botanical impurities and the genetic constitution of GM seeds are described and illustrated to better understand the complexity hidden behind this type of analysis. An overview of the different analytical methods based on DNA measurements and used for the expression of quantitative GM content results is provided, including the use of new techniques based on digital PCR (dPCR). A measuring system that allows for comparing results by making them traceable to the same reference system has been elaborated in detail. Needs and tools are described and a solution has been proposed to convert results expressing GM content to the required measurement unit, whenever this is needed. By following these recommendations, results obtained in GM copy number per haploid genome equivalent (cp/HGE) by dPCR can be converted into mass fraction percentage and compared to the results obtained by quantitative PCR (qPCR) either with a calibrant certified for its GM mass fraction or with a calibrant certified for its GM purity. The general principle is to relate a measurement result to a GM quantity embedded in a specified certified reference material (CRM) either directly or via one single conversion factor (CF) per event. This conversion factor and its related uncertainty need to be determined precisely for each CRM batch, preferably on the pure GM CRM (100 %), using, for example, dPCR. The estimated uncertainty associated with this conversion factor must be integrated into the measurement uncertainty of the final results expressed in GM mass fraction. CF are currently not yet established for most CRMs. CF values have been recently reported in a few pioneer dPCR studies. However, such proof of concept studies remain incomplete. Therefore, to avoid a gap between new technologies and current EU regulation, the working group recommends to launch a dedicated study to determine CF values. Such a study should involve a limited number of competent laboratories with a proven experience in dPCR. The study could be coordinated by the EURL-GMFF., JRC.F.5-Food and Feed Compliance

The European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF) organised a comparative test (CT) for National Reference Laboratories (NRLs) to support the official controls on food and feed in line with Regulation (EC) No 882/2004. Other official control laboratories were allowed to participate on a voluntary basis. Two test items were distributed: a soya milk powder spiked with soybean GM event DAS-444Ø6-6 (Test Item 1, T1) and a maize flour containing maize event VCO-Ø1981-5 (Test Item 2, T2). Participants were required to screen T1 and T2 for the presence of three GM soybean events and three GM maize events, respectively, and to quantify those events identified. The results had to be reported in GM mass/mass %. Eighty-three participants from 38 countries participated to this CT round, including 55 NRLs, of which 33 are designated in line with Regulation (EC) No 882/2004 (NRL/882) and 22 are nominated in Regulation (EU) No 120/2014 to support the EURL GMFF on method validation (NRL/120). The qualitative results, i.e. the correct identification of the GM events, were evaluated and scored as correct or incorrect. The assigned value for the 44406 soybean mass fraction in the soy milk material was derived as the robust mean of the data provided by NRLs, while for VCO-1981, the certified value was set as the assigned value. z and ζ scores were calculated to assess laboratory performance. The results reported indicate that all NRLs identified the correct GM events in both test items and most of the quantitative results were satisfactorily. Five laboratories, including one NRL/882, obtained an unsatisfactory z score for the quantification of 44406 soybean in soya milk powder. All z scores for VCO-1981 maize content in maize flour were satisfactory. A total of 19 and 7 unsatisfactory ζ scores were obtained for events 44406 and VCO-1981, respectively. At least for a number of laboratories this was the due to an underestimation or overestimation of the measurement uncertainty or a failure to report it. A root-cause analysis will be requested from NRLs having reported unsatisfactory results in this CT round and will be followed-up., JRC.F.5-Food and Feed Compliance