Detection of microorganisms, obtained by new genomic techniques, in food and feed products.
Date: 2025-10-02
Institutions: De Loose, Marc, Demsar, Tina, Fraiture, Marie Alice, Joint Research Centre, Roosens, Nancy, Zaoui, Xavier
Subjects: animal nutrition, food control, food industry, foodstuff, foodstuffs legislation, genetically modified organism, microorganism, report, research method

ISSN 1831-9424 

Detection of microorganisms, 
obtained by new genomic techniques, in 
food and feed products 

Sowa, S., Broothaerts, W., Burns, M., De Loose, M., 
Debode, F., Demsar, T., Fraiture, M.A., Prins, T.W., 
Roosens, N., van Der Berg, J.P., Zaoui, X. 

2025 

 EUR 40473 

 
 
  
 
 
 
This document is a publication by the Joint Research Centre (JRC), the European Commission’s science and knowledge 
service. It aims to provide evidence-based scientific support to the European policymaking process. The contents of this 
publication do not necessarily reflect the position or opinion of the European Commission. Neither the European 
Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of this 
publication. For information on the methodology and quality underlying the data used in this publication for which the 
source is neither Eurostat nor other Commission services, users should contact the referenced source. The designations 
employed and the presentation of material on the maps do not imply the expression of any opinion whatsoever on the 
part of the European Union concerning the legal status of any country, territory, city or area or of its authorities, or 
concerning the delimitation of its frontiers or boundaries. 

Contact information 
Name: Name: European Network of GMO Laboratories  
ENGL Secretariat  
Food & Feed Compliance Unit (F.5)  
Directorate F - Health and Food  
European Commission - Joint Research Centre (JRC)  
Via Enrico Fermi 2749. TP 201  
I-21027 Ispra (VA), Italy  

E-mail: JRC-ENGL-SECRETARIAT@ec.europa.eu 

EU Science Hub 
Address:  
https://joint-research-centre.ec.europa.eu 

Email:  
JRC143597 

Tel.: 
EUR 40473 

PDF  

ISBN 978-92-68-32179-9   ISSN 1831-9424   doi:10.2760/1846532  

KJ-01-25-499-EN-N 

Luxembourg: Publications Office of the European Union, 2025  

© European Union, 2025  

The reuse policy of the European Commission documents is implemented by the Commission Decision 2011/833/EU of 12 December 2011 
on the reuse of Commission documents (OJ L 330, 14.12.2011, p. 39). Unless otherwise noted, the reuse of this document is authorised 
under the Creative Commons Attribution 4.0 International (CC BY 4.0) licence (https://creativecommons.org/licenses/by/4.0/). This means that 
reuse is allowed provided appropriate credit is given and any changes are indicated.  

For any use or reproduction of photos or other material that is not owned by the European Union permission must be sought directly from 
the copyright holders. 

- Cover page illustration, © Marina 2024, image 979453103. Source: https://stock.adobe.com/. 

How to cite this report: Sowa, S., Broothaerts, W., Burns, M., De Loose, M., Debode, F. et al., Detection of microorganisms, obtained by new 
genomic techniques, in food and feed products, Publications Office of the European Union, Luxembourg, 2025, 
https://data.europa.eu/doi/10.2760/1846532, JRC143597. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contents 

Abstract ...................................................................................................................................................................................................... 2 

Authors ........................................................................................................................................................................................................ 3 

Acknowledgements .............................................................................................................................................................................. 4 

Executive summary .............................................................................................................................................................................. 5 

Scope ............................................................................................................................................................................................................ 7 

1  Introduction ....................................................................................................................................................................................... 8 

2  Genetic modification techniques applied to microorganisms ............................................................................ 10 

3  Applications of NGTs in microorganisms ....................................................................................................................... 12 

4  Potential detection strategies .............................................................................................................................................. 13 

5  Challenges and additional considerations associated with detection of NGT microorganisms ..... 16 

6  Conclusions ..................................................................................................................................................................................... 18 

References ............................................................................................................................................................................................. 20 

List of abbreviations and definitions ...................................................................................................................................... 25 

List of tables ......................................................................................................................................................................................... 26 

Annex 1. Summary of analytical challenges for NGT plants. .................................................................................... 27 

1 

 
 
 
 
 
 
Abstract  

New genomic techniques (NGTs) can be used for the targeted modification of the genome, not only 
of plants but also of microorganisms. Traceability of genetically modified microorganisms obtained 
with an NGT (NGT microorganisms) is currently a regulatory requirement under the EU GMO 
legislation. This report highlights the possibilities and challenges to detect NGT microorganisms 
(NGT-Ms) in food and feed using analytical technologies. The document builds upon the conclusions 
of the previously published report on the detection of plant products developed by targeted 
mutagenesis and cisgenesis (ENGL, 2023a) and adds issues specific for the detection of NGT-Ms. 

2 

 
 
 
 
Authors 

The European Network of GMO Laboratories (ENGL) Working Group (WG) on New Mutagenesis 
Techniques was established based on a mandate adopted at the 43rd meeting of the ENGL Steering 
Committee on 09 June 2021. The WG accepted an extension of its mandate in 2023 to develop a 
report on the possibilities and challenges for the detection of microorganisms obtained by an NGT 
in or as food and feed. 

The working group was chaired by Sławomir Sowa (Plant Breeding and Acclimatization Institute - 
National Research Institute, PL).  

The members of the working group developing this report on NGT-Ms were (in alphabetical order):  

Wim Broothaerts, European Commission, Joint Research Centre - EURL GMFF (BE)  

Malcolm Burns (invited expert), LGC (UK)  

Marc de Loose, Institute for Agricultural, Fisheries and Food Research (ILVO) (BE)  

Frédéric Debode, Walloon Agricultural Research Centre (CRA-W) (BE) 

Tina Demšar, National Institute of Biology (SI)  

Marie Alice Fraiture, Sciensano (BE)  

Lelde Grantina-Ievina, Institute of Food Safety, Animal Health and Environment "BIOR" (LV)  

Theo W. Prins, Wageningen Food Safety Research (WFSR) (NL)  

Nancy Roosens, Sciensano (BE)  

Jan Pieter van der Berg, Wageningen Food Safety Research (WFSR) (NL) 

Xavier Zaoui, European Commission, Joint Research Centre - EURL GMFF (IT) 

3 

 
 
 
 
 
Acknowledgements  

The WG would like to thank the ENGL members for their comments, in particular A. Barbante, J. 
Lämke and S. Pecoraro. 

4 

 
 
 
Executive summary 

This report, compiled by the European Network of GMO Laboratories (ENGL), investigates the 
analytical possibilities and challenges related to the detection of microorganisms modified using 
new genomic techniques (NGT-Ms). Extending the previous report on the opportunities and 
challenges for the detection of plant-based food and feed resulting from targeted mutagenesis and 
cisgenesis (ENGL, 2023a), this report focuses on NGT-Ms, excluding those with insertion of foreign 
recombinant DNA. 

New genomic techniques, such as CRISPR-Cas and other techniques of genome editing, have 
become instrumental in the modification of microbial strains for use in food and feed. 
Microorganisms obtained through NGTs are also used in other sectors, including enzyme production, 
probiotics, biofertilizers with many products being developed outside the EU. These microorganisms 
are subject to traceability and labelling requirements under the current EU GMO legislation. This 
implies the necessity for accurate and reliable detection methods. However, placing on the market, 
traceability and labelling of GM food and feed within the EU requires not only precise detection but 
also analytical identification and quantification methods.  

