Topic 1: New techniques for detecting and identifying LMOs
Dr. Raja Chalghoumi,
Tunisia#11945
Tunisia#11945
il y a 3 ans
Dear Colleagues,
Welcome to the online discussions of the Network of Laboratories for the Detection and Identification of Living Modified Organisms.
My name is Raja Chalghoumi, I am an agronomist with a Ph.D. in Agronomic Sciences and Bioengineering. Currently, I am an Associate Professor in Animal Sciences at the College of Agriculture - University of Carthage – Tunisia. Moreover, I am a certified professional in Biorisk Management and Biosecurity, a member of the Tunisian Biosafety Technical Commission, and a member of the Ad Hoc Technical Expert Group (AHTEG) on Risk Assessment and Risk Management.
Under this first topic, I would like to understand better the new techniques for detecting and identifying LMOs. From the discussions of the Network in 2019, next-generation sequencing and digital PCR were examples of two new techniques that were in use in some LMO analysis laboratories. Additionally, members of this Network identified isothermal techniques as being under development. Thus, it would be important to understand better developments since 2019. In this regard, I would like to ask you to keep in mind the following:
- What progress has been made with the implementation of next-generation sequencing and digital PCR in LMO analysis?
- Are there isothermal techniques now in use? What recent developments have been made with respect to these techniques?
- Are there any new analytical techniques that have been developed or implemented since 2019 that could be applied to detection and identification of LMOs? If so, what are they?
I look forward to your insights.
Raja Chalghoumi
Welcome to the online discussions of the Network of Laboratories for the Detection and Identification of Living Modified Organisms.
My name is Raja Chalghoumi, I am an agronomist with a Ph.D. in Agronomic Sciences and Bioengineering. Currently, I am an Associate Professor in Animal Sciences at the College of Agriculture - University of Carthage – Tunisia. Moreover, I am a certified professional in Biorisk Management and Biosecurity, a member of the Tunisian Biosafety Technical Commission, and a member of the Ad Hoc Technical Expert Group (AHTEG) on Risk Assessment and Risk Management.
Under this first topic, I would like to understand better the new techniques for detecting and identifying LMOs. From the discussions of the Network in 2019, next-generation sequencing and digital PCR were examples of two new techniques that were in use in some LMO analysis laboratories. Additionally, members of this Network identified isothermal techniques as being under development. Thus, it would be important to understand better developments since 2019. In this regard, I would like to ask you to keep in mind the following:
- What progress has been made with the implementation of next-generation sequencing and digital PCR in LMO analysis?
- Are there isothermal techniques now in use? What recent developments have been made with respect to these techniques?
- Are there any new analytical techniques that have been developed or implemented since 2019 that could be applied to detection and identification of LMOs? If so, what are they?
I look forward to your insights.
Raja Chalghoumi
New methods in the quantification analysis of LMO had been developed by nanoplate type digital PCR (ndPCR) in South Korea.
Please refer to PCT/KR2023/013652.
The characteristics and advantages of the methods compared to Real-Time PCR are as follows;
1. The quantification by ndPCR with a single copy gene as a built-in certified reference materials (CRM), meaning that CRM is not required for the quantification experiment.
2. The least discrepancy in the amplification efficiency between sample tested and CRM, meaning that the results are much more accurate and consistent.
3. Numerous options in the choice of CRMs. You may find many different one or two copy genes in higher plants.
4. The fast development of the quantification methods for newly approved LMOs without CRMs. You need only the information of the nucleotide sequences of ORFs or factors introduced in LMOs.
If necessary, more information will be given by comments or questions.
Jae Sun Moon
Please refer to PCT/KR2023/013652.
The characteristics and advantages of the methods compared to Real-Time PCR are as follows;
1. The quantification by ndPCR with a single copy gene as a built-in certified reference materials (CRM), meaning that CRM is not required for the quantification experiment.
2. The least discrepancy in the amplification efficiency between sample tested and CRM, meaning that the results are much more accurate and consistent.
3. Numerous options in the choice of CRMs. You may find many different one or two copy genes in higher plants.
4. The fast development of the quantification methods for newly approved LMOs without CRMs. You need only the information of the nucleotide sequences of ORFs or factors introduced in LMOs.
If necessary, more information will be given by comments or questions.
Jae Sun Moon
Dear Colleagues,
We believe that your expertise and contribution to the online forum would greatly enrich the discussions under the addressed topics. We will be truly grateful for your active participation.
Best regards
We believe that your expertise and contribution to the online forum would greatly enrich the discussions under the addressed topics. We will be truly grateful for your active participation.
Best regards
Dear colleagues,
My name is Daniela Wahler. I work for the German Federal Office of Consumer Protection and Food Safety (BVL) in the Unit traceability, detection methods, Biosafety Clearing-House. BVL is home to the competent national authority for the Cartagena Protocol and also to a working group that develops the official control methods for the detection and identification of LMO in Germany. I would like to thank the Secretariat for the opportunity to discuss here.
Regarding digital PCR, experience with this technique has increased since 2019 and a large number of official control laboratories in Germany are equipped to integrate digital PCR within their analyses. However, digital PCR methods are not yet part of routine analyses. The technique is mainly applied for the quantification of LMO in samples.
Next generation sequencing is a valuable tool that complements the toolbox for the detection and identification of LMO. Approaches using different highly-reproducible NGS techniques (Pallarz et al., 2023) in combination with continually improving NGS-data analysis tools and pipelines are currently evaluated. They are still at the stage of research and development. There are several fields of application:
1. The number of LMO is ever increasing and a proportion of LMO is not detectable by routinely used PCR-based screening methods. Here, NGS approaches with a capture enrichment, based on known elements, might be economically sensible, especially if the increased amount of information acquired with every run is taken into account.
