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Opening of the discussion: Overview of available detection methods [#5601]
Dear Forum Participants,

Welcome to the second round of discussions of the Network of laboratories for the Detection and Identification of LMOs. This discussion will focus on the topic of gathering information for an "Overview of available detection methods, including validated methods" for the detection and identification of LMOs.

There are many methodologies and techniques available to detect and identify LMOs ranging from simple yet effective protocols to more the more complex, all of which are valid options for use within laboratories. Thus, the aim of this forum is to compile a thorough selection of available methodologies in a single and accessible place to facilitate the availability of relevant information to national laboratories such that they can establish standard operating procedures that are satisfactory to their needs for the detection and identification of LMOs within their National Context.

Participants are invited to share and upload relevant methodologies that are currently in use in their laboratories. Methodologies for both DNA and protein (For example: strip tests or ELISA tests, end point PCR set up and product detection or quantitative Real-Time PCR) based detection at different levels of qualitative and quantitative detection are welcome.

We are looking forwards to a fruitful discussion!
Dina
posted on 2014-01-20 00:20 UTC by Dina Abdelhakim, SCBD
This is a reply to 5601 RE: Opening of the discussion: Overview of available detection methods [#5608]
Dear Forum Participants,
In contributing to the discussion, we would like to propose the following two papers to get the forum started.

The first is "Detection and traceability of genetically modified organisms in the food production chain" which discusses current detection and identification methodologies as well as highlights of new diagnostic methodologies.

Secondly is "Development of a Molecular Platform for GMO Detection in Food and Feed on the Basis of “Combinatory qPCR” Technology" more specifically discussed methodologies that are geared towards the development of a testing system that is based on qPCR to detect and identify LMOs.

We hope that you will find this information useful and we encourage you to contribute additional material to the discussion.

Best regards,
Dina
posted on 2014-02-04 15:48 UTC by Dina Abdelhakim, SCBD
This is a reply to 5608 RE: Opening of the discussion: Overview of available detection methods [#5619]
Dear Dina, dear participants,
many thanks for the materials sent. Here you can find the link to the overview "Detecting un-authorized genetically modified organisms (GMOs) and derived materials"
http://www.sciencedirect.com/science/article/pii/S0734975012000377,
which seems to me contains the helpful information and links on the topics under discussion.

Galina.
posted on 2014-02-13 15:14 UTC by Dr. Galina Mozgova, Belarus
This is a reply to 5619 RE: Opening of the discussion: Overview of available detection methods [#5622]
Thank you Galina for your contribution to the forum!
In addition I would also like to direct participant's attention to the following:

1. "Production of Certified Reference Materials for the Detection of Genetically Modified Organisms" (http://lib3.dss.go.th/fulltext/Journal/J.AOAC%201999-2003/J.AOAC2002/v85n3(may-jun)/v85n3p775.pdf) which discusses some steps in the production of reference materials. This information may be useful for those colleagues who are involved in in-house production of RM of. for example, regionally specific LMOs.

2. "PCR-Based Detection of Genetically Modified Soybean and Maize in Raw and Highly Processed Foodstuffs" (http://www.biotechniques.com/multimedia/archive/00011/01312pf01_11584a.pdf) which touches upon the isolation of DNA from processed food samples for the purposes of PCR analysis and detection as well as suggestions for circumventing potential sources of co-extracted PCR inhibitors.

I hope you will find these resources of use to you and hope to see your additional contributions to the forum soon.

Best regards,
Dina
posted on 2014-02-17 20:47 UTC by Dina Abdelhakim, SCBD
This is a reply to 5622 RE: Opening of the discussion: Overview of available detection methods [#5629]
Dear Forum participants,

Many thanks for the submitted materials.

I would also like to mention a very useful paper on Detection of Living Modified Organisms (LMOs) and Needs for Capacity Building by Christopher D. Viljoen, you can find at the link:

http://ris.org.in/images/RIS_images/pdf/vol7no3_article4.pdf

For those who may find useful the paper published in Romanian language I would suggest the following link - http://www.biosafety.md/public/209/ro/Aspecte_.pdf
ASPECTE METODOLOGICE ÎN TESTAREA PLANTELOR MODIFICATE GENETIC
by Maria DUCA, Angela LOZAN, Angela PORT, Alopna Glijin, Victor LUPAŞCU

Best regards,
Angela
posted on 2014-02-23 21:41 UTC by Ms. Angela Lozan, Republic of Moldova
This is a reply to 5608 RE: Opening of the discussion: Overview of available detection methods [#5639]
Dear Fellow Participants

There different methods that can be employed to detect LMOs. However the sensitivity of the method matters a lot. I say this because there are cases through experience that I have employed the use of protein based methods like lateral flow devices and got negative results in a grain sample but the same sample analyzed through PCR gave positive results. So in what instances should someone use protein based methods and in what cases should one use other methods. I think this should be clear because in most case laboratory staff with minimal experience and training are faced with such situations and one is unable to know where the problem lies.

