Topic 3: Experience and challenges with developing, using and maintaining reference materials.

Thank you for the opportunity to contribute to this issue:

Currently there are only two providers of CRMs for GM events that we are aware of: The JRC (crm.jrc.ec.europa.eu/en) and the AOCS (http://www.aocs.org/technical-products/certified-reference-materials-crms).

1. We have experienced a challenge in the cost of the CRMs which can add up when the laboratory needs to implement the identification of several GM events. We usually use the entire CRM sample when it is received for extracting DNA because the CRMs have an expiry date.
2. A further challenge is that the JRC and AOCS do not have CRM for all GM events which is understandable. It is currently a huge challenge to try and obtain positive control material from a developer when no other reference material is commercially available. We have not been successful to do this.
3. Because of the cost of CRMs, we have resorted to use synthetic oligonucleotides, ultramers or gBlocks, as positive controls where the target sequence is available for GM events in order to save on the cost of CRMs. We also published a paper regarding this: Viljoen et al. (2013). Stability of ultramer as copy number standards in real-time PCR. Gene 516(1): 143-145. Unfortunately, the complete target sequence is not always available in the public domain.

Thank you very much, Prof. Chris Viljoen, for your contribution from South Africa. Your comments highlight key practical issues regarding access to certified reference materials (CRMs):

Only two main CRM providers are currently available (JRC and AOCS), and the high cost of these materials is a major challenge when multiple GM events must be analysed.

Many GM events do not have corresponding CRMs, and obtaining positive control material directly from developers remains extremely difficult.

To reduce costs, your laboratory uses synthetic oligonucleotides and gBlocks as alternative positive controls when target sequences are available, although sequence access is sometimes limited.

We also appreciate you sharing the reference to your published work, which is highly valuable for the discussion.

Thank you once again for your important insights.

Dear Melina,
Thank you for the opportunity to contribute to this Topic.
My name is Dr. Wonkyun Choi, working at National Institute of Ecology in South Korea.
I have scientific experience for the development of LMO detection method using conventional PCR, LAMP and Lateral flow assay.

My response is below,
a) Are the reference materials available on the market sufficient for your laboratory's needs?
In my opinion, it is difficult to purchase RM from AOCS or IRMM immediately after approval. Also, the production date and manufacturing process of RM is very different.

b) What are the limitations in obtaining certified reference materials in your laboratory? Are the costs affordable?
I think that the price of RM is very expensive to use for analysis, and the expiration date is too short(less than 1 year).

c) Do you have experience in developing your own reference materials? Has it been possible to certify them?
No.

d) What alternatives can be used when certified reference materials (CRMs) are unavailable (e.g., in-house developed reference materials)?
No.

Dear Ms. Melina, Many thanks for the opportunity to share my experience on topic 3.

a) Are the reference materials available on the market sufficient for your laboratory's needs?
   - The reference materials for GMO analysis are insufficient on the market; especially positive and negative controls are not available.
b) What are the limitations in obtaining certified reference materials in your laboratory? Are the costs affordable?
   - The source and the way to get the materials
c) Do you have experience in developing your own reference materials? Has it been possible to certify them?
   - No; maybe the approved GMO crops like maize and cotton will be used as positive controls when testing the GMO status of the local one.
d) What alternatives can be used when certified reference materials (CRMs) are unavailable (e.g., in-house developed reference materials)?
    - A repeatedly tested non-GM local crop sample (tested by sequencing) can be used as a negative control, and the approved GM crop can be used as a positive control.
Amare Genetu

Dear Melina and colleagues,

Thank you for bringing this very important topic to the table, as CRMs are essential for ensuring traceability in LMO analysis and are often a source of difficulty for many laboratories. In our opinion, the availability of CRMs on the market is insufficient to meet our laboratory’s needs, since many LMO targets simply do not have CRMs available.
In such cases, we rely on the reference material provided by the applicants as positive and negative controls for that specific LMO. They provide the transformed line as the positive control and the isogenic untransformed line as the negative control, but only for LMOs that are approved in our country. However, there is a significant gap when CRMs are not commercially available and the LMOs are not approved here.
In these situations, we rely on screening strategies and confirm whether the screening results correspond to authorized LMOs in our country using event-specific methods. We also try to maintain event-specific methodologies and CRMs for LMOs that are authorized in countries we import from but unauthorized in Brazil.
We consider the purchase of CRMs to be a complex and expensive process, and the limited expiration date indicated in the certificate further increases the cost of maintaining CRMs in the laboratory. Although we extract DNA as soon as the material arrives, we still need to justify to our accreditation body the use of CRMs after the expiration date on the certificate.
We do not have experience developing our own reference materials; we use them as received from the developers, preparing only the necessary dilutions to obtain intermediate concentrations. One possible alternative for LMOs without available CRMs is synthesizing plasmids, provided that the LMO sequence is known. However, we must be very cautious with plasmids due to the risk of contamination in the laboratory. Other alternatives may include using material from proficiency tests or applying digital PCR strategies. Until now, we have not encountered a situation in which we needed to rely on any of these alternatives in our laboratory.