The report classifies NGT-M products into four categories and focusses on Categories 3 and 4, 
characterised by residual DNA or viable organisms which can theoretically be monitored using DNA-
based detection techniques. Nevertheless, detecting NGT-Ms poses considerable analytical 
challenges. Certain modifications made by NGTs — such as single nucleotide variants (SNVs) or 
small insertions/deletions — are indistinguishable from natural mutations or those resulting from 
conventional mutagenesis techniques. This makes it difficult to trace the origins of genetic 
modifications and to ensure compliance with current GMO legislation. 

Polymerase Chain Reaction (PCR)-based approaches, including digital PCR (dPCR), continue to play a 
key role in regulatory testing by official control laboratories, although they may struggle to identify 
very small genetic changes with required precision. High-throughput sequencing (HTS) methods, 
such as whole genome sequencing (WGS), provide higher resolution and can accurately determine 
specific strains or different genetic modifications, especially in single-ingredient microbial samples. 
Nevertheless, HTS still faces obstacles in routine testing of complex food or feed mixtures and in 
accurate quantification, due to technical limitations and problems with data interpretation. 
Additional challenges relate to the ability of microorganisms to exchange DNA material through 
horizontal gene transfer, coupled to their natural genomic mutability. In the absence of reference 
databases recording such genetic diversity, differentiating between natural and engineered 
mutations becomes highly difficult. 

Overall, the authors acknowledge the technological advancement offered by targeted (e.g., PCR) and 
untargeted (e.g., sequencing) methods to detect uniquely modified NGT-Ms. With the growing use of 
NGTs, forthcoming traceability will require further developments in sequencing detection 
techniques, flanked by comprehensive databases and supported by standardised approaches. 
However, in alignment with the conclusions drawn on NGT plants (ENGL, 2023a), this report 
highlights the inability of these methods to detect and unambiguously identify unknown or small 
mutations in microorganisms, especially in the context of routine laboratory control.  

From a regulatory perspective, persistent research gaps regarding the minimum performance 
parameters of detection and identification methods in both plants and microorganisms pose 
challenges for the consistent evaluation and oversight of NGT-derived products. These gaps may 

5 

 
 
 
affect the ability to establish harmonised standards for method validation, comparability, and 
traceability, which are essential for regulatory compliance. 

6 

 
 
 
Scope 

In 2023, the ENGL revised its previous report (ENGL, 2019) on the possibilities and challenges for 
the detection of food and feed plant products obtained by targeted mutagenesis and cisgenesis 
(ENGL, 2023a). Similar techniques, and NGTs in general, are also used for the targeted modification 
of microorganisms. 

The current report focusses on the possibilities and challenges for the detection of microorganisms 
obtained through NGTs (NGT-Ms) in or as food and feed. The major aim is to evaluate to what 
extent the conclusions of the corresponding report on plant products could be extended to the 
detection of NGT-Ms. The presence of an NGT-M could result either from the intentional inclusion of 
the NGT-M, or the product it produced, as an authorised ingredient in the food or feed or from the 
unintended presence of an (un)authorised NGT-M used as processing aid during the production of a 
food or feed additive. In line with the mandate of the WG, the focus of this report is on NGT-Ms that 
do not contain inserted recombinant foreign DNA.  

7 

 
 
 
1 

Introduction 

There are many conventional techniques for the genetic modification of microorganisms with a long 
history of use (basic microbial genetics). Unlike in plants, homologous recombination has been 
widely used in microorganisms for the targeted introduction of new genes or the replacement of 
endogenous genes with copies from a related organism. To facilitate such genetic modifications, 
microorganisms have historically been manipulated using mechanisms such as conjugative plasmid 
transfer and cell fusion, which allow the introduction of foreign genetic material into the host 
(Shitut et al., 2022; Vasileva et al., 2022). Newer techniques, including genome editing, create 
additional opportunities, not only for producing small mutations and knockouts, but also for 
inserting longer DNA fragments by homologous recombination or transposition. Such new genomic 
techniques (NGTs) have become standard tools for microbial strain modification along with 
established techniques including conventional mutagenesis and recombinant DNA technology 
(Broothaerts et al., 2021). In some cases, the analytical differentiation between genetically modified 
microorganisms (GMMs) produced by established or new genomic techniques may be challenging or 
impossible, and the occurrence of natural mutations and horizontal gene transfer (HGT) may add to 
the complexity of analytical detection and identification. It needs to be stressed that insertion of 
foreign DNA with the use of NGTs would lead to the generation of conventional GMMs, for which 
event-specific methods can usually be developed for enforcement analysis. However, because of 
asexual reproduction and HGT, i.e. the inheritance of genetic material by transfer between different 
species without reproduction, the term foreign DNA is more difficult to define in microorganisms 
compared to plants1. For instance, bacteria can take up DNA from the environment 
(transformation), through direct cell-to-cell contact (conjugation) or through the involvement of 
bacteriophages mediating gene transfer between donor and recipient cells (transduction) (Arnold et 
al., 2021). HGT is not limited to bacteria but also occurs in other microorganisms and is considered 
a fundamental evolutionary molecular mechanism in microbial genetics (Li et al., 2023; Del Duca et 
al., 2022). Therefore, identifying whether the origin of a specific gene in a microbial species was the 
result of a natural process or was induced by an NGT can be challenging. 

The terminology used in this document is compliant with the terminology used in the report on NGT 
plants (ENGL, 2019; ENGL, 2023a). 

In 2011 EFSA published a guidance document on the risk assessment of GMMs and their products 
intended for food and feed use (EFSA GMO Panel, 2011). Depending on the type of product and the 
degree of purification, GMM-derived products were classified according to four categories. The 
same classification could be applied to NGT-Ms. 

Category 1: Chemically defined purified compounds and their mixtures in which both NGT-Ms 
and their DNA have been removed (e.g., amino acids, vitamins); 

Category 2: Complex products in which both NGT-Ms and their DNA are no longer present (e.g., 
cell extracts, most enzyme preparations); 

1 In the Commission’s NGT proposal, foreign DNA is defined as “DNA produced by whatever means outside a recipient 

organism from a donor organism that is sexually incompatible (non-crossable)” 

8 

 
 
 
 
 
Category 3: Products derived from NGT-Ms in which NGT-Ms capable of multiplication or of 
transferring genes are not present, but in which their DNA is still present (e.g., heat-inactivated 
starter cultures, biomasses single-cell protein preparations, and cell extracts); 

Category 4: Products consisting of or containing NGT-Ms capable of multiplication or of 
transferring DNA (e.g., alive starter cultures for fermented foods and feed). 

These categories are suitable for differentiating NGT-Ms-derived products for the purpose of 
conducting the risk assessment required for authorisation of GM food and feed in the EU. From the 
perspective of GMO traceability and official control, DNA-based analytical methods are mainly 
applicable to products from Category 3 and Category 4 NGT-Ms as they may contain DNA from 
NGT-Ms. These methods can also be applied to verify if living microorganisms and their DNA have 
been removed from Category 1 and Category 2 products. Living NGT-Ms present in Category 4 
products may also be detected through in vitro culture techniques on suitable media, provided that 
they harbour detectable phenotypic characteristics. Growing NGT-Ms may then be identified through 
a variety of techniques, including DNA genotyping by PCR or sequencing. However, this is beyond the 
scope of the current report, which focusses on identification of the NGT-induced modifications 
rather than the type of microorganism. 

9 

 
 
 
 
2  Genetic modification techniques applied to microorganisms 

Microorganisms have been utilised for food and feed production for thousands of years, long before 
their role in these processes was fully understood. Traditional products such as bread, beer, wine, 
vinegar, yogurt, and cheese were developed without knowledge of the microorganisms driving the 
fermentation. For decades, bacteria, filamentous fungi and yeasts have been harnessed for the 
production of food and feed components, including additives, enzymes, and other valuable 
biomolecules such as vitamins and amino acids, and in nature, microbes also play a crucial role in 
biomass recycling. 