2. NGS allows for the simultaneous detection of many different LMO if the nucleotide sequence of the genetic modification is known.
3. Further molecular characterization and/or identification of unauthorized LMO may be achieved by NGS e.g. using primer-walking strategies that start from a common LMO element and elongating the sequence to reconstruct the entire LMO construct, or by whole genome sequencing approaches.
4. Furthermore, the continually collected NGS data allows for the development of AI models to detect even unknown LMO, which require many data sets to be properly trained
Next to digital PCR and NGS no new analytical techniques have been implemented. However, existing methods have been further optimized. For example, the use of locked nucleic acid (LNA) oligonucleotides or minor groove binder (MGB) probes allows for use of short oligonucleotides or probes.
Reference: Steffen Pallarz, Stefan Fiedler, Daniela Wahler, Jörn Lämke, Lutz Grohmann; 2023; Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape; https://doi.org/10.1016/j.fochms.2023.100182
My name is Daniela Wahler. I work for the German Federal Office of Consumer Protection and Food Safety (BVL) in the Unit traceability, detection methods, Biosafety Clearing-House. BVL is home to the competent national authority for the Cartagena Protocol and also to a working group that develops the official control methods for the detection and identification of LMO in Germany. I would like to thank the Secretariat for the opportunity to discuss here.
Regarding digital PCR, experience with this technique has increased since 2019 and a large number of official control laboratories in Germany are equipped to integrate digital PCR within their analyses. However, digital PCR methods are not yet part of routine analyses. The technique is mainly applied for the quantification of LMO in samples.
Next generation sequencing is a valuable tool that complements the toolbox for the detection and identification of LMO. Approaches using different highly-reproducible NGS techniques (Pallarz et al., 2023) in combination with continually improving NGS-data analysis tools and pipelines are currently evaluated. They are still at the stage of research and development. There are several fields of application:
1. The number of LMO is ever increasing and a proportion of LMO is not detectable by routinely used PCR-based screening methods. Here, NGS approaches with a capture enrichment, based on known elements, might be economically sensible, especially if the increased amount of information acquired with every run is taken into account.
2. NGS allows for the simultaneous detection of many different LMO if the nucleotide sequence of the genetic modification is known.
3. Further molecular characterization and/or identification of unauthorized LMO may be achieved by NGS e.g. using primer-walking strategies that start from a common LMO element and elongating the sequence to reconstruct the entire LMO construct, or by whole genome sequencing approaches.
4. Furthermore, the continually collected NGS data allows for the development of AI models to detect even unknown LMO, which require many data sets to be properly trained
Next to digital PCR and NGS no new analytical techniques have been implemented. However, existing methods have been further optimized. For example, the use of locked nucleic acid (LNA) oligonucleotides or minor groove binder (MGB) probes allows for use of short oligonucleotides or probes.
Reference: Steffen Pallarz, Stefan Fiedler, Daniela Wahler, Jörn Lämke, Lutz Grohmann; 2023; Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape; https://doi.org/10.1016/j.fochms.2023.100182
Dear colleagues,
My name is Margit Ross. I am a scientist at Corteva Agriscience and am a member of the CropLife International Detection Methods Experts Team.
Digital PCR by nature, allows more difficult PCR assays to perform well. By partitioning the DNA into thousands of single parallel ultra-low volume reactions, the signal to noise ratio is increased, reducing the negative impact of high-copy template DNA and/or PCR inhibitors. Benefits realized over real-time PCR are for distinguishing between LMOs or genetic elements that share a high homology, for genetic element assays that are reliant on only a few distinct nucleotides, for easier copy number, or zygosity determination, and for absolute quantification.
A recent capability with digital PCR is the ability to multiplex assays with some of the newer instrument platforms allowing for up to 4 reactions to be performed at once from a single sample reaction. While digital PCR in general is not high throughput, this functionality has greatly improved the output of the instrument as well as lowered the cost per sample. Additionally, it allows for analyses of an LMO (event-specific and member genetic elements) to be done at one timepoint. Another benefit is that accessibility and turnaround time for labeled probes with the newer dyes required by the instrument is not a problem. The required dyes, or their equivalents, are available through common oligonucleotide manufacturers.
My name is Margit Ross. I am a scientist at Corteva Agriscience and am a member of the CropLife International Detection Methods Experts Team.
Digital PCR by nature, allows more difficult PCR assays to perform well. By partitioning the DNA into thousands of single parallel ultra-low volume reactions, the signal to noise ratio is increased, reducing the negative impact of high-copy template DNA and/or PCR inhibitors. Benefits realized over real-time PCR are for distinguishing between LMOs or genetic elements that share a high homology, for genetic element assays that are reliant on only a few distinct nucleotides, for easier copy number, or zygosity determination, and for absolute quantification.
A recent capability with digital PCR is the ability to multiplex assays with some of the newer instrument platforms allowing for up to 4 reactions to be performed at once from a single sample reaction. While digital PCR in general is not high throughput, this functionality has greatly improved the output of the instrument as well as lowered the cost per sample. Additionally, it allows for analyses of an LMO (event-specific and member genetic elements) to be done at one timepoint. Another benefit is that accessibility and turnaround time for labeled probes with the newer dyes required by the instrument is not a problem. The required dyes, or their equivalents, are available through common oligonucleotide manufacturers.
Dear Ms. Daniela Wahler,
Thank you for the comments. I'm going to upload the ppt file describing the results of GM quantification by digital PCR. In my opinion, the most difficult and costly painful part for the quantification of unintentionally mixed GMO is the preparation and obtaining certified reference material (CRM). There is a new approach in the analysis of GM quantification by the combination of digital PCR + a single copy gene as a built-in CRM in the sample tested.
Whenever participants have questions how it work, leave comments below.
