Detection and Identification Online Discussions 2019

Dear Participants,

I am honoured to welcome you to the online discussions by the Network of Laboratories for the Detection and Identification of Living Modified Organisms in 2019.  This forum responds to decision 9/11 of the Conference of Parties to the Cartagena Protocol on Biosafety, which requested the Executive Secretary to continue facilitating online discussions of the Network of Laboratories for the Detection and Identification of Living Modified Organisms (LMOs).

Participants are invited to actively contribute to the forum discussions to provide a valuable pool of resources. Three topics would be discussed concurrently.

Under Topic 1, participants are requested to provide examples of the techniques currently in use, as well as to highlight any new technique or approach that is either in use or under development. Participants are encouraged to consider this with respect to LMOs that are currently on the market, as well as those that are under research and development. As well, participants are also encouraged to share their experiences in detection and identification of LMOs produced through genome editing.

Some points to consider are:
- New techniques that are in use for the detection and identification of LMOs;
- The accuracy, applicability and reliability of new methodologies;
- The applicability of current techniques for the detection and identification of LMOs with special consideration for genome editing

When posting this information; kindly provide the source, and indicate if it is coming from a publication, your own work, or any other source. In addition, if you would like to respond to another post, we request that you mention the # of the post you are responding to, for example: “in response to post # XXXX, …”. This will also facilitate the understanding of others. Please post your responses under this thread. We also advise you to review the general guidelines for the forum posted on the main webpage of the discussion.

I would like to thank you for your participation in this forum and look forward to an interesting discussion.

Kind regards,
Shakirat Ajenifujah-Solebo

Dear all

I am working in Costa Rica with the detection of LMO at the basic level of screening of the P35S and TNOS regions. I am using a commercial kit that includes the of P35S and TNOS, positive controls for  P35S, TNOS and a Plant specific region. It also includes the detection of a specific region for both CaMV and A. tumefaciens. These are intended to discriminate false positive results in cases where the virus or the bacteria could be in the analyzed sample. However, when I review some of the most common protocols I still have not found this as a common practice. In my case, I have found sone samples with a positive result for the specific region of A, tumefaciens.
So that, I would like to hear back some comments if the use of control for possible false positives is worth to continue.
I s appreciate your attention.

Dear colleague from Costa Rica, at CENAM Mexico we offer a PT to measure the p35S or t-NOS, if you consider that it could be useful for you to participate you can find our PT program at http://www.cenam.mx/servicios/paptitudtecnica/

Melina Pérez Urquiza

New techniques that are in use for the detection and identification of LMOs
The detection  of LMOs involves the screening test using qualitative PCR to target the regulator sequence of the foreign gene (transformation event) to determine its presence of absence in the food. At this level, the most authorized GMO on the market is the P35S promoter of the Cauliflower mosaic virus, the TNOS terminator of the nopaline solution, the T35S terminator of the Cauliflower mosaic virus and possibly genes of interest such as Cry1(b), pat or Bar.

After screening for LMOs, positive samples are analysed to identify the modified gene(s), a method approved by the national commission on Biosafety. For this, the modified gene primers in question are used. The DNA fragment amplified by the PCR should cover the region of the modified gene insertion in the plant genome, i.e; it comprised a portion of the modified gene and a portion of the plant genome

The accuracy, applicability and reliability of new methodologies:
These methods of screening of LMOs are very accurate and can be applied on a wide range of foodstuffs. However,  since new LMOs are developed very often, these methods may not detect new LMOs.

With respect to genome editing, I have no idea on how these methods can be applied.

DNA Technology Laboratory is one of the key laboratory providing standard LMO detection. Two limitations that we have are 1) quantitative detection: It is better if different labs have a similar type of standard and equivalent protocol, 2) specific ISO for quantitative detection of LMO has not yet been implemented, 3) while we are discussing traditional LMO, scientists are finding way to eliminate all footprints or markerless LMO. Actually, they have already found exactly what to do. Beyond that point, we do not need to detect LMO anymore.
Apichart Vanavichit,
Rice Science Center and DNATEC,
Kasetsart University, Thailand

Thank you Emmanuel #9974 (Costa Rica) and Stephen #9980 (Cameroon) for sharing your experiences on topic 1.

Emmanuel #9974 specifically requested for more insight on the use of false positives in LMO detection and identification. Forum participants are requested to provide more information on this that could assist other laboratories, based on their experiences and/or other reference documents.

Thank you.

Shakirat

Greetings from Bhutan!
I am working in National Food Testing Laboratory (NFTL) under Bhutan Agriculture and Food Regulatory Authority, Ministry of Agriculture and Forests. We have a GMO detection lab within NFTL wherein we carry out the qualitative screening of the GMOs for the regulatory elements like the p35S and the tnos along with few other GM elements. Further, we are also accredited for few basic parameters.