Microorganisms evolve naturally through both mutations and horizontal gene transfer, where 
genetic material is exchanged through mechanisms such as transformation - the uptake of DNA 
from the environment (Dalia et al., 2014). This DNA may integrate into the recipient’s genome or 
persist as an independent plasmid, contributing to genetic diversity. Advancements in microbial 
research have enabled the cultivation of pure strains for breeding and mutagenesis to enhance 
desirable production traits. Species used in industrial production often undergo rounds of 
spontaneous or induced mutagenesis followed by selection and may be further bred or genetically 
engineered to optimise production yield. 

Before the advent of recombinant DNA technology (discussed in the upcoming ENGL GMM report) 
and NGTs, microbial strain improvement relied on spontaneous random mutagenesis, directed 
evolution under selective pressure, or exposure to mutagens such as chemicals and ionizing 
radiations to increase mutation rates. These early methods laid the foundation for modern genetic 
engineering approaches, which offer more precise and efficient microbial strain development 
strategies. 

In recent years, genome editing tools have been widely applied in microorganisms (for a recent 
review, see Lv et al., 2023). Early techniques such as zinc-finger nucleases (ZFNs) and transcription 
activator-like effector nucleases (TALENs) required complex customisation, whereas techniques 
based on clustered regularly interspaced short palindromic repeats (CRISPR), particularly CRISPR-
Cas9, offer a simpler and more efficient approach (Yang et al., 2025). CRISPR-Cas9 enables precise 
DNA modifications via double-stranded breaks (DSBs), repaired through non-homologous end 
joining or homologous recombination. 

New methods of targeted mutagenesis are being continuously developed e.g., base-editing tools 
that allow targeted mutations without DSBs or prime editing that enables all possible nucleotide 
conversions along with targeted insertions and deletions with higher accuracy. These tools have 
been established in industrially relevant fungi such as Saccharomyces cerevisiae (Lv et al., 2023), 
Yarrowia lipolytica (Bae et al., 2019), and Aspergillus species (Huang et al., 2019). Multiple 
approaches were also developed for the introduction of the CRISPR-Cas9 tools into the cells of an 
organism of interest, based on stable DNA integration systems, or transient plasmid expression 
vectors or ribonucleoprotein complexes. Genome editing in microorganisms enables gene insertions, 
deletions, and multiplex modifications, for instance to enhance biomolecule production. 
Furthermore, unlike plants, many microorganisms can be efficiently edited without the use of 
methods classified as NGTs (e.g. using homologous recombination, transposon mutagenesis, etc.), 
though CRISPR remains valuable for engineering certain strains. 

Overall, the genetic modifications found in NGT-Ms are similar to those in plants obtained by 
targeted mutagenesis and cisgenesis, and the analytical challenges, therefore, will also be similar 
as well. A key challenge is detecting unauthorised genome-edited microorganisms, particularly 

10 

 
 
 
those with small, marker-free nucleotide changes, as these modifications are indistinguishable from 
natural mutations, complicating PCR-or sequencing-based detection methods. 

11 

 
 
 
3  Applications of NGTs in microorganisms  

In September 2023, EFSA published a horizon scanning report (Ballester et al., 2023), which 
provided an overview of microorganisms and their products obtained by NGTs. In 2024, this report 
was instrumental for publication of a scientific opinion by EFSA on the risk assessment of NGT-Ms 
and their products under Category 3 and 4 when these are placed on the market as or in food or 
feed or in the environment (EFSA, 2024). 

This horizon scanning report provided a systematic review based on key terms used in search 
engines and other online resources. Selection criteria included use of genomic techniques developed 
after 2001, Category 3 products derived from GMMs, Category 4 products consisting of or 
containing GMMs and usage for release into the environment or placing on the market as or in food 
and feed. In total, over 17,000 references were identified, of which only 35 cases met the selection 
criteria above. 

The majority of NGT-Ms were developed or were commercialised in China and in the USA. These 
applications involved CRISPR-based methods, sometimes in combination with homologous 
recombination. The main purpose of these techniques is to knock out genes encoding non-desirable 
characteristics or to introduce genes encoding desirable traits. However, newer techniques, based on 
such site-directed nucleases (SDN), were also used for introducing targeted mutations in 
microorganisms.  

Data from the report shows that the only CRISPR-Cas based SDN-1 mediated modification 
(introduction of a small mutation at a targeted location) was associated with a microalgae case. Of 
the remaining cases, approximately 50 % exhibited introduction of foreign genetic material 
(classified by the report as SDN-3 type changes), and 25 % exhibited introduction of small changes 
using a DNA template (classified by the report as SDN-2 type changes). The remaining 25 % of 
cases were predominantly associated with plasmid-mediated (Cas9) homologous recombination in 
yeast and were classified by the report as combinations of SDN-2 and/or SDN-3 type changes (no 
further information was provided on this grouping). Other NGTs, including phage-based approaches, 
oligonucleotide-directed mutagenesis, meganucleases, ZFNs and TALENS, were not identified as 
part of the study. 

From the 35 NGT-Ms, 22 of these were yeast (Saccharomyces cerevisiae), 11 were bacteria, and 
one each of a fungal endophyte and microalgae. Fifteen cases were classified as Category 3 and 20 
cases as Category 4 GMMs. Cited usage of the NGT-Ms were mainly as (or as a source of) food or 
food additives (30 cases) but also for (or as a source of) feed or feed additives (3 cases) or for use 
in agriculture as bacterial biofertilizer (2 cases). Products were either commercialised (8 cases), in 
pre-commercialisation status (9 cases) or under development (18 cases). Six of the cases had 
patents granted, whilst five others were undergoing a patent application process. We can expect 
that with the technological and scientific progress the number of NGT-Ms on the global market will 
increase in the near future. 

Unlike most NGT plants or animals, NGT-Ms may also be used as cellular factories to produce 
certain compounds. The end products are generally purified to remove the microorganism and the 
residual DNA as much as technically possible. However, this is not always the case. For instance, 
probiotics and brewing starter cultures may intentionally contain viable or unviable cells and traces 
of residual DNA may be detectable in certain end products. 

12 

 
 
 
4  Potential detection strategies 

It must be emphasised that analytical methods based on DNA detection are designed for the 
monitoring of Category 3 and Category 4 NGT-Ms. Such methods can, however, also be used to 
examine whether Category 1 and Category 2 products do not contain detectable DNA remaining 
from the NGT-Ms. This is similar to plant products that should not contain DNA, e.g., sugar or oil 
extracted from NGT plants. 

The challenges to detect and identify the NGT-Ms are similar to the challenges that were described 
for NGT plants. The term NGTs is an umbrella term to describe a variety of techniques that can alter 
the genetic material of a microorganism. As in the case of plants, applying genome editing, 
including targeted mutagenesis and cisgenesis, to modify microbial genomes can result in single 
nucleotide variants (SNVs), sequence substitutions, insertions or deletions (InDels) of various sizes 
and insertion of partial or whole genes from other microorganisms. Similar to NGT plants, if the 
same genomic modifications may also result from natural processes or conventional mutagenesis, 
identification of an NGT-M as the result of the application of an NGT may not be possible 
(Broothaerts et al., 2021; Guertler et al., 2023).  

In general, approaches for the detection and identification of NGT-Ms may be based on PCR or high-
throughput sequencing (HTS) or a combination of both. 

PCR-based methods 

Like for NGT plants (ENGL, 2019), the use of PCR-based methods, including real-time PCR and dPCR 
is relevant at the technical level to target the genetic modifications introduced by NGTs in 
microorganisms. Such techniques are widely used and mastered by the EU GMO enforcement 
laboratories. The development and validation workflow of such PCR-based methods to comply with 
the minimum performance requirements (MPR) for methods of GMO testing in the EU is harmonised 
and described in guidance documents (ENGL, 2015, 2023b). 