Thank you for the comments. I'm going to upload the ppt file describing the results of GM quantification by digital PCR. In my opinion, the most difficult and costly painful part for the quantification of unintentionally mixed GMO is the preparation and obtaining certified reference material (CRM). There is a new approach in the analysis of GM quantification by the combination of digital PCR + a single copy gene as a built-in CRM in the sample tested.
Whenever participants have questions how it work, leave comments below.
Dear colleagues,
My Name is Galina Mozgova, I work for the Institute of Genetics and Cytology, National Academy of Sciences of Belarus in the National Coordination Biosafety Centre accredited by the national system of accreditation for LMO detection and identification, as well as for species-specific identification. Geneticist by training, BCH NFP, and also a member of the AHTEG on LMO Risk Assessment.
Regarding dPCR, we use this method in our laboratory, it is reliable method for LMO detection and absolute quantification. The progress is made in relation to adapting it to the detection and quantification of more and more targets, which is important when you try to find ever-growing number of LMOs, especially unauthorized LMOs on the market of the country. Here are some articles of Slovenian laboratory on multiplex quantification of 15 lines of GM-soybean, and 12 GM-maize:
https://www.nature.com/articles/s41598-017-09377-w
https://pubs.acs.org/doi/10.1021/acs.analchem.5b01208
I also would like to share an article on interlaboratory analysis of reference materials, because as previously noted, this is a very sensitive question in the area of LMO detection: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748423/
However, in our laboratory we use dPCR usually for the identification and quantification of unauthorized LMOs, that we suppose present in the sample. In case of everyday routine analysis, we use RT-PCR, because dPCR is much more expensive due to the need to purchase specific plastic and reagents from one manufacturer. In addition, proficiency interlaboratory programs of collations in which we participated show that we can reliably quantify low level presence LMOs by the RT-PCR.
Both methods RT-PCR and dPCR are reliable for detection and quantification of new generations of LMOs in case if you know or suppose that you know what kind of LMOs are in the batch, and in case you have a reference materials. But in the case with the new breeding techniques, unfortunately, it can be a challenge. I agree with Ms. Daniela Wahler #11973 on cases in which NGS will be able to increase the detectability of known LMOs or new LMOs on which information is available. NGS could be also a method of choice when it is supposed that the new LM-line slightly differs from those normally detected in a laboratory, e.g. by a pair of nucleotides. With accumulating of the new sequences from NGS data it will be more easier to detect unauthorized LMOs, and NGS becomes a powerful tool for next generation LMOs. But, unfortunately, there are again a whole series of questions here. The availability of sequence data and the mandatory delivery of them to international databases, since the legislation of countries on what is considered LMOs or GMOs may differ; in addition, sequencing technologies are quite expensive and cannot be available, even after a certain number of years, for all laboratories, and reliable reference materials could be an obstacle. On the issue of capacity building, assistance will be needed both in training for many laboratories and in equipping them.
Also a very important question, is when you have this situation that you need preliminary quickly test a sample before sending to LDGMO, e.g. when monitoring unauthorized LMO in the field, or if it is an emergency situation in accordance with the Article 17 of the Cartagena Protocol. There must be such methods and equipment for rapid field research, but at the same time reliable. It could be also good to discuss such applications. I know one of such applications with equipment for field tests – KASP method which is adaptation of qPCR that is available for field tests commercially. KASP primers (typically three) are custom-designed to target the SNP or InDel of interest:
https://www.frontiersin.org/articles/10.3389/fmolb.2022.773956/full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751575/
Kind regards,
Galina
My Name is Galina Mozgova, I work for the Institute of Genetics and Cytology, National Academy of Sciences of Belarus in the National Coordination Biosafety Centre accredited by the national system of accreditation for LMO detection and identification, as well as for species-specific identification. Geneticist by training, BCH NFP, and also a member of the AHTEG on LMO Risk Assessment.
Regarding dPCR, we use this method in our laboratory, it is reliable method for LMO detection and absolute quantification. The progress is made in relation to adapting it to the detection and quantification of more and more targets, which is important when you try to find ever-growing number of LMOs, especially unauthorized LMOs on the market of the country. Here are some articles of Slovenian laboratory on multiplex quantification of 15 lines of GM-soybean, and 12 GM-maize:
https://www.nature.com/articles/s41598-017-09377-w
https://pubs.acs.org/doi/10.1021/acs.analchem.5b01208
I also would like to share an article on interlaboratory analysis of reference materials, because as previously noted, this is a very sensitive question in the area of LMO detection: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748423/
However, in our laboratory we use dPCR usually for the identification and quantification of unauthorized LMOs, that we suppose present in the sample. In case of everyday routine analysis, we use RT-PCR, because dPCR is much more expensive due to the need to purchase specific plastic and reagents from one manufacturer. In addition, proficiency interlaboratory programs of collations in which we participated show that we can reliably quantify low level presence LMOs by the RT-PCR.
Both methods RT-PCR and dPCR are reliable for detection and quantification of new generations of LMOs in case if you know or suppose that you know what kind of LMOs are in the batch, and in case you have a reference materials. But in the case with the new breeding techniques, unfortunately, it can be a challenge. I agree with Ms. Daniela Wahler #11973 on cases in which NGS will be able to increase the detectability of known LMOs or new LMOs on which information is available. NGS could be also a method of choice when it is supposed that the new LM-line slightly differs from those normally detected in a laboratory, e.g. by a pair of nucleotides. With accumulating of the new sequences from NGS data it will be more easier to detect unauthorized LMOs, and NGS becomes a powerful tool for next generation LMOs. But, unfortunately, there are again a whole series of questions here. The availability of sequence data and the mandatory delivery of them to international databases, since the legislation of countries on what is considered LMOs or GMOs may differ; in addition, sequencing technologies are quite expensive and cannot be available, even after a certain number of years, for all laboratories, and reliable reference materials could be an obstacle. On the issue of capacity building, assistance will be needed both in training for many laboratories and in equipping them.