However, we have few major challenges, which we currently face in the GMO Detection and Identification and they are:
1. Lack of expertise within the country on the GMO detection and identification or for any related technical discussions
2.No technical competence on the quantitative analysis
3. DNA Extraction kits/Reagents/consumables are not readily available within the country
4. Very Little knowledge on the method validation

So I would like to take this opportunity to explore on how Bhutan can benefit from rest of the countries in the above-mentioned areas.  Thanking you,

Dear Ms Dechen Wangmo
I am from the national reference Laboratory for GMO in Denmark. I understand your challenges, and for challenge no. 1 and probably also no.2, I can just say, that a Network in your area would be very useful. For me the European network for GMO laboratories has been very important for the competances I have today.
For your challenge no. 4:
In the European Network of GMO laboratories we have made a very practical guideline for verification og methods. It can be used when implementing qualitative or quantitative methods in the Laboratory.
You can find all guidelines here: https://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm
And the specific guideline for verification here: https://gmo-crl.jrc.ec.europa.eu/ENGL/docs/WG-MV-Report-version-2.pdf
You are welcome to ask for explanations.
Kind regards Lotte Hougs

Dear Lotte Hougs #9991, thank you for responding specifically to the questions posted by Dechen #9990. Yes, I agree that networking with other laboratories in your country and the region can assist with some of these challenges. Do you have or aware of any such network collaborations within your region?

And this forum also provides a platform for future collaborations!

Shakirat

Dear Dr. Lotte,
Thank you for your response (#9991) and for all the information shared. I agree on your idea of having a network in our region so looking forward to having one soon. In addition, I would like to take this opportunity to thank the European Network of GM Laboratories for making the Guiding documents/resources available for us online.  We are referring to the documents often and it is of immense help. And we will be contacting you for any explanations when required.
Thanking you

Hello everyone,
Greetings from Malaysia!

I am attached to the ASEAN GMO reference laboratory at the Department of Chemistry Malaysia. We routinely carry out GMO screening using the common screening elements such as 35s promoter, NOS terminator, pat, bar, cry1Ab and ctp2-cp4epsps. We then proceed with GM event identification based on the screening elements which are detected postive. We use the detection methods which have been validated by the EU Reference laboratory as a guide for most of our detection methods.

Our main issues at the moment which I would like some feedback on is:
1. How do you keep up with the development of new GM events and developing new detection methods for all these events that are currently available in the market?
2. What are the strategies employed for the detection of unknown GMO?

Any feedback is most appreciated.

Thank you

Yuva

The European Commission’s Joint Research Center has been involved in training on the detection – identification – quantification of genetically modified organisms and in related capacity building activities since the year 2000 on request of EU experts (later becoming members of the European Network of GMO Laboratories - ENGL). The activity was expanded over the years, going from an EU-centric to a more global initiative aimed to foster harmonisation in GMO analysis and to facilitate the alignment of testing procedures in non-EU countries. The project was carried out in fulfilment of the aims of DG SANTE Better Training for Safer Food (BTSF) Programme and complementary to the mandate of the European Union Reference Laboratory for GM Food and Feed (EURL GMFF) established within the JRC under Regulations (EC) No 1829/2003 on the placing on the Market of genetically modified and food and (EC) 882/2004 on food controls.
The Web site of the European Union Reference Laboratory for Genetically Modified Food and Feed accessible at https://gmo-crl.jrc.ec.europa.eu/default.htm provides a wealth of information on validated methods, measurement uncertainty, reference materials etc.
Along with the organisation of numerous practical training courses on laboratory procedures and on the different technical aspects  required for proper implementation of GMO control, several networking activities were organised, which contributed to the creation and/or consolidation of Regional GMO-laboratory Networks in Latin America and the Caribbean, Africa, Middle East and North Africa, Asia, New Member States and (potential) Candidate Countries.
The technical capacity of the participating laboratories has been progressively increasing in most countries and connections have been established among laboratories, within and among the regions, with consequent improved exchange and sharing of information. Overall, over the years, the project involved experts from more than 100 non-EU countries.
A large majority of these laboratories is involved and active in fields other than GMOs, from clinics to variety testing, to pathogen detection and security. Indeed, according to the information provided, while the experience and expertise developed is primarily devoted to the purpose of detection, identification and quantification of GMOs it is also used – by the same laboratories - in other contexts where competences in DNA analysis and molecular biology laboratory infrastructures are needed.
These laboratories, mostly interconnected through their national/regional networks, also form a sort of a "Global Network", enriching the potential network capacity.
In view of the growing global challenges, the JRC is considering to reshape its involvement and support to this "Global Network". The aim is to help laboratories to exploit the specific expertise and skills already in place to address new emerging challenges that need technical expertise within their remit.