The use of such PCR-based methods is expected to be satisfactory for the detection and 
identification of NGT-Ms containing sufficiently long and unique genetic modifications (ENGL, 2019). 
However, in case of NGT-Ms with SNVs or short mutations of a few nucleotides, the analytical 
methods may not always meet the requirements of the current MPR guidance (ENGL, 2015, 2023b) 
in terms of specificity and sensitivity. Additionally, in case the genetic modifications of interest are 
either small, arose spontaneously, or resulted from conventional mutagenesis techniques, their 
detection does not necessarily represent proof that they were introduced by an NGT. Consequently, 
such PCR-based detection methods cannot always be used to unambiguously identify a particular 
NGT-M (ENGL, 2019). 

PCR-based methods may be applied as first-line screening tools, complemented with further 
investigations using HTS in case of positive samples. Screening in this context may target species-
specific markers for bacteria, yeasts or microalgae (Deckers et al., 2020a, b) and common screening 
elements like antimicrobial resistance (AMR) genes (Berbers et al., 2020). AMR genes can be used 
for selection during the development of the GMMs, but nowadays companies mostly use alternative 
selection approaches.  We must, however, note that AMR genes can occur naturally in bacteria 
found in food and feed products (Ojha et al., 2023), as well as in the plasmids they harbour (Florez 
et al., 2021) since the AMR genes originate from bacteria. For detection of potential NGT-induced 
modifications, unique PCR screening methods would, therefore, usually need to be applied for each 

13 

 
 
 
potential genetic modification. This will make such screening approaches for routine practices both 
time- and resource-intensive in view of the increasing number of genetic targets.  

Sequencing-based methods 

Although their use is less widespread than PCR-based methods among the EU GMO enforcement 
laboratories, the interest for sequencing-based methods, such as HTS, in the GMO detection field is 
growing (Fraiture et al., 2017, 2019). The first considerations for quality criteria to assess 
sequencing data have been described in ENGL guidance documents (De Keersmaecker et al., 2025; 
ISO 20397-2:2021). HTS represents an attractive option for identifying a specific NGT-M through 
whole genome sequencing (WGS) and simultaneous detection of several key targets composing a 
unique genetic fingerprint associated to the specific NGT-M. Unlike for plants and animals, the WGS 
approach could be applied for microorganisms with a small genome size, such as viruses, bacteria 
and some fungi like yeast (genome size ~ 12 Mb) (Collins et al., 2021, Table 1). WGS is already 
commonly used on bacterial and viral genomes in the context of outbreak source-tracking, allowing 
specific identification of the line responsible for an outbreak and its origin (Barretto et al., 2021; 
Nouws et al., 2021; Vashisht et al., 2023; Berggreen et al., 2023). Similar WGS analysis and 
subsequent bioinformatics approaches may be applied for the identification of known and 
characterised NGT-Ms and may allow recognition of a specific line in which the NGT modifications 
were introduced.  

At the present time, the use of sequencing-based methods is still limited to pure products consisting 
of isolated lines on which WGS can be performed. Application of HTS to identify NGT-Ms is currently 
not feasible for food or feed samples composed of different species. Indeed, despite promising 
metagenomic studies, the analysis of mixed samples containing several microorganisms is still very 
complex, especially if the mixture is composed of different closely related species (Buytaers et al., 
2020; Ahlinder et al., 2022; Imanian et al., 2022; Delikanli-Kiyak et al., 2025; Xiao et al., 2025).  

Table 1. Genome size ranges for different organisms  

Type of organism  Genome size ranges  Reference 
Virus 
Bacterium 
Fungus 
Animal 
Plant 

~ 2 kb to ~ 2 Mb 
~ 100 kb to ~ 15 Mb 
~ 8 Mb to ~ 180 Mb 
~ 20 Mb to ~ 130 Gb 
~ 60 Mb to ~ 150 Gb 

Chaitanya (2019) 
Ochman and Caro-Quintero (2016) 
Mohanta and Bae (2015) 
Gregory (2005) 
Pellicer et al. (2018); Cranage (2022) 

Source: ENGL 

For screening purposes, targeted sequencing methods or enrichment procedures based on 
hybridisation capture could potentially be applied to detect many NGT-Ms with known modifications. 
Repeatability and transferability of sequencing-based methods has been demonstrated for an 
oilseed rape feed product spiked with genome-edited oilseed rape (Pallarz et al., 2023). However, 
research gaps persist regarding the minimum performance parameters of such methods in both 
plants and microorganisms, particularly in terms of their reliability, sensitivity, and applicability 
across diverse contexts.  

While quantification is not necessary for unauthorised NGT-Ms, the current sequencing-based 
methods, including those based on HTS, are not suitable for quantification of (authorised) NGT-Ms 
in food and feed. Most of the currently used HTS-based methods include a PCR step which has a 
direct impact on the quantification of the target sequences (Arulandhu et al., 2018; Debode et al., 

14 

 
 
 
2019, Fraiture et al., 2023). Therefore, it is expected that sequencing-based methods may be used 
for detection, but not for the quantification of NGT-Ms in or as food or feed in the near future.  

For PCR and sequencing-based methods, the above considerations are applicable only in case the 
NGT-Ms and the NGT-induced genetic modifications are known. Given the natural mutation rate in 
microorganisms, mutations that are detected in a microorganism cannot necessarily be explained 
as derived from application of an NGT. In addition, the whole genome sequence of the conventional 
microorganism should be available as reference genome during bioinformatics analysis (Lopera et 
al., 2023). If the NGT-M line is unknown, the same challenges for detection and identification exist 
as for unknown NGT plants. 

15 

 
 
 
 
5  Challenges and additional considerations associated with 

detection of NGT microorganisms 

Because analytical methods in the field of GMO analysis are based on detection, identification and 
quantification of specific target DNA sequences, similar challenges will apply for NGT plants and 
NGT-Ms. In case of NGT-Ms with SNVs and short InDels, the required specificity and sensitivity of 
the detection, identification and/or quantification method may not be achieved in every case. 
Despite individual successes, as described in the previous chapter, several challenges limit the 
general applicability of analytical methods targeting short mutations for enforcement purposes.  

Similar to NGT plants, the mutation size highly restricts the flexibility (in terms of positioning the 
primers and probe along the genetic sequence) needed to design a suitable PCR method targeting 
mutations in NGT-Ms. The design of a PCR-based method to distinguish sequences that differ by 
one or a few nucleotides from a natural sequence may not in all cases be sufficient to deliver the 
required specificity of the method (Shillito et al., 2021; Hommelsheim et al., 2014). In addition, 
while the specificity of a PCR method can be increased by Locked Nucleic Acid modified primers or 
Minor Groove Binder probes, this does not resolve this problem in case the mutation occurs in 
repetitive DNA or in DNA made up mostly of certain bases (e.g. either G and C or A and T bases). 
Furthermore, the development of a fit for purpose detection method would require prior knowledge 
of the mutations to detect. 

Whilst NGT-Ms present similar detection challenges compared to both NGT plants and conventional 
GMMs, these challenges are further amplified in NGT-Ms. Some of these challenges, applicable also 
to conventional GMMs, are linked to their higher propensity to mutate compared to higher 
organisms. Indeed, due to microbes’ fast evolving rate, pan-genomes, representing the genetic 
variation in the genome found within a species (Medini et al., 2005; Aggarwal et al., 2022), will 
continuously expand with newly identified mutations. The speed and diversity at which mutations 
emerge in microorganisms, paired with the ability of NGTs to introduce small mutations, can result 
in two specific challenges. First, constantly expanding microbial pangenomes lead to increased 
difficulties in identifying the origin of a mutation (natural or targeted), compared to NGT plants. 
Second, the possibility that living GM microorganisms, once they are authorised and introduced into 
the food or feed chain, may accumulate further mutations, creates detectability and interpretability 
issues. Furthermore, as most microorganisms do not rely on sexual compatibility, but instead on 
horizontal gene transfer to exchange genetic material, the boundary between cisgenesis and 
transgenesis becomes less clear. 