Also a very important question, is when you have this situation that you need preliminary quickly test a sample before sending to LDGMO, e.g. when monitoring unauthorized LMO in the field, or if it is an emergency situation in accordance with the Article 17 of the Cartagena Protocol. There must be such methods and equipment for rapid field research, but at the same time reliable. It could be also good to discuss such applications. I know one of such applications with equipment for field tests – KASP method which is adaptation of qPCR that is available for field tests commercially. KASP primers (typically three) are custom-designed to target the SNP or InDel of interest:
https://www.frontiersin.org/articles/10.3389/fmolb.2022.773956/full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751575/
Kind regards,
Galina
Dear Ms. Gallina Mozgova,
We appreciate the time you committed to providing us with detailed feedback and valuable bibliographic references.
Kind regards
We appreciate the time you committed to providing us with detailed feedback and valuable bibliographic references.
Kind regards
Dear Mr. Jae Sun Moon,
Thank you for the interesting information on quantification of LMO in samples by nanoplate digital PCR. In the EU, for the initial method development and validation we rely on (certified) reference material. Moreover, even during quantification analyses (certified) reference materials are tested in separate partitions/compartments of dPCR-runs as a positive control if possible and reasonable.
May I ask whether there is a publication on your work that you would be happy to share? Please find attached a link to a report by the Joint Research Center (JRC) of the EU COM on how digital PCR is handled in the EU (Overview and recommendations for the application of digital PCR; https://gmo-crl.jrc.ec.europa.eu/doc/WG-dPCR-Report.pdf).
Kind regards,
Daniela
Thank you for the interesting information on quantification of LMO in samples by nanoplate digital PCR. In the EU, for the initial method development and validation we rely on (certified) reference material. Moreover, even during quantification analyses (certified) reference materials are tested in separate partitions/compartments of dPCR-runs as a positive control if possible and reasonable.
May I ask whether there is a publication on your work that you would be happy to share? Please find attached a link to a report by the Joint Research Center (JRC) of the EU COM on how digital PCR is handled in the EU (Overview and recommendations for the application of digital PCR; https://gmo-crl.jrc.ec.europa.eu/doc/WG-dPCR-Report.pdf).
Kind regards,
Daniela
Dear colleagues,
Our laboratory, as a unit of the Ministry of Agriculture and Livestock of Brazil, has fifteen years of experience in Living Modified Organism analysis. We work mainly with Real Time PCR methods published by the International Organization for Standardization (ISO) or European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF). We also have expertise and facilities in analysis based on Digital PCR and Next Generation Sequencing technologies. These technologies have been used for pest identification and food fraud detection. Currently, we don’t have any implemented method for analysis of products obtained by New Genomic Techniques (NGT).
Our laboratory has almost ten years of experience with Sanger Sequencing and, ultimately, we have implemented Next Generation Sequencing and Digital PCR technologies, but not devoted to LMO analysis yet.
Best regards,
Cairo Oliveira
Our laboratory, as a unit of the Ministry of Agriculture and Livestock of Brazil, has fifteen years of experience in Living Modified Organism analysis. We work mainly with Real Time PCR methods published by the International Organization for Standardization (ISO) or European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF). We also have expertise and facilities in analysis based on Digital PCR and Next Generation Sequencing technologies. These technologies have been used for pest identification and food fraud detection. Currently, we don’t have any implemented method for analysis of products obtained by New Genomic Techniques (NGT).
Our laboratory has almost ten years of experience with Sanger Sequencing and, ultimately, we have implemented Next Generation Sequencing and Digital PCR technologies, but not devoted to LMO analysis yet.
Best regards,
Cairo Oliveira
Dear colleagues,
Thank you for your insightful inputs and for Dr. Raja Chalghoumi for moderating this session.
My name is Sarah Agapito and I am a Research Professor at NORCE Norwegian Research Centre. NORCE hosts the competence centre for biosafety in Norway since 2009. The centre offers governmental advice and performs research within all the matters related to the implementation of The Gene Technology Act which regulates GMOs in Norway. This includes, among others, detection and identification of GMOs and new technologies issues.
Recently, I have also dedicated my research to developing novel analytical methods and new methodological approaches to the detection and identification of NGT products. We are finalizing some experiments under a research project funded by The Research Council of Norway. We have tested some improved real-time PCR systems but also worked towards multi-target approaches using sequencing strategies. In the latter case, target deep sequencing. Whereas our projects are mainly research projects, they are always carried out in light of the European regulations in order to provide useful and fit-for-purpose methods.
1) I would also like to call attention to the recent effort by the European Commission to fund research dedicated to develop new methods for NGT detection, identification and quantification under the EC Horizon Europe Program Cluster 6 (10M EUR). Under this topic, two projects were awarded to develop analytical which also include next-generation sequencing and digital PCR in LMO analysis and digital methods (traceability). I can also refer to the work of colleagues:
Fraiture MA, et al. (2022) ddPCR strategy to detect a gene-edited plant carrying a single variation point: Technical feasibility and interpretation issues. Food Control 137, 108904. https://doi.org/10.1016/j.foodcont.2022.108904.
2) New methods are being developed since 2019 that could be applied to the detection and identification of new LMOs. However, they differ in the degree they would meet regulatory criteria and this still needs to be assessed. Some of these methods are untargeted and since no prior enrichment of key targets is required, this open strategy offers the opportunity to characterize both known and unknown NGT organisms. I also recognize that some progress has been done in other areas of knowledge in term of detection and identification of analytical molecules and some of these methods could also be adjusted to new LMOs. Few examples of published papers:
Fraiture, M. A., et al. (2023). Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms. Foods (Basel, Switzerland), 12(3), 455. https://doi.org/10.3390/foods12030455
D'aes J, et al. (2022) Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products. Life 12(12), 1971. https://doi.org/10.3390/life12121971.