Thank you Guy (#9995) for the information you have shared on the efforts and available capacities in the EU-JRC and the EU Network of GMO Laboratories. I am aware of the global outreach of the EU-JRC to strengthen capacities in other regions and many countries have benefited from this including Nigeria. The  link provided by Guy to the EU-JRC website is a source of valuable information on detection and identification of LMOs. However, there might be need for opportunities for practical experiences and interactions for full utiilisation of these resources for some of the forum participants

There are on-going efforts at establishing regional networks, and the EU-JRC, as at 2018, had  facilitated three international workshops on the harmonisation of GMO detection and analysis for African countries. This has stimulated the establishment of the sub-regional networks in Africa, they are still fledgling but some sort of network does exist. The challenge is to make the networks more functional to address the gaps in capacity in the regions.

It will be good to know if there are similar efforts in other regions.

I thank everyone who has made some contributions on this, and invite more discussions

Shakirat

Hullo Participants,

Greetings from Kampala, Uganda.

I work under the National Agricultural Research Organization (NARO). In NARO, we have been involved in LMOs research and development since 2003. We have conducted LMOs research in 7 plant species for 14 traits, and some livestock vaccines. Within my organization, we have four laboratories with basic capacity for detection and  identification of LMOs. Currently, we are using PCR-based and DNA-amplification-based methods, which have been applied to identify LM materials developed locally, or those whose sequence information is known.

We need to build capacity for newer methods that can be used for composite samples, and new methods detecting for genome-edited organisms (both qualitative and quantitative methods).

Barbara M. Zawedde, PhD
Chairperson, NARO Institutional Biosafety Committee

The European Commission’s Joint Research Center has been involved in training on the detection – identification – quantification of genetically modified organisms and in related capacity building activities since the year 2000 on request of EU experts (later becoming members of the European Network of GMO Laboratories - ENGL). The activity was expanded over the years, going from an EU-centric to a more global initiative aimed to foster harmonisation in GMO analysis and to facilitate the alignment of testing procedures in non-EU countries. The project was carried out in fulfilment of the aims of DG SANTE Better Training for Safer Food (BTSF) Programme and complementary to the mandate of the European Union Reference Laboratory for GM Food and Feed (EURL GMFF) established within the JRC under Regulations (EC) No 1829/2003 on the placing on the Market of genetically modified and food and (EC) 882/2004 on food controls.
The Web site of the European Union Reference Laboratory for Genetically Modified Food and Feed accessible at https://gmo-crl.jrc.ec.europa.eu/default.htm provides a wealth of information on validated methods, measurement uncertainty, reference materials etc.
Along with the organisation of numerous practical training courses on laboratory procedures and on the different technical aspects  required for proper implementation of GMO control, several networking activities were organised, which contributed to the creation and/or consolidation of Regional GMO-laboratory Networks in Latin America and the Caribbean, Africa, Middle East and North Africa, Asia, New Member States and (potential) Candidate Countries.
The technical capacity of the participating laboratories has been progressively increasing in most countries and connections have been established among laboratories, within and among the regions, with consequent improved exchange and sharing of information. Overall, over the years, the project involved experts from more than 100 non-EU countries.
A large majority of these laboratories is involved and active in fields other than GMOs, from clinics to variety testing, to pathogen detection and security. Indeed, according to the information provided, while the experience and expertise developed is primarily devoted to the purpose of detection, identification and quantification of GMOs it is also used – by the same laboratories - in other contexts where competences in DNA analysis and molecular biology laboratory infrastructures are needed.
These laboratories, mostly interconnected through their national/regional networks, also form a sort of a "Global Network", enriching the potential network capacity.
In view of the growing global challenges, the JRC is considering to reshape its involvement and support to this "Global Network". The aim is to help laboratories to exploit the specific expertise and skills already in place to address new emerging challenges that need technical expertise within their remit.

Greetings Participants,

And regards from Abuja, Nigeria.
I work with the National Biosafety Management Agency (NBMA) that serves as the Competent National Authority on all biosafety issues in Nigeria. Please recall that Nigeria signed and ratified the Cartagena Protocol on Biosafety in 2000 and 2003 respectively after which she has progressively developed the nation’s biosafety framework that culminated in 2015 with the signing of the National Biosafety Law. We have so far given approvals for confined field trials and general release of GM crops with the most current being the Pod-Borer Resistant Cowpea in February 2019.
Within my Agency, we have the GM Detection and Analysis Laboratory with the capacity for detection and identification of LMOs. The lab is also actively involved in the West African Network of GMO Laboratories (WANGL). We employ both PCR-based and protein – based methods of detection.

Capacity strengthening is however required with the real-time PCR technique. Capacity building is also required as it relates to techniques employed in detecting products from gene editing and synthetic biology.