In microorganisms with relatively small genomes, some of these constraints could be resolved by 
sequencing-based approaches, such as targeted sequencing or WGS. However, identification of 
NGT-Ms could be achieved only in a simple product consisting of or containing a specific NGT-M 
strain. Furthermore, as previously explained, HTS cannot always unambiguously identify whether a 
mutation occurred spontaneously or is a result of conventional mutagenesis techniques. For the 
time being, application of HTS for food/feed composed of many ingredients including 
microorganisms and plants would not be feasible in routine market control. Moreover, up to now 
methods based on HTS do not allow for precise quantification of NGT-Ms in food/feed products.  

Application of HTS, which is still a new technology for many official laboratories, would require both 
investment in laboratory equipment and training not only on the analytical procedure and 
bioinformatics pipeline, but also on the interpretation of the results. Additionally, a laboratory must 
be equipped with the relevant infrastructure and knowledge base appropriate for the handling and 

16 

 
 
 
analysis of living microorganisms, which can present further challenges when these may be 
genetically modified (e.g., biosafety requirements according to Directive 2009/41/EC). For this 
purpose, also wildtype reference microbial genome sequence databases would need to be 
developed for bioinformatic sequence comparisons (Hurel et al., 2020; Lopera et al., 2023). 
However, various initiatives have already demonstrated that WGS can be routinely applied to 
isolated living microorganisms for monitoring in the food industry, particularly to determine whether 
contamination by a pathogen is due to the presence of a new strain or the persistence of an 
existing strain within the facility (Allard et al., 2016; Gomes et al., 2025). 

Sequencing approaches afford further developmental opportunities in order to provide 
demonstratable evidence of their applicability for routine detection of NGT-Ms. While progress is 
being made in standardising laboratory protocols, there is a lack of consensus on standardised 
bioinformatic methods for data analysis and results interpretation, highlighting the need for 
guidelines and consensus among institutions (Barretto et al., 2021). In addition, the current deficit in 
the provision of proficiency tests for the identification (and quantification) of GMMs (and NGT-Ms) 
provides a barrier to demonstrating competency in this area. 

Under the current regulatory framework, applicants have to provide an event-specific detection 
method for the NGT-M for which authorisation is requested. Such method will be validated by the 
EURL GMFF and made available for the NRLs and routine laboratories. Currently, there are no 
minimum performance requirements established for methods based on HTS and it is not clear if 
such methods will ever fulfil the basic requirements under the GMO regulations, e.g., with regard to 
quantification of the NGT-Ms. It is rather foreseen that HTS methods may in the future be used by 
more experienced enforcement laboratories for a first-line screening of products that may contain 
NGT-Ms, thereby targeting many potential targets at once. 

17 

 
 
 
 
6  Conclusions 

This report aims to inform the European Commission on analytical aspects related to the detection, 
identification and quantification of NGT-Ms and food/feed products derived thereof. This report 
builds upon an ENGL report from 2023 on the detection of food and feed plant products obtained 
by targeted mutagenesis and cisgenesis (ENGL, 2023a). The major challenges for the detection of 
various NGT-Ms in or as food or feed that were identified by the working group are similar to the 
challenges described for NGT plants. The same new genomic techniques, introducing different types 
of genetic modifications, can be used in both NGT-Ms and NGT plants. Application of NGTs in 
microorganisms can result in deletions, insertions or substitutions of diverse sizes. Therefore, the 
analytical challenges for the development and validation of methods for detection, identification 
and quantification of NGT plants summarised in Table 2 of the ENGL (2023a) report still remain 
valid for NGT-Ms (Annex 1).  

Like in NGT plants, the shortest DNA sequence alterations (SNVs) are the most challenging when 
developing a detection method that is specific for an NGT-M. This is partly related to technical 
difficulties for targeting short mutations within an unmodified DNA sequence context, and partly to 
the fact that similar short mutations may occur naturally in the target microorganisms.  Methods 
developed for some SNVs or short mutations may, therefore, fail to meet the current minimum 
performance requirements for analytical methods of GMO testing. In case of known, larger 
modifications that do not already exist in the microbial species, detection, identification and 
quantification would normally be feasible using event-specific PCR-based methods.  

Enforcement laboratories usually apply screening methods in a first phase when analysing a 
sample for the presence of GM plants. Such screening methods target common foreign genetic 
elements present in various conventional GM plants. This approach cannot be applied to NGT-
products (plants or microorganisms) containing only alterations of endogenous DNA or sequences 
present in the pan-genome of the species. Instead, specific methods for every possible mutation will 
need to be applied, increasing the complexity of the analytical work. Overall, detection of unknown 
products of targeted mutagenesis or cisgenesis, without prior knowledge of the modified DNA 
sequences, will not be possible with the current technology used by most of the enforcement 
laboratories, especially in complex samples.  

Some NGT-Ms with small genomes could be identified by HTS if pure microbial samples would be 
available. However, even applying HTS techniques would not easily allow for the detection of 
unknown and unauthorised NGT-Ms (especially in case of small mutations). Targeted sequencing 
could be used to detect and identify multiple targets of known larger sequences specific to 
particular NGT-Ms. This could be combined in the next step with selected PCR-based techniques for 
quantification purposes.  

Theoretically, HTS-based approaches may also be developed for a semi-targeted approach on genes 
that are known to be a potential target for NGT-induced modifications. However, this has not been 
investigated so far. It would require access to a knowledge database containing information on all 
potential endogenous genomic targets in the various microbial families that, when modified by an 
NGT, could create beneficial characteristics. Similarly, a database of all described NGT-Ms with their 
modified sequences, and one containing the reference (pan-)genomes, would be required to 
compare any given putatively mutated sequence to. If analytical detection of large amounts of 
diverse NGT-Ms would become an important enforcement requirement in the future, a dataspace 
linking all these databases, and (informatics or machine learning-based) tools to interrogate them, 

18 

 
 
 
may be useful to develop. Currently, identification of a few specific NGT-Ms is mostly achieved in 
the form of research studies by some enforcement laboratories. 

More likely than with plants, applications of NGTs to microorganisms may result in a genomic 
sequence already present in the pan-genome of a specific species. Such a DNA sequence can also 
occur naturally in the future as microorganisms can multiply very quickly, so new genetic diversity 
in microorganisms is developed much faster when compared to plants. Therefore, it may be 
impossible to analytically evaluate whether a particular genetic modification is produced by 
targeted mutagenesis or cisgenesis rather than resulting from conventional breeding or natural 
mutagenesis.  

Importantly, as some microorganisms are capable of natural transformation and exchanges of 
genetic material through horizontal gene transfer, defining NGT-Ms as microorganisms that do not 
contain inserted foreign DNA might not sufficiently describe the nature of these organisms. This 
would provide additional complexity to the detection and identification of both NGT-Ms and 
conventional GMMs compared to GM plants.  

It is concluded that analytical testing may support traceability and labelling of NGT-Ms with known 
and unique sequence modifications. Further developments at both the level of the available 
technologies and their validation, may further expand the enforcement actions to more known NGT-
Ms in the future. 