Saltykova, A. et al. (2022). Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database. Food chemistry. Molecular sciences, 4, 100096. https://doi.org/10.1016/j.fochms.2022.100096
D'aes J, et al. (2021) Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products. Foods 10(11), 2637. https://doi.org/10.3390/foods10112637.
3) Finally, I can echo the colleagues who have highlighted the importance of databases in support of analytical methods.
Thank you.
Best regards,
Sarah
Thank you for your insightful inputs and for Dr. Raja Chalghoumi for moderating this session.
My name is Sarah Agapito and I am a Research Professor at NORCE Norwegian Research Centre. NORCE hosts the competence centre for biosafety in Norway since 2009. The centre offers governmental advice and performs research within all the matters related to the implementation of The Gene Technology Act which regulates GMOs in Norway. This includes, among others, detection and identification of GMOs and new technologies issues.
Recently, I have also dedicated my research to developing novel analytical methods and new methodological approaches to the detection and identification of NGT products. We are finalizing some experiments under a research project funded by The Research Council of Norway. We have tested some improved real-time PCR systems but also worked towards multi-target approaches using sequencing strategies. In the latter case, target deep sequencing. Whereas our projects are mainly research projects, they are always carried out in light of the European regulations in order to provide useful and fit-for-purpose methods.
1) I would also like to call attention to the recent effort by the European Commission to fund research dedicated to develop new methods for NGT detection, identification and quantification under the EC Horizon Europe Program Cluster 6 (10M EUR). Under this topic, two projects were awarded to develop analytical which also include next-generation sequencing and digital PCR in LMO analysis and digital methods (traceability). I can also refer to the work of colleagues:
Fraiture MA, et al. (2022) ddPCR strategy to detect a gene-edited plant carrying a single variation point: Technical feasibility and interpretation issues. Food Control 137, 108904. https://doi.org/10.1016/j.foodcont.2022.108904.
2) New methods are being developed since 2019 that could be applied to the detection and identification of new LMOs. However, they differ in the degree they would meet regulatory criteria and this still needs to be assessed. Some of these methods are untargeted and since no prior enrichment of key targets is required, this open strategy offers the opportunity to characterize both known and unknown NGT organisms. I also recognize that some progress has been done in other areas of knowledge in term of detection and identification of analytical molecules and some of these methods could also be adjusted to new LMOs. Few examples of published papers:
Fraiture, M. A., et al. (2023). Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms. Foods (Basel, Switzerland), 12(3), 455. https://doi.org/10.3390/foods12030455
D'aes J, et al. (2022) Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products. Life 12(12), 1971. https://doi.org/10.3390/life12121971.
Saltykova, A. et al. (2022). Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database. Food chemistry. Molecular sciences, 4, 100096. https://doi.org/10.1016/j.fochms.2022.100096
D'aes J, et al. (2021) Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products. Foods 10(11), 2637. https://doi.org/10.3390/foods10112637.
3) Finally, I can echo the colleagues who have highlighted the importance of databases in support of analytical methods.
Thank you.
Best regards,
Sarah
Dear Ms Daniela Wahler,
Thank you for your comments and suggestions.
I'm preparing the manuscript and waiting for the permission from the company having right on intellectual property. I'll give you the publication just after an acceptance letter from a journal.
As you realize, all I try to do is to eliminate CRMs in the systems of the relative quantification of GMO. Including South Korea, many countries are not able to properly inspect approved GM crops due to the lack of CRMs.
You may open the attached ppt file in my posting whenever you need to obtain further information.
Best regards
Jae Sun.
Thank you for your comments and suggestions.
I'm preparing the manuscript and waiting for the permission from the company having right on intellectual property. I'll give you the publication just after an acceptance letter from a journal.
As you realize, all I try to do is to eliminate CRMs in the systems of the relative quantification of GMO. Including South Korea, many countries are not able to properly inspect approved GM crops due to the lack of CRMs.
You may open the attached ppt file in my posting whenever you need to obtain further information.
Best regards
Jae Sun.
Dear Mr. Jae Sun Moon,
Thank you for sharing the slides regarding your new digital PCR platform.
I am quite interested in how the performance of the selected built-in CRM as a taxon-specific reference gene, if physically extracted from each specific crop, performed and were quantified on the new platform? Additionally, can you provide comment on how a quantitative detection method developed on this new platform may easily transfer to another digital PCR platform that relies on the physical use of a taxon-specific reference gene?
Recently, CropLife International industry members published taxon-specific reference assays for maize, soybean, cotton and canola that are proven to amplify a single copy gene as well as demonstrate no allelic variation in the host plant genome (https://detection-methods.com/endogenous-methods/). These reference genes are also the most prevalently used in the development of quantitative detection methods and have been fully evaluated by digital PCR (https://doi.org/10.1016/j.foodcont.2018.06.013).
Best regards,
Margit Ross
Thank you for sharing the slides regarding your new digital PCR platform.
I am quite interested in how the performance of the selected built-in CRM as a taxon-specific reference gene, if physically extracted from each specific crop, performed and were quantified on the new platform? Additionally, can you provide comment on how a quantitative detection method developed on this new platform may easily transfer to another digital PCR platform that relies on the physical use of a taxon-specific reference gene?
Recently, CropLife International industry members published taxon-specific reference assays for maize, soybean, cotton and canola that are proven to amplify a single copy gene as well as demonstrate no allelic variation in the host plant genome (https://detection-methods.com/endogenous-methods/). These reference genes are also the most prevalently used in the development of quantitative detection methods and have been fully evaluated by digital PCR (https://doi.org/10.1016/j.foodcont.2018.06.013).