Josephine Amedu
Head,  GMO Detection and Analysis Laboratory (National Biosafety Management Agency), Nigeria

Dear Josephine (#9998),

Thank you for sharing information on the experiences with LMO detection and identification in Nigeria, including some of the authorised GM activities. And bringing to the fore the existence of the West African Network of  GMO Detection Laboratories (WANGL) for strengthening capacities within the region.


Shakirat

Dear colleagues,

I appreciate the opportunity to participate and share important information related to GMO detection

In this regard, I think that in Mexico the detection of GMOs is mainly carried out through the qPCR technique, which has proven to be a reliable, reproducible and precise technique, although there are now methods such as new generation sequencing and that is usually more accurate. However, the use of this technique is more expensive, and costs increase significantly due to the number of samples. Therefore, I think that qPCR technique is the best option for the identification of GMOs in both food and grains or crops through the identification of P35S and T-NOs and specific events.

In this sense, INECC conducted a consultancy in which the main transgenic events, P35S and T-NOS, were identified, in addition to the specific events TC1507, NK603 and MON810, through the qPCR technique in some states of the country in Corn samples. Therefore, I consider that the qPCR technique is by far the most recommended technique for the identification of GMOs.

Finally, I believe that more laboratories are lacking this technique for the identification of GMOs in different crops because the use of this type of crops (GMOs) is increasingly common. This makes identification increasingly necessary. On the other hand, it is crucial to join efforts to identify these types of crops and gather information about the adverse effects on the environment and the human.

In this same sense, I consider this forum a great opportunity to add experiences and work together to share relevant techniques, findings, efforts and information for the identification of GMOs. In addition, I think that is necessary to focus efforts on the identification of new technologies

Best regards,

Deputy Director of Biosafety, Mariana Ayala
INECC, Mexico

Thank you Mariana in your post #10005 and the information you shared on the capacities that have been developed in Mexico for LMO detection and identification.

Your post also highlighted the need to focus on identifying new technologies which is one of the targets of the discussions on Topic 1, their accuracy, reliability, and possible applicability to detection of LMOs derived from genome editing.

Participants are encouraged to share available information on these. Are there specific products of genome editing that can be referenced in these discussions?

Shakirat

Hello! My greetings for all the participants

I’m Eva Bermúdez from the Executive Secretary of the Inter-Secretarial Commission of Biosafety of Genetically Modified Organisms (CIBIOGEM) that gathers all the National Competent Authorities in biosafety issues. One of our functions is to coordinate a National Network from LMO Detection to reunite laboratories from the Competent Secretaries of State but also laboratories from Research Centers and Universities which have been certified by some of the Federal Secretaries that have the capacity to perform detection and identification of LMOs. In this Network we have conducted assays to implement validated methods as qPCR. In this regard, Mexico was the first Latin-American country which developed its own certified materials from soy and maize in a project carried out the National Center of Metrology with the financing of the CIBIOGEM.

As previously mention, the most used technique in the Network to detect and identify LMO is real time PCR (qPCR) either using Sybr Green methods or fluorescent probes methods. In addition, some of the laboratories still using final point PCR of some protein-based methods as a complement. Until now, qPCR remains a sensitive and reliable technique that allows to detect common GM elements as P35S or TNOS or, if the specific sequence is known, also permits the identification of a specific events.

The past year, the National Institute of Ecology and Climate Change (INECC) carried out a study to detect and identify the presence of transgenes in samples of native maize from five states where the maize biodiversity is very important. In this work, duplex qPCR was used to detect in the same reaction the 35S promoter and the NOS terminator. This modification results in the saving of resources and working time.

Actually, some other methodologies begin to be used and seem to be have lower detection limits as digital PCR or the protocols derived from the Next Generation Sequencing methods. These techniques could be helpful also in the detection of products from the synthetic biology or from the gene editing   However; in our country there are no validated protocol established for the new biotechnologies.

Finally, I think this forum is a great opportunity to share experiences and to cooperate in order to obtain information related with LMO identification that will be helpful to protect our biodiversity and to try to minimize the negative impacts of LMO in it and in human health.

Best regards:

Eva Bermúdez
Deputy Director of Scientific and Technological Development and Innovation
Executive Secretary of CIBIOGEM

Dear colleagues,

First, I would like to thank Shakirat Ajenifujah-Solebo for chairing these discussions and also thank the colleagues who have already made very good contributions to the topic.
I can only agree with other who expressed the idea that most developed countries have validated qPCR methods, as the EU; at the same time that most developing countries do not have complete training and facility capacity to perform validated qPCR methods.
I acknowledge the efforts of the JRC in capacity building and training for the global community as well.
At the same time, I would like to highlight the lack of detection and identification methods, either harmonized or not, for the detection and identification of organisms arising from new technologies, like gene-editing. This is in line with the report from the 29th ENGL meeting & 14th workshop of GMO National Reference Laboratories under Regulation (EU) 2017/625 (Part 2) October in Ispra. In addition, the challenge has been also raised in the last AHTEG meeting in synthetic biology in June in Montreal, 2019. The report is available under BCH.
I can share with you a review paper we published last December (open access) in which we list many methodological strategies for gene-editing detection as food for thought as I tend to think that most of the currently available methods will not be easily validated and implemented.