Technical advancements will, however, not resolve the problem of distinguishing identical DNA 
sequences generated naturally, by mutation or homologous recombination, or by an NGT. 
Furthermore, unknown modifications created by an NGT will remain extremely difficult to detect and 
identify as the product of an NGT. In case of small mutations and any modification that is not 
unique in the microbial gene pool, it is expected that analytical tools will not lead to a solution 
supporting enforcement. An integrated data-driven approach, including comprehensive sources of 
knowledge from literature, company websites and patent databases, as well as genome sequencing 
data for various species and strains, may be needed to allow enforcement for a broad range of 
NGT-Ms.  

19 

 
 
 
 
References 

Aggarwal, S.K., Singh, A., Choudhary, M., Kumar, A., Rakshit, S., Kumar, P., Bohra, A., Varshney, R.K. 
(2022) Pangenomics in microbial and crop research: progress, applications, and perspectives. Genes 
13, 598 (https://doi.org/10.3390/genes13040598). 

Ahlinder, J., Svedberg, A.-L.., Nystedt, A., Dryselius, R., Jacobsson, K., Hägglund, M., Brindefalk, B., 
Forsman, M., Ottoson, J., Troell, K. (2022) Use of metagenomic microbial source tracking to 
investigate the source of a foodborne outbreak of cryptosporidiosis. Food and Waterborne 
Parasitology 26, e00142 (https://doi.org/10.1016/j.fawpar.2021.e00142). 

Allard, M. W., Strain, E., Melka, D., Bunning, K., Musser, S. M., Brown, E. W., Timme, R. (2016) Practical 
value of food pathogen traceability through building a whole-genome sequencing network and 
database. Journal of Clinical Microbiology 54(8), 1975-1983. 

Arnold, B.J., Huang, IT., Hanage, W.P. (2022) Horizontal gene transfer and adaptive evolution in 
bacteria. Nature Reviews on Microbiology 20, 206–218 (https://doi.org/10.1038/s41579-021-
00650-4). 

Arulandhu, A.J., van Dijk, J., Staats, M., Hagelaar, R., Voorhuijzen, M., Molenaar, B., et al. (2018) NGS-
based amplicon sequencing approach; towards a new era in GMO screening and detection. Food 
Control 93, 201-210. 

Bae, S-J., Park, B.G., Kim, B-G., Hahn, J-S. (2019) Multiplex gene disruption by targeted base editing 
of Yarrowia lipolytica genome using cytidine deaminase combined with the CRISPR/Cas9 system. 
Biotechnology Journal 15, 1900238 (https://doi.org/10.1002/biot.201900238). 

Ballester, A.R., Roqué, M., Ricci-Cabello, I., Rotger, A., Malih, N. (2023) Horizon scanning on 
microorganisms and their products obtained by new developments in biotechnology. EFSA 
supporting publication 2023:EN-8503, 65 pp. (doi:10.2903/sp.efsa.2023.EN-8503). 

Barretto, C., Rincón, C., Portmann, A.C., Ngom-Bru, C. (2021) Whole genome sequencing applied to 
pathogen source tracking in food industry: key considerations for robust bioinformatics data 
analysis and reliable results interpretation. Genes (Basel) 12(2):275 (doi: 10.3390/genes12020275). 

Berbers, B., Saltykova, A. a., Garcia-Graells, C., Philipp, P., Arella, F., Marchal, K., et al. (2020) 
Combining short and long read sequencing to characterize antimicrobial resistance genes on 
plasmids applied to an unauthorized genetically modified Bacillus. Scientific Reports, 10, 4310. 

Berggreen, H., Løvestad, A.H., Helmersen, K., Jørgensen, S.B., Aamot, H.V. (2023) Lessons learned: 
use of WGS in real-time investigation of suspected intrahospital SARS-CoV-2 outbreaks. Journal of 
Hospital Infection 131, 81-88 (https://doi.org/10.1016/j.jhin.2022.10.003). 

Broothaerts, W., Jacchia, S., Angers, A., Petrillo, M., Querci, M., Savini, C., Van den Eede, G., Emons, H. 
(2021) New genomic techniques: state-of-the-art review. Publications Office of the European Union, 
Luxembourg (doi:10.2760/710056). 

Buytaers, F.E., Saltykova, A., Denayer, S., Verhaegen, B., Vanneste, K., Roosens, N.H.C., Piérard, D., 
Marchal, K., De Keersmaecker, S.C.J. (2020) A practical method to implement strain-level 
metagenomics-based foodborne outbreak investigation and source tracking in routine. 
Microorganisms 8, 1191 (https://doi.org/10.3390/microorganisms8081191). 

20 

 
 
 
Chaitanya, K.V. (2019) Structure and organization of virus genomes. Genome and Genomics 18:1–
30 (doi: 10.1007/978-981-15-0702-1_1). 

Collins, J.H., Keating, K.W., Jones, T.R., Balaji, S., Marsan, C.B., Çomo, M., Newlon, Z.J., Mitchell, T., 
Bartley, B., Adler, A., Roehner, N., Young, E.M. (2021) Engineered yeast genomes accurately 
assembled from pure and mixed samples. Nature Communications 12, 1485 
(https://doi.org/10.1038/s41467-021-21656-9). 

Cranage, A. (2022) Genomes great and small: The diversity of plants 
(https://www.darwintreeoflife.org/news_item/genomes-great-and-small-the-diversity-of-plants/).  

Dalia, A.B., McDonough, EK., Camilli, A. (2014) Multiplex genome editing by natural transformation. 
Proceedings of the National Academy of Sciences 111, 8937–8942 
(doi/full/10.1073/pnas.1406478111). 

Debode, F., Hulin, J., Charloteaux, B., Coppieters, W., Hanikenne, M., Karim, L., et al. (2019) Detection 
and identification of transgenic events by next generation sequencing combined with enrichment 
technologies. Scientific Reports 9, 15595 (https://doi.org/10.1038/s41598-019-51668-x). 

Deckers, M., Vanneste, K., Winand, R., De Keersmaecker, S.C.J., Denayer, S., Hendrickx, M., Deforce, D., 
Fraiture, M.-A., Roosens, N.H.C. (2020a) Strategy for the identification of micro-organisms producing 
food and feed products: Bacteria producing food enzymes as study case. Food Chemistry 305, 
125431 (10.1016/j.foodchem.2019.125431). 

Deckers, M., Vanneste, K., Winand, R., Hendrickx, M., Becker, P., De Keersmaecker, S.C.J., Deforce, D., 
Fraiture, M.-A., Roosens, N.H.C. (2020b) Screening strategy targeting the presence of food enzyme-
producing fungi in food enzyme preparations. Food Control 117, 107295 
(https://doi.org/10.1016/j.foodcont.2020.107295). 

De Keersmaecker, S.C., Jacchia, S., Kok, E.J., Roosens, N., Zaoui, X. et al. (2025) Sequencing strategies 
for the traceability of GMOs - methods and related quality aspects, Publications Office of the 
European Union, Luxembourg (https://data.europa.eu/doi/10.2760/890142).  

Delikanli-Kiyak, B., Yilmaz, I., Guldas, M. (2025) Can metagenomics analyses be used effectively in 
safe food production? Journal of Food Science and Nutrition 13, e70772 (doi: 10.1002/fsn3.70772). 

Del Duca, S., Vassallo, A., Mengoni, A., Fani, R. (2022) Microbial genetics and evolution. 
Microorganisms 10, 1274 (https://doi.org/10.3390/ microorganisms10071274). 

EFSA Panel on Genetically Modified Organisms, Mullins, E., Bresson, J.-L., Dewhurst, I.C., Epstein, 
M.M., Firbank, L.G. et al. (2024) New developments in biotechnology applied to microorganisms. 
EFSA Journal, 22(7), e8895 (https://doi.org/10.2903/j.efsa.2024.8895). 