Dear Melina, please find below our responses for Topic 3.
a) Are the reference materials available on the market sufficient for your laboratory's needs?
Yes. We currently use certified reference material (included in the GMO detection kit) alongside our lab-developed reference material (uncertified).

b) What are the limitations in obtaining certified reference materials in your laboratory? Are the costs affordable?
Our main difficulty is sourcing certified reference materials (CRMs). They are expensive, not easily accessible locally, and require international procurement, which increases cost due to shipment and taxes.

c) Do you have experience in developing your own reference materials? Has it been possible to certify them?
Yes, we have experience in developing our own reference material, however, we haven't been able to certify it.

d) What alternatives can be used when certified reference materials are unavailable?
Currently, we don't have any alternatives, as we always make sure to use the certified reference material alongside our lab-developed reference material.

Thank you!

Dr. Wonkyun Choi, I appreciate your contribution from the National Institute of Ecology in South Korea. You have highlighted several important points.

Availability of reference materials: Purchasing RMs from AOCS or IRMM immediately after approval is difficult, and differences in production dates and manufacturing processes add further complexity.

Limitations and cost: The high price of reference materials and their short expiration period (often less than one year) represent significant challenges for routine analysis.

In-house development: Your laboratory has not developed its own reference materials, and therefore no certification has been pursued.

Alternatives: At present, no alternative reference materials are used when CRMs are unavailable.

Thank you for sharing these insights, which contribute to a clearer understanding of the practical constraints laboratories face.

Mr. Amare Genetu, thank you for sharing your experience from Ethiopia, which highlights several important points regarding the availability and usage of reference materials.
• Availability: Reference materials for GMO analysis remain insufficient on the market, particularly positive and negative controls.
• Limitations: Access challenges relate mainly to sourcing and obtaining the materials, rather than affordability alone.
• In-house development: Your laboratory has not developed its own reference materials, though approved GMO crops such as maize and cotton may serve as positive controls when testing local varieties.
• Alternatives: Repeatedly tested non-GM local crop samples (confirmed by sequencing) can be used as negative controls, and approved GM crops can act as positive controls when CRMs are not available.
Thank you again for your valuable input and for clearly outlining practical alternatives used in your context.

Thank you very much, Maria da Glória Trindade, for your detailed contribution from Brazil. Your comments highlight several key challenges related to the availability and use of certified reference materials (CRMs):
• Insufficient availability: Many LMO targets do not have commercial CRMs, requiring your laboratory to rely on applicant-provided materials as positive and negative controls—an option that works only for LMOs approved in Brazil.
• Gaps for unapproved LMOs: When neither CRMs nor approved materials are available, your laboratory depends on screening strategies and maintains event-specific methods and CRMs for LMOs authorized in exporting countries.
• Cost and short expiration: CRM acquisition is complex and expensive, and short validity periods increase the overall cost of maintaining them, even when DNA is extracted upon arrival. Additional justification to accreditation bodies is also needed for use beyond the expiry date.
• Alternatives: Although your laboratory does not need to use them, you identify potential alternatives such as plasmid synthesis (with caution due to contamination risks), materials from proficiency tests, and digital PCR strategies.
Thank you for presenting these practical considerations, which are crucial for the discussion.

Dear Dr. Belinda Akomeah, thank you for sharing your experience and highlighting several important points regarding the use of reference materials.
• Availability: Your laboratory successfully uses certified reference materials included in commercial GMO detection kits, complemented by lab-developed (but uncertified) reference materials.
• Limitations: The main challenge is access, as CRMs are expensive, not locally available, and require international procurement—raising costs due to shipping and taxes.
• In-house development: Your laboratory has experience producing its own reference materials, though certification has not yet been possible.
• Alternatives: No additional alternatives are currently used, as CRMs are always employed alongside your in-house materials.
I appreciate your clear and valuable contribution to Topic 3.