Best regards,
Margit Ross
Dear Dr. Sarah Agapito-Tenfen,
Thank you for providing us with valuable comments and relevant references.
All the best
Thank you for providing us with valuable comments and relevant references.
All the best
Dear Ms. Ross and Mr. Moon,
Thank you both for the fruitful exchange regarding digital PCR.
Warm regards
Thank you both for the fruitful exchange regarding digital PCR.
Warm regards
Dear Colleagues,
I would like to gently remind you that the forum will close on Tuesday, 28 November 2023 at 17:00 EST (UTC -5).
We look forward to your contributions and exchanges!
Best regards
I would like to gently remind you that the forum will close on Tuesday, 28 November 2023 at 17:00 EST (UTC -5).
We look forward to your contributions and exchanges!
Best regards
Dear colleagues,
I am Litao Yang, a professor from Shanghai Jiao Tong UNiversity. In my group, we are working on developing new techniques for identifiation and quantification of LMOs. As to those three questions, my opinions are listed as below:
- What progress has been made with the implementation of next-generation sequencing and digital PCR in LMO analysis?
Digital PCR showed the advantages of accuracy and CRM-independence, whcih has been accepted and used in GMO quantification in sevral labs in China, However, the disadvantages of the high costs and throughput for dPCR might limit its application in routine GMO analysis currently.
We explored the potenial of NGS in GMO detection ang quantification, and we have achieved in unathorized GMOs analysis, GMO content screening, and molecular characterization of GM events. However, it is still challege for developing the suitable NGS stretagies for GM content quanrification. Although the NGS cost decreased dramastcally, the sequencing depth requirement matching the quantitative limit of 0.1% or 0.01% is still very high.
- Are there isothermal techniques now in use? What recent developments have been made with respect to these techniques?
Isotherml techniques, such as LAMP, RPA, and RCA is acceptable for rapid GMO screening in China. The MOA of China are seeking rapid screening methods for GMOs. Recently, we have combined the CRISPR/Cas with LAMP and RCA to develope new POCT assays for GM contents screening, and these methods have been confirmed by MOA.
- Are there any new analytical techniques that have been developed or implemented since 2019 that could be applied to detection and identification of LMOs? If so, what are they?
Since 2019, several isothermal methods and amplification free methods combined with CRISPR/Cas were reported and used for GMO analysis. Also, the NGS with various pre-enrichment strategies were also developed, such as LIFE-Seq. In our group, we also esatblised serval methods for gene-editing crops identification and geno-typing, such as qPCR-HRM, cc-qPCR, SMART, etc.
I am Litao Yang, a professor from Shanghai Jiao Tong UNiversity. In my group, we are working on developing new techniques for identifiation and quantification of LMOs. As to those three questions, my opinions are listed as below:
- What progress has been made with the implementation of next-generation sequencing and digital PCR in LMO analysis?
Digital PCR showed the advantages of accuracy and CRM-independence, whcih has been accepted and used in GMO quantification in sevral labs in China, However, the disadvantages of the high costs and throughput for dPCR might limit its application in routine GMO analysis currently.
We explored the potenial of NGS in GMO detection ang quantification, and we have achieved in unathorized GMOs analysis, GMO content screening, and molecular characterization of GM events. However, it is still challege for developing the suitable NGS stretagies for GM content quanrification. Although the NGS cost decreased dramastcally, the sequencing depth requirement matching the quantitative limit of 0.1% or 0.01% is still very high.
- Are there isothermal techniques now in use? What recent developments have been made with respect to these techniques?
Isotherml techniques, such as LAMP, RPA, and RCA is acceptable for rapid GMO screening in China. The MOA of China are seeking rapid screening methods for GMOs. Recently, we have combined the CRISPR/Cas with LAMP and RCA to develope new POCT assays for GM contents screening, and these methods have been confirmed by MOA.
- Are there any new analytical techniques that have been developed or implemented since 2019 that could be applied to detection and identification of LMOs? If so, what are they?
Since 2019, several isothermal methods and amplification free methods combined with CRISPR/Cas were reported and used for GMO analysis. Also, the NGS with various pre-enrichment strategies were also developed, such as LIFE-Seq. In our group, we also esatblised serval methods for gene-editing crops identification and geno-typing, such as qPCR-HRM, cc-qPCR, SMART, etc.
Dear colleagues,
My name is Emmanuel González-Ortega. I’m trained in Biotechnology (Engineering, Ms Sc, PhD), and I have expertise in LMO detection and identification, biosafety governance, molecular biology, risk assessment, socioeconomic considerations of LMO, synthetic biology. Our research group have investigated and published on the presence of unauthorized genetically modified maize in several Mexican regions, and on the presence of genetically modified maize in highly consumed food manufactured with maize in Mexico. I have previously participated in several fora in the Cartagena Protocol of the CBD. I would like to thank the Secretariat of the CBD for the organization of the on-line forum of the Network of Laboratories for the Detection and Identification of Living Modified Organisms and for the opportunity to participate in this discussion.
Regarding the first topic [#11945] (New techniques for detecting and identifying LMOs), I have some considerations.
We are witnessing a speed race in the field of technological innovations in molecular biology applied, in this case, to the detection and identification of LMOs (i.e. next generation sequencing, digital PCR, biostatistical methods and protocols for genome analysis). Of course, multilateral discussion on these new techniques is very necessary and pertinent, and even more so considering the new applications and products of biotechnology and synthetic biology, the generation of LMOs through genome editing, the application of iRNA, gene drives, etc. However, it is necessary to mention that there are still inequities and lack of access to knowledge and daily application in many countries (mainly in the so-called “Global South”) of the basic techniques historically used as protocols for the detection and identification of LMOs, which, in addition, have proven to be robust and reliable over time, and are progressively more economically accessible. There is also discontinuity in the training or specialization of technical personnel in many countries, and importantly, there is little availability of economic resources and infrastructure (equipment, supplies) that could allow the continued activities on LMO monitoring, detection and identification in the countries (which, in many cases, are regions of high biological diversity).
