Past Activities 2016-2018

Dear Participants,

Welcome to the online discussions of the Network of Laboratories for the Detection and Identification of Living Modified Organisms.

At their last meeting, on Decision CP VIII/16,  the Parties to the Cartagena Protocol on Biosafety requested the Executive Secretary to continue working on the draft training manual, in collaboration with the Network of Laboratories for the Detection and identification of Living Modified organisms, and make a draft version available for consideration at their ninth meeting. Accordingly, an updated version of the draft manual has been prepared by the Secretariat, in collaboration with a small group of experts from the Network, and it is available for review by all participants of the Network.

This discussion will focus on module 4 which provides details regarding methods that are commonly used for LMO detection. This includes information on protein-based methods such as the lateral flow assay and the ELISA Technique as well as a discussion on the limitations of such methods. Furthermore, information on DNA-based methods are also provided and cover details on the nature of DNA followed by the principles of PCR and considerations for the analysis of PCR Products. Specific information relating to LMO screening is also addressed including an overview of some of the limitations of DNA-based methods. Finally information on novel technologies for LMO Detection is also introduced.

Participants are invited to review and provide feedback on the contents for each module during the course of the next 2 weeks. We request that you review the manual from both a technical and editorial perspective focusing on edits and comments that  would provide additional clarity to the manual. In providing your comments, please refer to specific line numbers or highlight the text directly in the Word document containing the module and upload it together with your message.

We look forward to two weeks of constructive and fruitful discussions which will bring us a step closer to the goal of developing and improved version of the manual for the consideration of the COPMOP.

Kindest regards,
Secretariat

Table 3 in line 578
Table 3: Comparison of genome size of some plant species and corresponding genome copies in defined amount of DNA
shows genome size of maize as 5 x10^9 bp, but do not specify if it is in 2C or 1C, in the last case it will be 2.3 to 2.7 x10^9 Mbp/1C from Plant Molecular Biology Reporter Volume 9(3) 1991 K. Arumuganathan and E.D. Earle

Dear all participants

This a very comprehensive document that compiles techniques for detection and identification. Thanks to all people that worked on it. It may take several working hours to carefully review the document. So, I would like to recommend if among the 128 participants the revision may be splitted according to the different expertises.

Best wishes,
Emanuel.

Dear Forum Participants,

I would like to thank everyone who has shared their comments on this module so far.

As previously indicated, the main objective of this discussion is to review and provide feedback on the contents of module 4 which can be accessed at http://bch.cbd.int/detectionlabs/trainingmanual/module%204_27mar18_posted.docx.

We request that you review the manual from both a technical and editorial perspective focusing on edits and comments that  would provide additional clarity to the manual. In providing your comments, please refer to specific line numbers or highlight the text directly in the Word document containing the module and upload it together with your message.

I hope you will be able to contribute to this discussion in the coming week and share your knowledge with us in order to contribute to the development of a through and comprehensive training manual.

Kindest regards
Dina

Thank you for compiling a document for this area of detection and identification, and especially the excellent illustrations.  The document is somewhat lacking in information in the use of protein-based testing.  I have made a number of edits in tracking mode and comments that provide additional clarity to the module as requested.  As other participants have done I have attached the document with my tracked suggested changes and related comments to this reply, as compiling each comment with the line to which it refers to would take much longer than the limited two week time frame that has been allowed for review of all of these documents. 

This document and the other documents here described focus solely on the detection and identification of plant LMOs (except for one mention of salmon in module 2).   This limitation to the scope of the document should be stated in the title, and in the scope statement for the document.

Key suggestions:

As the document is to be globally relevant, I added the source document links for Codex CXG_074e, and for the ISO committee that sets standards in this subject area: TC34 SC16.

I have made a considerable number of suggestions in the area of protein-based methods, as it appears that the text is out of date.  This is an area where I have considerable expertise, having been responsible for the development of such tests from 1997 until recently.  The statement that only tests for a few proteins are available is misleading.  Antibodies per se are not sold as antibodies, but they are available as kits.    I thus added a section on Availability of protein-based methods to clarify this area.  In the service of balance I changed the section headed ‘Limitations of protein-based methods’ to cover both ‘Advantages and Limitations of protein-based methods’ and reorganized it.   I added  a similar section to the PCR methods section, so as to provide balance.

If we are to refer to kits then we should make a list of the providers, rather than referring to the site of one provider.  This is consistent with a doctrine of fairness.
Sensitivity can be a particular challenge when attempting to detect a small number of LMO seeds in a bulk sample.  I have added text to reflect that as the original did not discriminate between use of strips for single plants and for single seed or bulk seed.

Throughout this document there are references to GMO (possibly due to use of text from elsewhere).  I have changed them to LMO as the Protocol refers to LMO not GMO.

The section on DNA-based detection also has a large number of really great explanations and provides a great deal of clarity. 
I did add some text to clarify that the 35S promoter sequence originates in the Cauliflower mosaic virus, which is found widely in brassicas and can cause erroneous conclusions. 

One point is that everything is based on PCR, so the section should refer to PCR-based rather than DNA-based.

As the Protein portion contains sections on availability and advantages and disadvantages, then the PCR portion should too.  I have added some suggested text.
The inclusion of a number of protocols is useful for the reader. As these are examples of protocols, we must make clear that they are examples, and not BCH approved protocols.