Dear Ms. Perez Urquiza,

Thank you for moderating this foundational topic and for sharing your profound expertise from CENAM. The availability and reliability of reference materials are the bedrock upon which all credible LMO detection and quantification are built. Our experiences in Iran directly reflect the critical challenges you have outlined.

Please find below our laboratory's perspective on your guiding questions.

a) Are the reference materials available on the market sufficient for your laboratory's needs?
No, the commercially available Certified Reference Materials (CRMs) are insufficient for our needs. While they cover major, globally commercialized "classical" GM events (e.g., MON810, Roundup Ready soybeans), our requirements are more specific and constrained. The range is inadequate for:

Region-specific or unauthorized GMOs that may enter through informal channels.

New Genomic Technique (NGT) products, for which CRMs are virtually non-existent.

Local crop varieties that require validated reference methods for species-specific genes (e.g., for Camelina sativa).

b) What are the limitations in obtaining certified reference materials in your laboratory? Are the costs affordable?
The limitations are severe and multi-faceted:

Procurement and Sanctions: The primary barrier is geopolitical. International sanctions severely complicate financial transactions and direct shipping from major CRM suppliers in Europe and North America. This often makes procurement impossible, regardless of cost.

Prohibitive Cost: When a procurement route is available, the costs are unaffordable for our operating budget. A single vial of a CRM can cost hundreds of Euros, and a full suite for a screening matrix is financially out of reach. The high cost also prohibits the purchase of the large quantities needed for robust method development and validation.

Logistical Delays: Any successful order is subject to extreme delays due to complex customs procedures, often rendering materials unusable upon arrival if specific storage conditions are compromised.

c) Do you have experience in developing your own reference materials? Has it been possible to certify them?
Yes, out of necessity, we have extensive experience in developing in-house reference materials (IHRMs). Our process involves:

Sourcing: Using verified seed material from controlled environments or international research collaborators.

Processing: Milling and homogenizing materials to a fine, consistent powder under controlled conditions.

Characterization: Using digital PCR (ddPCR) for absolute quantification to assign a copy number value to the IHRM, as it provides superior accuracy and precision compared to qPCR for this purpose.
However, formal certification through an internationally recognized body like ISO Guide 34 has not been possible. The process is prohibitively expensive and requires a quality management infrastructure that is difficult to sustain under our constraints. Therefore, our IHRMs are validated for "in-house use only," which limits their acceptance for official control purposes but is indispensable for our research and internal quality control.

d) What alternatives can be used when certified reference materials (CRMs) are unavailable?
Our strategy is a multi-pronged approach to mitigate the absence of CRMs:

In-House Reference Materials (IHRMs): As described, these are our primary alternative. We maintain a small bank of characterized materials for the most critical targets.

Cross-Laboratory Calibration: We engage in collaborative exchanges with trusted international academic partners. We can send our IHRMs to their laboratory for analysis on their accredited equipment, and vice-versa, to verify our assigned values and ensure a degree of inter-laboratory comparability.

Leveraging Digital PCR: The advent of dPCR has been a game-changer. Its ability for absolute quantification without a standard curve significantly reduces our dependence on CRMs for routine quantification once a method is validated. We use dPCR as the primary tool to value-assign our own IHRMs.

Genomic DNA as a Calibrant: For qualitative screening, we sometimes use purified genomic DNA from a known positive control as a temporary calibrant, though this is not suitable for quantification.

In conclusion, the global disparity in access to CRMs is a critical weakness in the international biosafety framework. We strongly advocate for initiatives that could support laboratories in our situation, such as:

The establishment of an international repository for non-certified but well-characterized reference materials for research and method development.

Programs to facilitate the affordable certification of IHRMs developed by national laboratories.

Enhanced training and resource-sharing on dPCR-based value-assignment protocols.

Sincerely,

Danial Kahrizi

Dear Mr. Kahrizi,
Thank you for your contribution to Topic 3. Your observations regarding the limited availability of certified reference materials, the challenges associated with regional procurement, and the absence of structured collaborative mechanisms are highly valuable to this discussion. Your insights help highlight persistent constraints shared by laboratories and reinforce the need for coordinated regional and international support.
Your description of the in-house reference materials developed and used in your laboratory is particularly valuable, as these practical approaches provide essential insight into how laboratories maintain analytical reliability in the absence of commercially available CRMs.
Your contribution meaningfully enriches our discussion and helps us gain a clearer understanding of the regional and international context and its technical requirements.