Secondly on the  screening methods used. I had an opportunity to learn from the workshop that screening GM can be done through the Waiblinger table. This is not known to some labs in developing countries such as mine so you realize mostly screening kits are used which are very expensive and still the do not cover all the major elements. In other cases only 35S and NOS are used which may leave out other events. Hence the proper way of screening GMOs should be described for labs which little experience to be able to do the right thing.

The following are the methods that can be useful to contribute to the material that has already been provided.

Thanks,

Lilian
(edited on 2014-02-25 18:29 UTC by Ms Lilian Okiro)
posted on 2014-02-25 18:00 UTC by Ms Lilian Okiro, Egerton University
This is a reply to 5639 RE: Opening of the discussion: Overview of available detection methods [#5645]
Dear Participants, Dear Dina.
Please, find attached two presentations from the Workshops, that were held in Slovenia and South Africa, I have found on the BCH Site. It seems to me they are very illustrative, contain good schemata and links to the methods and scientific papers under the topics discussed and would be helpful.
http://bch.cbd.int/forum/art18/customs%20officers/cee/sampling.pdf
http://gmo-crl.jrc.ec.europa.eu/capacitybuilding/docsworkshops/SouthAfrica-Feb-2012/2012-02-08%20new%20approaches%20detection%20unapproved.pdf

Also I would like to add the Methodical Guidelines (detection and identification) developed and approved in Russian Federation that were approved  in our country last year. Unfortunately in Russian.

With best wishes, Galina
(edited on 2014-02-26 08:36 UTC by Dr. Galina Mozgova)
posted on 2014-02-26 08:26 UTC by Dr. Galina Mozgova, Belarus
This is a reply to 5645 RE: Opening of the discussion: Overview of available detection methods [#5646]
Thank you Galina for sharing The Methodical Guidelines
I'm sure they will be of great use to Russian speaking colleagues!
Best regards
Dina
posted on 2014-02-26 13:23 UTC by Dina Abdelhakim, SCBD
This is a reply to 5601 RE: Opening of the discussion: Overview of available detection methods [#5679]
Dear all,

Thank you for posting such interesting information. I would like to add some helpful resources.

1. The “Training Manual on the Analysis of Food Samples for the Presence of Genetically Modified Organisms” (Querci, M., et al., 2006) provides theoretical and practical information on methodologies used in GMO analysis. It is available at
http://gmocrl.jrc.ec.europa.eu/capacitybuilding/manuals/Manual%20EN/User%20Manual%20EN%20full.pdf.
In order to disseminate the available information to the Arabic speaking countries, this manual was used as a basis to develop an Arabic manual http://www.fao.org/biotech/biotech-add-edit-section/biotech-add-edit-news/biotech-news-detail/en/c/168577/
The manual was prepared in collaboration with colleagues from Syria in the context of a training course that was organized as a part of the Technical Cooperation Programme (TCP) project by FAO.

2. The report “Compendium of reference methods for GMO analysis” (JRC 2011) lists qualitative and quantitative reference methods for GMO analysis that have been validated according to international standards. The methods target various types of DNA sequences in GMO such as promoters, gene, construct and/or event. It is therefore of great interest to the testing laboratories at different testing levels. It is available at this link
http://publications.jrc.ec.europa.eu/repository/bitstream/111111111/22754/1/gmo-jrc_reference%20report_2011_publ.pdf

3. The review articles:
“Advances in molecular techniques for the detection and quantification of genetically modified organisms” (Elenis, D., et al., 2008) focuses on the developments in molecular techniques for GMO detection and quantification.
“New approaches in GMO detection” (Querci, M., et al., 2010) reviews some testing strategies which are developed in order to cope with the complexity of GMO analysis such as the stepwise matrix approach.

4. The ISO standards 21569 (Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods) and 21570 (Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods). These standards include several testing protocols that can be easily implemented by new testing laboratories.

Hope these resources are of help to you.