Thank you

Sarah

Link to paper: https://www.frontiersin.org/articles/10.3389/fpls.2018.01874/full

Dear colleagues,

I would like to thank Shakirat Ajenifujah-Solebo for chairing this discussion and to all of you for sharing your experience.
I agree with Sarah #10021 and with previous participants who mentioned that the most accurate, reliable and at the same time, which is also important, cost-effective method for LMO detection and identification is qPCR.  With a constantly increasing number of LMOs on the world market in routine laboratory practice, such as in our laboratory (National Coordination BiosafetyCentre), it is necessary to constantly look for the possibility of identifying all possible authorized and unauthorized events in country, but at the same time look for the opportunity not to increase the costs of analyses to boundless amounts. And qPCR method still allows to resolve this issues by application of multiplex qPCR and screening schemas.
We are very grateful to JRC and European Network of LDGMOs (ENGL) for the opportunity to participate in the trainings since I think 2013 mentioned by Mr. Guy Van Den Eede in #9996. It gave us knowledge on how to apply screening approach in our daily routine to low costs and cover as much GM-lines as possible. Thank you very much for constant development of JRC webpage, it is really helpful and useful capacity building source and it could be really used by those laboratories who want to identify GM-lines and have need to identify not only authorized but unauthorized lines.
Also for our lab we decided to introduce the practice to screen every half of year for new LMOs that could have new screening elements that we can`t cover by our screening schemes and detection kits and then decide do we need new primers and probes. For doing that we using BCH database bch.cbd.int and GenBit database https://www.genbitgroup.com/en/gmo/gmodatabase/index.php
The other quantitative and reliable method for accurate LMO quantification is droplet digital PCR. It also could be multiplexing and it is easy to transfer qPCRmethod to ddPCR. Some references are here   https://www.nature.com/articles/srep35451, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561262/. Its advantage is “the capacity of dPCR for absolute quantification of a target without reference to a standard/calibration curve. This minimizes the effect of matrix differences between the calibrant and the test sample, which could cause different amplification efficiencies. Because of the principle of high-level sample partitioning, the results obtained with dPCR are very precise and accurate even at very low target copy numbers. Sample partitioning also allows reliable detection of rare targets in a high background of non-target DNA, which is important for GMO analysis, where a transgene (GMO event) might be present at much lower concentrations than the reference gene (endogene)” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010488/.
Regarding detection of new LMOs developed by genome editing I agree with Sarah`s previous post, it is not easy to develop especially cost-effective techniques for genome editing as well as appropriate screening schemes. Sequencing unfortunately at this moment is not a solution for many countries. First of all because of the cost. Secondly it may need combination of methods depending on what changes will be in genome (change could look like mutation, deletion, insertion).  By sequencing you will not always will prove that this organism was not received by conventional breeding. You always need to develop new method for new gene edited LMO as you will not prove it by using just primers for promoter, terminator, marker gene, you can't firstly apply simple scrining and then identification.
Thanks Sarah for sharing with us paper on strategies. In any case, we must thinking on how to detect new organisms most effectively.

Dear Participants,

Thank you for the activity under Topic 1.

Due to the increased activity, the Secretariat would like to extend the forum to 1:00 GMT on Monday 11 November to give all participants more time to respond to recent posts and continue to share their experiences with the techniques for detecting and identifying living modified organisms.

We look forward to the continuation of fruitful discussions.

Kind regards,

Austein McLoughlin

Dear Participants,
I would like to share my experience on gm detection. My lab used to use the screening method by PCR for plant genes and further used Simplex Real-time PCR for CaMV 35S promoter (FAM-TAMRA) and Nos terminator (FAM-TAMRA). Due to the quantity of many samples, we did validate the triplex Real-time PCR for detection CaMV 35S promoter (FAM-BHQ), Nos terminator (HEX-BHQ) and plant gene (Cy5-BHQ) (HMG for maize, Lectin for soybean, PLD for rice, Papain for papaya and tRNA for others) using LightCycler480 platform. This method is effective, save cost and time.
Since we may face the quantitative detection, I would like to request sharing information of validation method by ddPCR platform (simplex and multiplex ddPCR).
Kind regards,
Piyarat  Thammakijjawat
Biotechnology Research and Development Office,
Department of Agriculture
Thailand

Dear Austein,

Thanks to the Secretariat for this extension. This I am sure will enable more participants make valuable contributions to the discussions.