Dear Dr. Perez Urquiza,

Thank you for your gracious feedback and for acknowledging the challenges we highlighted regarding reference materials. I truly appreciate you taking the time to recognize our contribution and the practical aspects of our in-house approaches.

The constraints we face with CRMs are indeed significant, and I believe that sharing these practical experiences from different regional contexts is crucial for developing meaningful, collaborative solutions. Our in-house methods, while born out of necessity, have proven to be a reliable stopgap, and we are continuously working to refine them.

I am pleased to hear that our insights were found valuable for understanding the broader technical requirements. I remain fully committed to supporting this important discussion and any subsequent initiatives aimed at improving access to reference materials through regional and international cooperation.

Thank you once again for your leadership in moderating this critical topic.

Sincerely,

Danial Kahrizi

Hello Melina and other members.

My name is Rabi Ahmed working with the GMO detection and analysis laboratory of the National Biosafety Management Agency.

We understand that the development, use and maintenance of certified reference materials are central to the credibility and reproducibility of results in any GMO detection Laboratory in ensuring the validation, proficiency testing and routine quality assurance. However procuring and managing these materials in standard conditions present several operational challenges. For example, sourcing and accessibility to certified reference materials where Nigeria’s local production is non existent and only internationally sourced, results in lengthy procurement time, unsustainable costs and limited availability of certain events. This causes us to face delays in project timeliness and compromises in its capacity to respond to national surveillance. Secondly, the laboratory has an inconsistent power supply which leads to fluctuations in temperature in freezers used to preserve  reference materials, ultimately reducing its viability and affecting our ability to provide consistent results.

Thank you.

Dear colleagues,

My name is Daniela Wahler. I work for the German Federal Office of Consumer Protection and Food Safety (BVL). We are home to the competent national authority for the Cartagena Protocol, a working group that has the legal mandate to publish an official collection of methods of sampling and analysis of LMOs in Germany (§ 28b GenTG Working Group) and the national reference laboratory for genetically modified organisms (NRL GMO). I would like to thank the Secretariat for the opportunity to discuss here and Ms. Melina Perez Urquiza for her efforts to moderate the topic on reference materials.

a) and b): In Germany, as in all Member States of the European Union, certified reference material (CRM) is used by official control laboratories in routine analyses for the detection and identification of LMOs. By using CRMs in conduction with standardized detection methods, official controls are harmonized within the European Union what enables to act as a common economic area. According to Regulation (EU) No. 503/2013, applicants for genetically modified food and feed have to make sure that CRM is provided. The availability of CRM for authorised LMOs is thus ensured.

As pointed out by other participants of this forum, there are (only) the accredited producers AOCS (USA) and the Joint Research Centre (JRC) of the European Commission that sell CRMs. This is one bottleneck in acquiring reference material. Another, even more important factor, impeding detection and identification of LMOs is the limited access to reference material for unauthorised LMOs.

c): Therefore, in Germany, the NRL GMO might produces reference material for the official control laboratories on an ad hoc basis and with regard to
i) the relevance for the official control,
ii) whether the presence of the LMO in question is detectable with a routine screening method
iii) whether biomass or genomic DNA can be obtained from the producer of the LMO in question and
iv) whether information about the genetic modification is available.

Similarly, within the European Union, this task is also carried out by the European Reference Laboratory for genetically modified food and feed (EURL GMFF). However, as the production of reference material requires trained staff, appropriate infrastructure and resources and is a time-consuming task, it is only conducted if needed to guarantee consumer protection. It must be noted that strictly speaking, reference materials must comply with the requirements of ISO 33405, such as ensuring homogeneity and monitoring the stability of the material over time under various conditions. The resulting reference material should be as similar as possible to the control sample material in terms of material characteristics such as DNA extractability, homogeneity and zygosity of insertions. Thus, such reference material is preferentially produced from biomass of the LMO.

d): If reference material is not available, control samples made from genomic DNA of the LMO or artificial plasmids containing the sequence of the known genetic modification are also used.

With a view to the ever-increasing number and diversity of LMOs today, availability of (certified) reference materials or appropriate control samples is one of the biggest challenges for official control laboratories.