A number of different methods have been developed using an isothermal DNA amplification to detect DNA sequences. I therefore added a small paragraph listing these and then stating that LAMP is described here as an example.

I find that the references in this and other documents are very out of date – often being from 20 years ago.  Statements and assumptions made at that time may no longer be applicable. We should make a strong effort to use more up to date citations, especially as this field has developed significantly in the last 20 years.   I thus added a list of more recent citations to the reference section.

As with the previous post from Dr. Melina Perez-Urquiza, I would like some more clarification on genome sizes.  This reference though widely used, is more than 25 years old, and based on out of date techniques. Have these numbers been checked for accuracy against the consensus sequence for those organisms that have been fully sequenced.

Please accept the enclosed marked up draft for module #3. Thank you for permitting me to look at the document.

Dear colleagues,

many thanks to the authors for writing this module as a basis for the development of training materials, which is a very good starting point.

Module 4 should provide details regarding methods that are commonly used for LMO detection. This includes information on protein-based and DNA-based methods. Specific information relating to LMO screening, finally information on novel technologies for LMO Detection is also introduced.

As said concerning the other modules, the limitations of protein-based methods are not completely provided. In addition, the content of the existing ISO standards (valid more than 10 years and globally accepted) need far more consideration.
These standards provide on far less pages more valuable information than the current draft module 4, which is too lengthy in many parts and will require a more clear structure. Particularly the screening strategy is not made clear and added at the end of the doc. Screening strategies are nowadays the most important starting point before LMO detection experiements should be performed. It is a cosequence of the increasing number of LMOs globally used.

My apologies, not providing comments  to specific line numbers. I added comments and changes/additions directly in the Word document containing the module 4, which is hereby uploaded together with my message.

I am looking forward for a further and fruitful online discussion towards developing and improving the manuals for the consideration of the COPMOP. Please contact me, if further support and expertise is required.

Kind regards and have a nice weekend,
Lutz

Thank you Lutz and the BVL for a lot of excellent and thorough edits in the documents, which can be combined with those we suggested to improve the document.  The replacement of Plant by LMO is a good edit.
We (the GIC) agree that nested PCR is unsuitable for LMO detection, as it is prone to excessive risk of contamination, and is not normally employed.  We believe that teh use of multiplex PCR should be retained in the document as this can provide a less expensive approach in terms of the reagent costs.
We agree that the section on interpretation (Analysis of PCR Products ) would be more valuable and appropriate if moved to a position prior to the PCR protocols.
After line 606 there are no line numbers in the document to refer to.
We are also supportive of the comments made in section on LMO Screening/Detection using PCR.  However, I am not familiar and cannot find CEN/TS 16577 (which would be a regional standard) and so must assume that this refers to  CEN/TS 16707:2014 (this is indeed the reference given in the references).  As I do not have a copy of this regional standard I cannot judge whether this is correct and appropriate.
Regarding teaching the structure of DNA (comment WDD20), I do wonder whether this is appropriate and necessary as those using this manual would be expected to have a training in biology and thus an understanding of DNA structure.

Dear colleagues,
I would like to say many thanks for developers of this module. The information is rather comprehensive and huge work was done. Thanks to Dr Raymond Shillito for input on protein based methods. Because not all laboratories or even countries use this analysis for LMO detection. At the same time it is valuable for monitoring purposes and your experience is very good for this training module.
From my point of view I think that principle of LMO screening becomes more and more important as the best approach to low cost LMO identification. That`s why this chapter should be developed more thorough and more examples should be given. It will be very good to add real comprehensive screening schemas from reference laboratories. And this chapter is better put at the beginning, but not at the end of the modules to show from the very beginning the modern approach to LMO detection and identification.
As to the text of the module 4 it is really very good, but it is too long. In some places it seems to me it will be good to omit some book explanations regarding the general principles of molecular analysis, e.g. delete or make shorter lines 380-403, put all things of quality control to module 5 (e.g. Lines 486-522). I don’t know do we need explanations 523-534? And probably some lines regarding overall considerations for molecular techniques also could be made shorter or given illustrative schemas rather than a lot of text. I think we need additional work to make the text shorter. Also some work should be done with references, we need to try to give latest references where applicable and give links to the references in the text (this comment for all modules).
Again I would like to say many thanks to the team who developed all modules. It was a hard task, overall expression from modules is very good, we have information of a very good quality.
Thanks a lot,
Kind regards,
Galina.

POSTED ON BEHALF OF SARAH AGAPITO TENFEN
(this message was received prior to the closing of the online forum)
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Dear colleagues,

Thanks to the Secretariat for providing such a comprehensive document on LMO detection and identification, all modules. And I would also like to thank our colleagues who were able to revise the document and provide insightful comments to them. Unfortunately, I havent had time to properly revise and provide suggestions.

I have just a few and brief comments to this module and I am sorry again I do this here and not within the document.

I think it would be much useful to have a topic on low level presence since this might be well the case for escape and env monitoring scenarios, e.g. transgene flow into landraces or wild relatives. There are methodological specificities when dealing with LLP samples. For example, threshold levels for real time PCR. In an attempt to contribute in this very last minute, I attach two recent papers we have published in which me mention our difficulty in detecting LLP in maize landraces in Mexico. I also felt the lack of methods for RNA detection.

If there is again a schedule for a second round revision of the document, which I strongly support, then I would be happy to contribute with text.

Best regards,

Sarah