Best Regards,
posted on 2014-03-03 14:04 UTC by Dr Gretta Abou-Sleymane, Lebanon
This is a reply to 5679 RE: Opening of the discussion: Overview of available detection methods [#5685]
Dear all,
I would like to thank all the participants to this forum which is of a major importance since in my country (TUNISIA) the work is ongoing to the establishment of a national network of GMO laboratories. Thus, all the methodologies and techniques are welcome.
I would like also to share my experience in this domain since my phd was involved in the European Co-Extra program (finalized in France 2009) and we have developed in the frame of this project new methods and approaches for the detection and quantification of GMO in different matrixes (attached and pubmed address). You will find also all my recent publications developed in our laboratory and reviews in this domain.

With my best wishes

http://www.ncbi.nlm.nih.gov/pubmed/19053386?dopt=AbstractPlus
http://www.ncbi.nlm.nih.gov/pubmed/18303841?dopt=AbstractPlus
posted on 2014-03-04 13:44 UTC by Dr MAHER CHAOUACHI, Institut Superieur de Biotechnologie de Monastir
This is a reply to 5601 RE: Opening of the discussion: Overview of available detection methods [#5722]
Dear Forum Participants,
Attached are two papers describing methods for the DNA based identification of LMOs.

The first "Four new SYBRGreen qPCR screening methods for the detection of Roundup Ready, LibertyLink, and CryIAb traits in genetically modified products" describes trait specific qPCR methods of detection.

Second "An innovative and integrated approach based on DNA walking to identify unauthorised GMOs" details a methodology for for strategically approaching the detection of possible unauthorised events.

I hope you will find this information useful and hope you will also be able to further contribute additional resources to the forum.

Best regards,
Dina
posted on 2014-03-27 20:56 UTC by Dina Abdelhakim, SCBD
This is a reply to 5601 RE: Opening of the discussion: Overview of available detection methods [#5723]
POSTED ON BEHALF OF BOLDIZSÁR VAJDA
---------------------------------------------------------

Dear Dina and forum participants,

We are developing a "screening matrix" for use in our laboratory. For that we have collected articles describing real-time PCR methods, mainly element specific, but some construct specific too and a duplex method. Attached I'm sending our collection. I hope it will be useful for all of the colleagues. The method described in the Lee paper, theoretically works also in 88017. Two of the papers are in German, but still useful.

Best regards,

Boldizsár Vajda
from Hungary
posted on 2014-03-28 14:00 UTC by Dina Abdelhakim, SCBD
This is a reply to 5601 RE: Opening of the discussion: Overview of available detection methods [#5734]
Dear Forum Participants,

Further to the postings that have been made to this forum, I would like to further add to the contributions the paper "Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods."

Best regards,
Dina
posted on 2014-04-03 20:59 UTC by Dina Abdelhakim, SCBD
This is a reply to 5639 RE: Opening of the discussion: Overview of available detection methods [#5739]
Dear Lilian, and forum participants,

You had posted a question on appropriate use of PCR and protein-based methods on February 25th.
Indeed, as you say, the sensitivity of the method matters a lot.
For a method to be fit for use (Fit for purpose) it must be validated as it is to be used.  A method should only be used on a size of sample that is appropriate - thus if is has been proven to be valid on a sample of 400 seeds, then it should not be used on a sample of 4000, unless you can validate it for that purpose.  Each lateral flow strip device has a limit of detection that is given in the instructions.  Similarly a PCR method should not be used to try and detect something in a sample size that it has not been validated for.
In teh case where teh sample size is larger that the validated detection level for the test, the sample can be split into subsamples.  This sub-sampling approach not only makes it possible to use a low cost test (e.g. LFS) but it also gives a quanatitative value for the presence of the characteristic detected.
The principles of acceptance sampling is also describe din the article by Kobilinsky and Bertheau (which you posted). This paper explains acceptance sampling as a means of quantifying GMOs in grain.  Other publications on the use of acceptance testing include Mano et al. 2011.

Acceptance sampling is used very extensively in testing for low levels of GMO’s throughout the seed and grain businesses.  ISTA has developed a useful Microsoft Excel spreadsheet tool (Seedcalc) for applying acceptance testing, including the development of efficient sampling and testing plans.  This tool is widely used.  It is described in These principles are described in Remund et al 2001, and Laffont et al 2005.  These articles are open access.  Seedcalc8 and other tools are freely available online at http://seedtest.org/en/stats-tool-box-_content---1--1143.html.

Best regards,

Ray Shillito

Refs (not open access): Practicable Group Testing Method to Evaluate Weight/Weight GMO Content in Maize Grains.  Junichi Mano eta al., J. Agric. Food Chem., 2011, 59 (13), pp 6856–6863.  DOI: 10.1021/jf200212v
posted on 2014-04-04 19:57 UTC by Dr Raymond Shillito, Bayer CropScience