Additionally, countries that due to various situations (i.e. economic, scientific and technological) are not capable of carrying out innovation activities or to implemen new LMO detection and identification techniques in their laboratories, see their role perpetuated as adopters of the technologies that were designed in different contexts or technological backgrounds, they become dependent on the expensive inputs (as briefly commented in the post [#11981]), necessary to carry out LMO detection and identification activities, and perpetuate their role as potential victims of technological obsolescence and hegemonically proposed technologies for LMO monitoring activities.
In Mexico, few demonstrative workshops have been performed on dPCR for detection of LMO, however, the analyzes related to the biosafety of LMO, which are carried out by very few public universities and research centers in the country, are based on RT-PCR (Taq- Man or Sybr Green). There are even some laboratories that have published studies detecting genetically modified corn using endpoint PCR, or lateral flow strips.
Next Generation Sequencing (NGS) is not being used for the purposes of detection or identification of LMO in Mexico in a consistent or routine manner. However, given the eminent introduction into the environment of genetically modified organisms through genome editing and their products (i.e. CRISPR-Cas), it will be essential to make accessible methodologies, specialized training, technical and scientific discussion on the most appropriate NGS technique, even considering the relationship costs/benefits. It is urgently needed the organization of courses and workshops related to updating or capacity-building on the molecular techniques for LMO detection, even considering the well-established techniques for detecting LMO for the less favored countries, as well as information and discussion forums on the governance of the biosafety of the new LMOs from the Biosafety Clearing House.
Best regards,
Emmanuel.
My name is Emmanuel González-Ortega. I’m trained in Biotechnology (Engineering, Ms Sc, PhD), and I have expertise in LMO detection and identification, biosafety governance, molecular biology, risk assessment, socioeconomic considerations of LMO, synthetic biology. Our research group have investigated and published on the presence of unauthorized genetically modified maize in several Mexican regions, and on the presence of genetically modified maize in highly consumed food manufactured with maize in Mexico. I have previously participated in several fora in the Cartagena Protocol of the CBD. I would like to thank the Secretariat of the CBD for the organization of the on-line forum of the Network of Laboratories for the Detection and Identification of Living Modified Organisms and for the opportunity to participate in this discussion.
Regarding the first topic [#11945] (New techniques for detecting and identifying LMOs), I have some considerations.
We are witnessing a speed race in the field of technological innovations in molecular biology applied, in this case, to the detection and identification of LMOs (i.e. next generation sequencing, digital PCR, biostatistical methods and protocols for genome analysis). Of course, multilateral discussion on these new techniques is very necessary and pertinent, and even more so considering the new applications and products of biotechnology and synthetic biology, the generation of LMOs through genome editing, the application of iRNA, gene drives, etc. However, it is necessary to mention that there are still inequities and lack of access to knowledge and daily application in many countries (mainly in the so-called “Global South”) of the basic techniques historically used as protocols for the detection and identification of LMOs, which, in addition, have proven to be robust and reliable over time, and are progressively more economically accessible. There is also discontinuity in the training or specialization of technical personnel in many countries, and importantly, there is little availability of economic resources and infrastructure (equipment, supplies) that could allow the continued activities on LMO monitoring, detection and identification in the countries (which, in many cases, are regions of high biological diversity).
Additionally, countries that due to various situations (i.e. economic, scientific and technological) are not capable of carrying out innovation activities or to implemen new LMO detection and identification techniques in their laboratories, see their role perpetuated as adopters of the technologies that were designed in different contexts or technological backgrounds, they become dependent on the expensive inputs (as briefly commented in the post [#11981]), necessary to carry out LMO detection and identification activities, and perpetuate their role as potential victims of technological obsolescence and hegemonically proposed technologies for LMO monitoring activities.
In Mexico, few demonstrative workshops have been performed on dPCR for detection of LMO, however, the analyzes related to the biosafety of LMO, which are carried out by very few public universities and research centers in the country, are based on RT-PCR (Taq- Man or Sybr Green). There are even some laboratories that have published studies detecting genetically modified corn using endpoint PCR, or lateral flow strips.
Next Generation Sequencing (NGS) is not being used for the purposes of detection or identification of LMO in Mexico in a consistent or routine manner. However, given the eminent introduction into the environment of genetically modified organisms through genome editing and their products (i.e. CRISPR-Cas), it will be essential to make accessible methodologies, specialized training, technical and scientific discussion on the most appropriate NGS technique, even considering the relationship costs/benefits. It is urgently needed the organization of courses and workshops related to updating or capacity-building on the molecular techniques for LMO detection, even considering the well-established techniques for detecting LMO for the less favored countries, as well as information and discussion forums on the governance of the biosafety of the new LMOs from the Biosafety Clearing House.
Best regards,
Emmanuel.
My name is Guadalupe Barrera, since 2006 work at GMO laboratory from ministry of agriculture in Mexico. The laboratory has accreditation since 2011, for GMO detection, identificación, and quantification. We work with qPCR and dPCR both techniques have accreditation. We have projects with isothermal amplification for use in field, and microarrays.