EFSA Panel on Genetically Modified Organisms (2011) Scientific Opinion on Guidance on the risk 
assessment of genetically modified microorganisms and their products intended for food and feed 
use. EFSA Journal 9(6):2193 (https://doi.org/10.2903/j.efsa.2011.2193). 

European Network of GMO Laboratories (ENGL, 2015) Definition of minimum performance 
requirements for methods of GMO testing (http://gmo-
crl.jrc.ec.europa.eu/doc/MPR%20Report%20Application%2020_10_2015.pdf). 

21 

 
 
 
European Network of GMO Laboratories (ENGL, 2019) Detection of food and feed plant products 
obtained by new mutagenesis techniques, 26 March 2019 (https://gmo-
crl.jrc.ec.europa.eu/ENGLabs#inline-nav-engl-reports). 

European Network of GMO Laboratories (ENGL, 2023a) Detection of food and feed plant products 
obtained by targeted mutagenesis and cisgenesis. Publications Office of the European Union, 
Luxembourg (doi:10.2760/007925). 

European Network of GMO Laboratories (ENGL, 2023b) Definition of minimum performance 
requirements for analytical methods of GMO testing, part 2. Publications Office of the European 
Union, Luxembourg (doi:10.2760/63656). 

Florez, A. B., Vazquez, L., Rodriguez, J., Mayo, B. (2021) Directed recovery and molecular 
characterization of antibiotic resistance plasmids from cheese bacteria. International Journal of 
Molecular Sciences, 22(15), 7801. 

Fraiture, M.A., Herman, P., Papazova, N., De Loose, M., Deforce, D., Ruttink, T., et al. (2017) An 
integrated strategy combining DNA walking and NGS to detect GMOs. Food Chemistry 232, 351-
358. 

Fraiture, M.-A., Ujhelyi, G., Ovesná, J., Van Geel, D., De Keersmaecker, S., Saltykova, A., et al. (2019) 
MinION sequencing technology to characterize unauthorized GM petunia plants circulating on the 
European Union market. Scientific Reports 9, 7141. 

Fraiture, M.-A.; D’aes, J.; Guiderdoni, E.; Meunier, A.-C.; Delcourt, T.; Hoffman, S.; Vandermassen, E.; De 
Keersmaecker, S.C.J.; Vanneste, K.; Roosens, N.H.C. (2023) Targeted high-throughput sequencing 
enables the detection of single nucleotide variations in CRISPR/Cas9 gene-edited organisms. Foods 
12, 455 (https://doi.org/10.3390/foods12030455). 

Gomes, E., Araújo, D., Nogueira, T., Oliveira, R., Silva, S., Oliveira, L. V. (2025) Advances in whole 
genome sequencing for foodborne pathogens: implications for clinical infectious disease 
surveillance and public health. Frontiers in Cellular and Infection Microbiology, 15, 1593219. 

Gregory, T.R. (2005) Animal genome size database, release 2.0 (http://www.genomesize.com). 

Guertler, P., Pallarz, S., Belter, A., Eckermann, K.N., Grohmann, L. (2023) Detection of commercialized 
plant products derived from new genomic techniques (NGT)-Practical examples and current 
perspectives. Food Control 152, 109869 (https://doi.org/10.1016/j.foodcont.2023.109869). 

Hommelsheim, C.M., Frantzeskakis, L., Huang, M., Ülker, B. (2014) PCR amplification of repetitive 
DNA: a limitation to genome editing technologies and many other applications. Scientific Reports 23, 
5052 (doi: 10.1038/srep05052). 

Huang, L., Dong, H., Zheng, J., Wang, B., Pan, L. (2019) Highly efficient single base editing in 
Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion. Microbiological Research 223-225, 
44-50 (https://doi.org/10.1016/j.micres.2019.03.007). 

Hurel, J., Schbath, S., Bougeard, S., Rolland, M., Petrillo, M., Touzain, F. (2020) DUGMO: tool for the 
detection of unknown genetically modified organisms with high throughput sequencing data for 
pure bacterial samples. BMC Bioinformatics 21, 284 (https://doi.org/10.1186/s12859-020-03611-
5). 

22 

 
 
 
Imanian, B., Donaghy, J., Jackson, T., Gummalla, S., Ganesan, B., Baker, R.C., et al. (2022) The power, 
potential, benefits, and challenges of implementing high-throughput sequencing in food safety 
systems. NPJ Science of Food 6, 35 (https://doi.org/10.1038/s41538-022-00150-6). 

ISO 20397-2:2021 (2021) Biotechnology - General requirements for massive parallel sequencing - 
Part 2: Quality evaluation of sequencing data. International Organization for Standardization 
(https://www.iso.org/standard/67895.html). 

Li, L., Liu, Y., Xiao, Q., Xiao, Z., Meng, D., Yang, Z., Deng, W., Yin, H., Liu, Z. (2023) Dissecting the HGT 
network of carbon metabolic genes in soil-borne microbiota. Frontiers in Microbiology 14, 1173748 
(doi: 10.3389/fmicb.2023.1173748). 

Lopera, J., Frank, A., McCluskey, A., King, S., Fenn, S., Kindig, K., Riojas, M., Sohn, J.-W., Sadural, H., 
Briana, B., Wilder, C. (2023) Microbial genome databases. Existing challenges and the need for 
authenticated reference genomes. ATCC Application Note (https://www.atcc.org/-
/media/resources/application-notes/microbial-genome-
databases.pdf?rev=3db0596e37cb45b1b270daa42b6e5925). 

Lv, X., Li, Y., Xiu, X., Liao, C., Xu, Y., Liu, Y., Li, J., Du, G., Liu, L. (2023) CRISPR genetic toolkits of 
classical food microorganisms: Current state and future prospects. Biotechnology Advances 69, 
108261 (doi.org/10.1016/j.biotechadv.2023.108261). 

Medini D., Donati C, Tettelin H., Masignani V., Rappuoli R., (2005) The microbial pan-genome. Current 
Opinion in Genetics & Development 15(6), 589-594 (https://doi.org/10.1016/j.gde.2005.09.006). 

Mohanta, T.K., Bae, H. (2015) The diversity of fungal genome. Biological Procedures Online 17:8 (doi: 
10.1186/s12575-015-0020-z). 

Nouws, S., Bogaerts, B., Verhaegen, B., Denayer, S., Laeremans, L., Marchal, K., Roosens, N.H.C., 
Vanneste, K., De Keersmaecker, S.C.J. (2021) Whole genome sequencing provides an added value to 
the investigation of staphylococcal food poisoning outbreaks. Frontiers in Microbiology 12:750278 
(doi: 10.3389/fmicb.2021.750278). 

Ochman, H., Caro-Quintero, A. (2016) Genome size and structure, bacterial.  In: R. M. Kliman (ed.), 
Encyclopedia of Evolutionary Biology, Academic Press, pp. 179-185 (doi.org/10.1016/B978-0-12-
800049-6.00235-3). 

Ojha, A.K., Shah, N.P., Mishra, V., Emanuel, N., Taneja, N.K. (2023) Prevalence of antibiotic resistance 
in lactic acid bacteria isolated from traditional fermented Indian food products. Food science and 
biotechnology 32(14), 2131-2143.  

Pallarz, S., Fiedler, S., Wahler, D., Lämke, J., Grohmann, L. (2023) Reproducibility of next-generation-
sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape. Food Chemistry: 
Molecular Sciences 7, 100182 (https://doi.org/10.1016/j.fochms.2023.100182). 

Pellicer, J., Hidalgo, O., Dodsworth, S., Leitch, I.J. (2018) Genome size diversity and its impact on the 
evolution of land plants. Genes (Basel) 9(2):88 (doi: 10.3390/genes9020088). 