Kind regards,
Daniela

Dear Ms. Urquiza, thank you for the opportunity  to share on Experiences and challenges in the development, use, and maintenance of reference materials. My name is Mulubwa Sunday, from the National Biotechnology Laboratory of the National Institute for Scientific and Industrial Research (NISIR) Zambia.
to answer your specific questions, a), the reference materials that are commercially  available are now proving to be insufficient especially that we have seen a number of matrices for GMOs/LMOs testing for which we couldn't find CRMs, except for maize and soya that are widely studied. Regarding b) limitations in obtaining CRMs, bureaucratic constraints have  been at the helm of the limitations and the slow procurement process also serves as a limitation. And regarding c), we do not have any experience in developing references materials. and d) in an event that CRMs are unavailable, we could use samples that have been confidently tested as alternative references. Thank you.

Dear Mr. Mulubwa,

Thank you for your contribution to Topic 3. Your observations regarding the limited availability of certified reference materials beyond maize and soy, as well as the procurement challenges caused by administrative procedures, are highly appreciated. We also take note of your comments on the absence of in-house reference material development and your approach of using well-characterized samples as alternatives when CRMs are unavailable.

Thank you, Mr. Ahmed, for your contribution to Topic 3.
You highlight key challenges related to the procurement and maintenance of certified reference materials, particularly the absence of local production, long international procurement times, high costs, and limited availability of certain events. You also note the impact of inconsistent power supply, which affects proper storage conditions and compromises the long-term viability of reference materials. Project timelines and the laboratory's capacity to support national surveillance efforts are directly impacted by these operational constraints.

Thank you for sharing these important insights with the forum.

Thank you very much, Ms. Wahler, for your comprehensive contribution.
Your detailed overview of Germany’s structured framework particularly the functions of the §28b GenTG Working Group, the NRL GMO, and the regulatory mechanisms ensuring the availability of CRM for authorized LMOs provides the forum with a valuable reference model for effective national coordination.
Your explanation of the challenges related to accessing reference material for unauthorized LMOs, as well as the criteria and rigor involved in producing ad hoc reference materials, highlights the complexity of ensuring reliable controls. The clarification on the roles of both the NRL GMO and the EURL GMFF in supporting official laboratories is especially relevant for countries seeking to strengthen their own systems.
Your contribution to the discussion on reference material availability and the practical considerations that official control laboratories must navigate is greatly appreciated.

Dear Ms Melina Pérez,
Here are our comments on the topic:
a) Certified reference materials are not sufficient, which hinders the accreditation of testing methods because the accreditation authority in Peru requires their use. If they are not available, they must be produced from positive material, which is almost impossible to obtain from GMOs developers, as we have a moratorium law in Peru.
b) The following limitations have been identified with regard to certified reference materials:
•    Certified reference materials for few crops
•    Few developers of certified reference materials
•    Few suppliers of certified reference materials in Peru
•    High cost of reference materials in Peru
•    Very short shelf life of reference materials. It is suggested that the validity period of refrigerated and frozen DNA be indicated on the certificates.
c) We have no experience in the development of reference materials.
d) Prepare internal reference materials, but this is difficult because we do not have access to positive materials.

Sincerely,

Yeny Aquino

Dear Ms. Aquino,
Thank you very much for sharing the perspectives and challenges faced in Peru. Your contribution highlights several critical issues that strongly resonate with the broader discussion on the availability and use of certified reference materials (CRMs) in GMO/LMO detection.

Key points include limited access to certified reference materials, which directly affect accreditation and quality-system implementation, and regulatory restrictions or lack of access to positive raw materials can prevent laboratories from producing their own internal reference materials.

Thank you once again for this clear and valuable contribution. It adds essential context to our collective understanding of the challenges laboratories face and reinforces the need for coordinated regional solutions, diversified CRM production sources, and mechanisms that support access to reference materials.

The main problem is the expiration date of reference materials, most of which are annual. Furthermore, the Mexican government has austerity policies that restrict the acquisition of certified reference materials and, consequently, compliance with ISO 17025 criteria.
Efforts are underway to develop certified reference materials; however, because these are proprietary developments, access to them is limited.
One point of agreement among the laboratories of the Latin American Network is that the certificate should include information stating that the materials can still be used after their expiration date.
According to Mexican legislation, it is not possible for our institution to certify the reference materials we generate. Therefore, we identify an opportunity to find an alternative that supports the reliability of the results.
Finally, access to technical training to begin production and accreditation is also an important aspect.

Dear Participants,

Thank you very much for your invaluable contributions and active participation in the Online discussions of the Network of Laboratories for the Detection and Identification of Living Modified Organisms. Your thoughtful insights and shared experiences have greatly enriched the exchange.
The Secretariat will carefully review and analyse the information provided and will prepare a comprehensive synthesis to inform and support future work of the network.

The online forum is now closed for comments.

Kind regards,
The Secretariat