In this link you can consult more information about the events and matrices that we analyze
https://www.gob.mx/senasica/acciones-y-programas/departamento-de-analisis-molecular-y-nuevas-tecnologias-y-el-area-de-secuenciacion-y-bioinformatica
In this link you can consult more information about the events and matrices that we analyze
https://www.gob.mx/senasica/acciones-y-programas/departamento-de-analisis-molecular-y-nuevas-tecnologias-y-el-area-de-secuenciacion-y-bioinformatica
Dear Colleagues,
My name is Lilian Okiro from Egerton University, Kenya. Kenya has modern labs for detection of LMOs mainly using conventional PCR and qPCR. Since LMOs have not yet been approved for commercialization, most advanced detection techniques have not been implemented for use by regulators except for research purposes. The lab techniques currently in use are basically based on screening of the 35S promoter and the NOS terminator. Quantification based on CRS has also been implemented on a trial basis by some regulatory labs. It is good to hear that more sensitive methods using digital PCR and NGS are currently in use which can also be implemented in Kenya for regulatory purposes based on availability of resources.
Lilian
My name is Lilian Okiro from Egerton University, Kenya. Kenya has modern labs for detection of LMOs mainly using conventional PCR and qPCR. Since LMOs have not yet been approved for commercialization, most advanced detection techniques have not been implemented for use by regulators except for research purposes. The lab techniques currently in use are basically based on screening of the 35S promoter and the NOS terminator. Quantification based on CRS has also been implemented on a trial basis by some regulatory labs. It is good to hear that more sensitive methods using digital PCR and NGS are currently in use which can also be implemented in Kenya for regulatory purposes based on availability of resources.
Lilian
Dear participants and esteemed colleagues,
My name is Nancy Serrano-Silva, and I am affiliated with the Executive Secretariat of the Interministerial Commission for the Biosafety of Genetically Modified Organisms in Mexico. I am pleased to join this forum on new techniques for detecting and identifying Genetically Modified Organisms (GMOs) within the framework of the Conference of the Parties to the Cartagena Protocol on Biosafety.
As my colleague Emmanuel González-Ortega highlighted earlier, we find ourselves amidst a rapid race of technological innovations in molecular biology, particularly in the realm of GMO detection and identification. Techniques such as next-generation sequencing (NGS), digital PCR, and bioinformatic and biostatistic analyses have indeed transformed the landscape of biosafety. It is undeniable that a multilateral discussion on these technologies is crucial, especially in light of the new applications and products emerging from biotechnology and synthetic biology. The generation of GMOs through genome editing, the application of iRNA, and other innovations present significant challenges that necessitate a collaborative and coordinated approach.
However, it is imperative to underscore that despite technological progress, disparities and a lack of access to knowledge persist in many countries. While historically established techniques, such as traditional protocols for GMO detection and identification, remain robust and increasingly economically accessible, the shortage of financial resources, infrastructure, and adequate training creates a substantial gap in the effective implementation of these technologies.
In the Mexican context, there is a noticeable disparity in the adoption of new techniques. While some demonstrative workshops on digital PCR for GMO detection have taken place, the majority of biosafety analyses in the country still rely on more conventional methods like RT-PCR. The imminent introduction of genetically modified organisms through genome editing demands a swift adaptation to more advanced technologies, such as Next-Generation Sequencing (NGS).
Addressing the lack of consistency in the application of these techniques in Mexico and other resource-limited countries is essential. There is an urgent need for the organization of courses and workshops focusing on training and updating in molecular techniques for GMO detection. Additionally, forums for discussing the governance of biosafety for new GMOs are crucial, ensuring equitable participation from all nations, irrespective of their economic or technological development.
We advocate for an inclusive approach that recognizes the importance of established techniques while promoting the responsible adoption of innovations. Only through global collaboration and equitable access to technology can we ensure a sustainable and secure future in the field of biosafety.
Thank you for your attention, and the valuable contributions of participants in this forum.
Sincerely,
Nancy
My name is Nancy Serrano-Silva, and I am affiliated with the Executive Secretariat of the Interministerial Commission for the Biosafety of Genetically Modified Organisms in Mexico. I am pleased to join this forum on new techniques for detecting and identifying Genetically Modified Organisms (GMOs) within the framework of the Conference of the Parties to the Cartagena Protocol on Biosafety.
As my colleague Emmanuel González-Ortega highlighted earlier, we find ourselves amidst a rapid race of technological innovations in molecular biology, particularly in the realm of GMO detection and identification. Techniques such as next-generation sequencing (NGS), digital PCR, and bioinformatic and biostatistic analyses have indeed transformed the landscape of biosafety. It is undeniable that a multilateral discussion on these technologies is crucial, especially in light of the new applications and products emerging from biotechnology and synthetic biology. The generation of GMOs through genome editing, the application of iRNA, and other innovations present significant challenges that necessitate a collaborative and coordinated approach.
However, it is imperative to underscore that despite technological progress, disparities and a lack of access to knowledge persist in many countries. While historically established techniques, such as traditional protocols for GMO detection and identification, remain robust and increasingly economically accessible, the shortage of financial resources, infrastructure, and adequate training creates a substantial gap in the effective implementation of these technologies.
In the Mexican context, there is a noticeable disparity in the adoption of new techniques. While some demonstrative workshops on digital PCR for GMO detection have taken place, the majority of biosafety analyses in the country still rely on more conventional methods like RT-PCR. The imminent introduction of genetically modified organisms through genome editing demands a swift adaptation to more advanced technologies, such as Next-Generation Sequencing (NGS).
Addressing the lack of consistency in the application of these techniques in Mexico and other resource-limited countries is essential. There is an urgent need for the organization of courses and workshops focusing on training and updating in molecular techniques for GMO detection. Additionally, forums for discussing the governance of biosafety for new GMOs are crucial, ensuring equitable participation from all nations, irrespective of their economic or technological development.
We advocate for an inclusive approach that recognizes the importance of established techniques while promoting the responsible adoption of innovations. Only through global collaboration and equitable access to technology can we ensure a sustainable and secure future in the field of biosafety.
Thank you for your attention, and the valuable contributions of participants in this forum.
Sincerely,
Nancy