Regards

Shakirat

Dear colleagues,
I’m from the Institute of Food Safety, Animal Health and Environment «BIOR», Latvia. Latvia is Member State of European Union (EU).
Our institute is National reference Laboratory for GMO detection in food, feed and seeds. Since the year 2002 the Institute of Food Safety, Animal Health and Environment "BIOR“ (former National Diagnostic Centre) is a member of The European Network of Genetically Modified Organisms Laboratories (ENGL). We use ISO standard methods and methods validated by Joint Research Centre (JRC) of EU. Our laboratory is accredited by Latvian National Accreditation Bureau (LATAK) according to ISO 17025 since 2002.
All samples first are screened for the most common elements. For the quality control of the extracted DNA we amplify the trnl intron gene according to ISO 21569:2005, A.2. Screening is done to check whether the sample contains screening elements. Following PCR methods are used:
CaMV 35S promoter and T-nos according to ISO 21569:2005, B.9.;
Pat - „Quantitative PCR method for detection of phosphinothricin N-acetyltransferase gene”, JRC Compendium of Reference Methods for GMO Analysis;
Bar - ISO 21569:2005, B.8.;
nptII - ISO 21569:2005, B.4.,
FMV 34S promoter - ISO/TS 21569-5:2016.
If one or several screening elements are positive tests are continued with Event-specific Pre-Spotted Plates (Eve-PSP) produced by JRC. According to the results of Event-specific Pre-Spotted Plates (Eve-PSP) quantification of particular GM lines is done according to real time PCR event specific methods. If all screening elements are negative no other tests are done.
We have started the implementation of digital PCR in the GMO detection, and we plan to use next generation sequencing as well especially for the new LMOs developed by genome editing technologies.
We are carrying out research activities as well. Recent publications in the GMO field are:
1) Grantina-Ievina L., Ievina B., Evelone V., Berga S., Kovalcuka L., Bergspica I., Jakovele A., Malisevs A., Valcina O., Rodze I., Rostoks N. 2019. Potential risk evaluation for unintended entry of genetically modified plant Propagating material in Europe through import of seeds and animal feed – the experience of Latvia. GM Crops & Food, DOI: 10.1080/21645698.2019.1638721 https://www.tandfonline.com/doi/abs/10.1080/21645698.2019.1638721?journalCode=kgmc20
2) N. Rostoks, L. Grantiņa-Ieviņa, B. Ieviņa, V. Evelone, O. Valciņa, I. Aleksejeva. Genetically modified seeds and plant propagating material in Europe: potential routes of entrance and current status, Heliyon, vol. 5, no. 2, art. no. e01242, 2019. https://www.sciencedirect.com/science/article/pii/S2405844018383932

Best ragards,
Lelde Grantina-Ievina, Dr. biol.
Senior Researcher,
Department of Risk Asessment and Epidemiology,
Institute of Food Safety, Animal Health and Environment “BIOR”,
Lejupes Street 3,
Riga, LV1076,
Latvia

Thank you Lelde for your contributions and sharing the links


Shakirat

Dear Forum Participants,

I wish to acknowledge and thank you for your contributions to Topic 1. It has been very interesting and engaging. More participants are encouraged to avail the opportunity that the extension of time by the Secretariat provides to make their input into the discussions.

Sarah's post (#10021) and the link to the journal she shared is an interesting read, participants may wish to look this up. Thank you Sarah.

Galina's post (#10027) also shared some links, such as information on the JRC-GMO matrix or pre-spotted plates and BCH website for identifying unauthorised LMOs, and the use of droplet digital PCR. Thank you Galina.

And thanks to everyone for sharing your experiences. Looking forward to more interesting discourse.

Kind regards

Shakirat

Dear colleagues,

First I like to thank Shakirat and the secretariat for the excellent work and preparation of this topic. I am grateful for having the opportunity to participate in this forum.

In the EU (and Germany as Member State) we have a long history of experience with the most efficient and accurate methodology which is PCR. Mainly, real-time PCR is the preferred approach. In general for detection of EU authorized and unauthorized LMO advanced screening strategies are combined with subsequent identification of the event. The methods commonly used are validated. They are compiled in the reference method database hosted by the EU Reference Laboratory (EU-RL GMFF; https://gmo-crl.jrc.ec.europa.eu/default.htm). Additional methods are established on demand and are fully validated before routinely used in official control laboratories. This guarantees accuracy, reliability, and, also noteworthy, comparable results among laboratories. In specific cases, e.g. for detection of unauthorized LMO, in-house validated methods are applied. Many of these methods are provided in another valuable database (http://www.euginius.eu).

New techniques being tested for applicability are mainly digital PCR and NGS approaches (targeted and untargeted). Digital PCR is a promising new tool for precise and reliable LMO quantification. Approaches to detect and identify plants derived from new breeding techniques are at an early stage and no specific method is available at the moment.

l fully support the posts of my EU colleagues Lotte Hougs [#9991] and Guy van den Eede [#9995]. The webpage of the EURL-GMFF offers a wealth of information and guidance on the detection and identification of LMO.