Shillito, R.D., Whitt, S., Ross, M., Ghavami, F., De Vleesschauwer, D., D’Halluin, K., Van Hoecke, A., 
Meulewaeter, F. (2021) Detection of genome edits in plants - from editing to seed. In vitro Cellular 
& Developmental Biology - Plant 57, 595–608 (https://doi.org/10.1007/s11627-021-10214-z). 

23 

 
 
 
Shitut, S., Shen, M.J., Claushuis, B., Derks, R.J.E., Giera, M., Rozen, D., Claessen, D., Kros, A. (2022) 
Generating heterokaryotic cells via bacterial cell-cell fusion. Microbiology Spectrum 31, 
10(4):e0169322 (doi: 10.1128/spectrum.01693-22). 

Vashisht, V., Vashisht, A., Mondal, A.K., Farmaha, J., Alptekin, A., Singh, H., Ahluwalia, P., Srinivas, A., 
Kolhe, R. (2023) Genomics for emerging pathogen identification and monitoring: prospects and 
obstacles. BioMedInformatics 3, 1145-1177 (https://doi.org/10.3390/biomedinformatics3040069). 

Vasileva, D.P., Streich, J.C., Burdick, L.H., Klingeman, D.M., Chhetri, H.B., Brelsford, C.M., Ellis, J.C., 
Close, D.M., Jacobson, D.A., Michener, J.K. (2022) Protoplast fusion in Bacillus species produces 
frequent, unbiased, genome-wide homologous recombination. Nucleic Acids Research 50, 6211–
6223 (https://doi.org/10.1093/nar/gkac025). 

Xiao, L., Li, Z., Dou, X., Long, Y., Li, Z., Liu, Y., Dong, Q. (2025) Advancing microbial risk assessment: 
perspectives from the evolution of detection technologies. NPJ Science of Food 28, 157 (doi: 
10.1038/s41538-025-00527-3). 

Yang, J., Song, J., Feng, Z., Ma, Y. (2025) Application of CRISPR-Cas9 in microbial cell factories. 
Biotechnology Letters 47, 46 (doi: 10.1007/s10529-025-03592-6). 

24 

 
 
 
 
 
List of abbreviations and definitions  

Abbreviations 

Definitions 

AMR 

CRISPR-Cas9 

Antimicrobial Resistance 

Clustered Regularly Interspaced Short Palindromic Repeats 
and CRISPR-associated protein 9 

DNA 

DSB 

dPCR 

EFSA 

ENGL 

EU 

Deoxyribonucleic acid 

Double-Stranded Breaks 

digital Polymerase Chain Reaction 

European Food Safety Authority 

European Network of GMO Laboratories 

European Union 

EURL GMFF 

European Union Reference Laboratory for GM Food and Feed  

GM 

GMM 

GMO 

HGT 

HTS 

InDels 

JRC 

MPR 

NGT 

NGT-M 

PCR 

SDN 

SNV 

TALENs 

WG 

WGS 

ZFNs 

Genetically Modified 

Genetically Modified Microorganism 

Genetically Modified Organism 

Horizontal Gene Transfer 

High-Throughput Sequencing 

Insertions and Deletions 

Joint Research Centre 

Minimum Performance Requirements 

New Genomic Technique 

NGT microorganism (microorganism that is genetically    
modified through an NGT) 

Polymerase Chain Reaction 

Site-Directed Nucleases 

Single Nucleotide Variant 

Transcription Activator-Like Effector Nucleases 

Working Group 

Whole Genome Sequencing 

Zinc-Finger Nucleases 

25 

 
 
 
 
List of tables 

Table 1. Genome size ranges for different organisms ................................................................................................ 14 

Table 2. Analytical challenges for the development and validation of methods for detection, 
identification and quantification of plant products developed by targeted mutagenesis or cisgenesis.
 ....................................................................................................................................................................................................................... 28 

26 

 
 
 
 
 
Annex 1. Summary of analytical challenges for NGT plants.  

Table copied from the ENGL (2023a) report on the “Detection of food and feed plant products 
obtained by targeted mutagenesis and cisgenesis” 

Table 2. Analytical challenges for the development and validation of methods for detection, 
identification and quantification of plant products developed by targeted mutagenesis or cisgenesis. 

Genomic 
technique 

Type of modification  Method development and validation 

Method implementation for enforce-
ment 

SNV 

Targeted 
mutagene-
sis 

Short mutation 

Long insertion or dele-
tion 

Targeted sequence in-
sertion or substitution 

Cisgenesis 

Technical feasibility depends on se-
quence context (case-by-case), but 
event-specificity, robustness and quanti-
tative use of the method generally diffi-
cult to demonstrate  

Technical feasibility depends on se-
quence context (case-by-case), but 
event-specificity, robustness and quanti-
tative use of the method may in some 
cases be difficult to demonstrate 

Technically feasible, but event-specific-
ity depends on whether the modification 
created a new, unique sequence junction 
or not 

Technically feasible, but event-specific-
ity of the method depends on whether 
the altered new sequence is different 
(and by how many nucleotides) from 
similar sequences already existing in the 
species 

Difficult/impossible to implement such meth-
ods for analytical enforcement, when the re-
sults are not reliable 

Difficult/impossible to implement such meth-
ods for analytical enforcement, when the re-
sults are not reliable 

Feasible, when event-specificity is demon-
strated 

Feasible when event-specificity is demon-
strated. Feasible, but implementation issues 
may occur in case the new sequence only 
differs from existing sequences by a SNV or 
short mutation  

Random whole gene in-
sertion 

Random whole gene in-
sertion or targeted se-
quence insertion or 
substitution 

Feasible  

Feasible 

Intragenesis 

Feasible 

Feasible 

Various 
techniques 

Multiple modifications 

Requires development and validation of 
one method per modified site, signifi-
cantly increasing the workload; technical 
feasibility depends on the types of mod-
ifications (see above) 

Source: ENGL, 2023a 

Analysis of each plant product with all indi-
vidual methods known for the species pre-
sent in the product will enormously increase 
the analytical workload and will rapidly make 
analytical enforcement impossible to con-
tinue in the same way. Also difficult to eval-
uate product quantity in case of multiple 
modifications derived from a single plant, 
some of which may also segregate out. 

27 

 
 
 
 
 
 
Getting in touch with the EU 

In person 

All over the European Union there are hundreds of Europe Direct centres. You can find the 
address of the centre nearest you online (european-union.europa.eu/contact-eu/meet-us_en). 

On the phone or in writing 

Europe Direct is a service that answers your questions about the European Union. You can 
contact this service: 

— by freephone: 00 800 6 7 8 9 10 11 (certain operators may charge for these calls), 

— at the following standard number: +32 22999696, 

— via the following form: european-union.europa.eu/contact-eu/write-us_en. 

Finding information about the EU 

Online 

Information about the European Union in all the official languages of the EU is available on the 
Europa website (european-union.europa.eu). 

EU publications 

You can view or order EU publications at op.europa.eu/en/publications. Multiple copies of free 
publications can be obtained by contacting Europe Direct or your local documentation centre 
(european-union.europa.eu/contact-eu/meet-us_en). 

EU law and related documents 

For access to legal information from the EU, including all EU law since 1951 in all the official 
language versions, go to EUR-Lex (eur-lex.europa.eu). 

EU open data 

The portal data.europa.eu provides access to open datasets from the EU institutions, bodies 
and agencies. These can be downloaded and reused for free, for both commercial and non-
commercial purposes. The portal also provides access to a wealth of datasets from European 
countries. 

28 

 
 
 
 
 
 
 
 
The Joint Research Centre (JRC) provides 
independent, evidence-based knowledge and 
science, supporting EU policies to positively 
impact society 

EU Science Hub 
Joint-research-centre.ec.europa.eu 

29