Thanks for hosting this interesting online forum for exchange among experts on a global scale!

Best regards,
Lutz Grohmann

Thank you Lutz (#10050) for sharing your experience and knowledge with us. Good to meet you again on this platform!

And in the forum discussions so far, the EU-JRC Database of GMO detection methods is a recurring reference for most countries.


Best regards

Shakirat

Greetings from Brazil!

I am Maria da Glória Trindade from National Official Laboratory in Goiânia / Ministry of Agriculture Livestock and Food Supply of Brazil. Ministry of Agriculture Livestock and Food Supply together with Ministry of Environment and Ministry of Health is responsible for the oficial GMO control in Brazil. The Brazil surveillance system is structured in a national network involving two official laboratories and three private ones. There are two more privates laboratories in process of accreditation by the Ministry of Agriculture Livestock and Food Supply of Brazil. As an official laboratory our unit located in Goiás State is responsible for the official GMO control and monitoring of private laboratories in the national network. As routine activities we perform analysis of detection, identification and quantification of GMO in food and feed products mainly by Real Time PCR methods. We follow the guidelines published by JRC related to methods criteria acceptance: Definition of minimum performance requirements for analytical methods of GMO testing available at https://gmo-crl.jrc.ec.europa.eu/doc/MPR%20Report%20Application%2020_10_2015.pdf, methods validation: Verification of analytical methods for GMO testing when implementing interlaboratory validated methods - Version 2, available at https://gmo-crl.jrc.ec.europa.eu/ENGL/docs/WG-MV-Report-version-2.pdf and uncertainty measurement: Guidance Document on Measurement Uncertainty for GMO Testing Laboratories, available at https://ec.europa.eu/jrc/sites/jrcsh/files/eur22756en.pdf. We also follow the standards published by ISO related to GMO analysis. To identify a specific GMO we perform a screening analysis by seven sequences common to the majority of  GMO events released on the market (P35S, T-NOS, CTP2-EPS-PS, PAT, BAR, PFMV, NPTII) plus one plant specific sequence as an endogenous control (tRNA-Leu). Based on the matrix approach we search the GMOs present in the sample and, if necessary, we quantify the GMOs detected based on a standard curve stablished by CRMs. In the case of some events that are not detect by the screening approach, we perform event specific detection methods.  In the GMO field we follow all the quality controls for quality assurance like use of certified reference materials, participation in proficiency programs (FAPAS, USDA), equipment calibration, personnel training and etc to comply with ISO/IEC 17025:2017 (we have all the scope related to GMO analysis accredited based on this standard). We also perform other kind of analysis like plant pest identification by sequencing and qPCR, animal species identification and quantification by qPCR to combat fraud by species addition. Fish species identification by SANGER sequencing aiming to combat fraud by species substitution and Plant Genotyping by fragment analysis. We are in process to implement Next Generation Sequencing in our routine analysis aiming metagenomics analysis among other applications.  I think the biggest challenge is to implement detection methods for the new GMO obtained by the New Breeding Techniques, Like genome editing. We need standardized methods which nowadays are not available in the current scientific databases. Another challenge is the lack of CRMs and Proficiency Program for specific species, like cotton and Beans.

Thank you all!

Dear participants,

Thank you for all the great contributions.

As already mentioned some of the new techniques such as genome editing will present a challenge as no common sequences for screening are presented and the altered sequences may mimic naturally occurring sequence variants or are not distinguishable from alterations of conventional mutagenesis.

This is a challenge but also a point of reflection for countries regulations since if the changes are so similar to natural variations those would be considered a conventional product and not a LMO. And in those situations, as in the case of Brazil, we would rather spend resources to detect LMO that may have adverse effects on the conservation and sustainable use of biological diversity.

Thank you

Luciana Ambrozevicius
Ministry of Agriculture, Livestock and Food Supply - Brazil

Dear participants,
In response to posts #9987, #9988, #10053 and other posts regarding animal and fish species identification and quantification to combat fraud, I am glad to inform you that ISO/TC 34/SC16 https://www.iso.org/committee/560239.html has a working group specifically working on standards for such purposes and has recently published a standard ISO 20813:2019.  Its scope also includes fish.
In addition the Codex document CAC/GL 74-2010 http://www.fao.org/fileadmin/user_upload/gmfp/resources/CXG_074e.pdf provides some guidance that may be useful.

Dear all, Dear Shakirat,

Thank you Shakirat for moderating this online forum.
With respect to Topic 1, I would like to provide the following information;
Since 2011, LMO research team in National Institute of Ecology (NIE) which is affiliated to the Ministry of Environment in Republic of Korea have been developing LMO detection methods. We developed simplex (single-event specific identification) and multiplex (simultaneous detection) PCR methods for 4 major LM crops (corn, soybean, canola, and cotton) which were approved in Republic of Korea. Although advance of PCR techniques force to us to develop LMO identification method using new tools (such as digital PCR etc), we have pursued conventional PCR method for public applicability of the LMO detection method. Most of developed method are event specific PCR method (one primer is for plant genome and the other primer is for introduced vector). Moreover, we have been developing novel detection methods to increase the amplifying efficiency. Normally, many validated methods are for real time PCR, so the band intensity for conventional PCR reaction is very low and the PCR product size is limited (100~150 bp). Therefore, to establish the multiplex PCR method for identifying LMO, we need to develop new method and all newly developed methods are validated by comparing JRC method. Finally, all NIE LMO detection methods have been using for LMO identification of suspicious samples collected from the LMO monitoring project in natural environment. After performing PCR using NIE detection method, we sequenced the PCR product to confirm the PCR result.
Relevant to Topic 1, the main points to consider are new techniques and methodologies for LMOs or genome editing products. In case of NIE, Republic of Korea, we used conventional PCR method for LMO detection and identification. As many scientists reported that most product made by Synthetic biology such as genome editing does not have foreign gene, therefore the detection for these products is not easy compare to conventional GMO. I also would like to ask to participants to share your own experience about LMO detection.

Wonkyun Choi, PhD
Researcher, National Institute of Ecology

Thanks Luciana ($10059) and Wonkyun (#10062) for your contributions.

You also expressed the need for more research and information on synthetic biology and genome editing techniques to aid in the detection and identification of the products.


Kind regards

Shakirat

Dear Forum Participants,

Its been a great interaction on this platform. I thought I should contribute to the previously shared experiences from Nigeria as well....for Topics 1, 2 and 3

In Nigeria, the National Biotechnology Development Agency (NABDA) is the focal point for promoting the sustainable and safe application of biotechnology for food security, sustainable environment and quality healthcare delivery. A number of GM Crops have been approved for confined field trials, multi-locational trials and commercial release. Some GM crops have been approved for introgression of the GM trait into local varieties. Approvals and regulation of GMOs are under the purview of the National Biosafety Management Agency (NBMA). The NBMA also certifies and accredits biotech research facilities as a measure to monitor activities within the country.

Biotech research and academic facilities such as NABDA and others contribute technical support for regulatory activities including the drafting of legislations and implementation guidelines; building local capacity for GMO detection and identification. There are capabilities in the research and academia to support LMO detection and identification. Most referenced methods are the EU-JRC validated methods and end-point PCR capabilities are widely applied.
For the assessment of foods and feeds, NABDA employs the use of taxon specific primers to screen for presence of known GM crops used as ingredients in products, element and event specific primers such as TNOS, P35S, CaMV, EPSPS, Cry genes depending on the materials being tested. Aside from the activities of the NBMA in enforcing compliance, pockets of surveys have been undertaken by individual researchers/teams to assess the presence of GM products in the Nigerian markets

For capacity development in NABDA, we are looking at optimizing the qPCR techniques for quantification of GM content to meet regulatory requirements. There might be a need to certify some reference laboratories and standardize and validate methods to support GM regulations in Nigeria. The development of reference materials for the GM crops being tested in Nigeria such as sorghum, cowpea, cassava is another consideration.

There are recent efforts by the NBMA to amend the Biosafety Laws to accommodate synthetic biology and genome editing techniques. And as such I agree with other forum participants that more knowledge and capacities should be built and shared on the subject of synthetic biology and genome editing techniques.

Organisations that are involved in building capacity in biosafety include the International Centre for Genetic Engineering and Biotechnology (ICGEB), NABDA has also benefitted from EU-JRC capacity building, the Secretariat for the Convention of Biological Diversity (SCBD) among others.

NABDA is also involved in building networking initiatives, such as the West African Network of GM Detection Laboratories (WANGL), to impact on regional capabilities in detection and identification of GMOs; and to harmonise standards.


Shakirat Ajenifujah-Solebo, PhD
Deputy Director (Research)
National Biotechnology Development Agency, Nigeria

Dear Participants,
As commented several times previously in the participation in the CBD synthetic biology programme of work, the Global Industry Coalition agrees that most organisms “assigned to synthetic biology” are LMOs – with the exception of organisms developed using genome editing. I have addressed the challenges of detecting certain organisms developed using genome editing in my post # under Topic 1.

Dear Forum Participants,

Thank you very much for all your contributions in the past 2 weeks under Topic 1. It has been very engaging and interactive. And I hope that all participants will find the information shared here useful.

Kind regards

Shakirat

Dear Participants,

Thank you all for your vibrant discussions and insightful input.

The forum is now closed for comment.

Thank you very much and kind regards,

The Secretariat