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Past Discussions 2013

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Compilation of laboratory methods for the detection and identification of LMOs [#5278]
Dear Participants of the Laboratory Network,

Welcome to this ongoing discussion forum open with the view to allowing participants to share and discuss relevant documents outlining methodologies that are currently in use in their laboratories. These documents from the participants will be compiled and published in a Biosafety Technical Series in line with a request from the COP-MOP compile relevant laboratory methods and current protocols that are currently in use for the detection of LMOs.

Participants can upload methodologies on various topics that can range from best practices for subsampling techniques, extraction methods, use of automated sample preparation, end point PCR set up and product detection or quantitative Real-Time PCR methods amongst the various other techniques that are used in your laboratories. Documents can be attached to the posts or uploaded using the “Document Exchange” function below.

We thank you for your contributions and hope for an enthusiastic exchange of technical knowledge and expertise.

Best regards,
The Secretariat
posted on 2013-05-20 02:52 UTC by Dina Abdelhakim, SCBD
This is a reply to 5278 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5356]
Dear collegues,
Quality in a GMO laboratory is crucial in order to ensure reliable results for qualitative and quantitative analysis and the validation of methods is necessary to confirm them. For this reliable international guidelines are obligatory in the platforms of LMO detection and quantification. In fact, the general field of analytical chemistry today benefits from an overwhelming supply of guidelines and norms on quality assurance (QA). To the contrary, there is a lack of clear insight in and guidance on how to set up and implement a quality system for bioanalyses such as GMO analysis. For this it is recommanded to set up such a quality system, starting from relevant information from the field of analytical chemistry on the one hand, and from an extensive study of the difficulties faced for GMO analysis on the other hand. First, the analytical system as such is described. An in-house validation study is carried out for the analyte determination
step, which is the real-time PCR quantification of a GM-specific target, relative to an
endogenous, species-specific target. Second, internal quality control (IQC) procedures are
implemented, ensuring statistical control of the data obtained over extended periods of time.
Finally, traceability to stated references is assured and a component-by-component approach
to MU estimation is applied. The different QA principles frame within accreditation conform
to ISO/IEC 17025.
All the methods used in our laboratories are carried using the guidelines of ISO21569, ISO21570, ISO21571, ISO21572 and ISO24276.
We can also use the validated methods used for LMO identification found in the web databases such as those of EURL or IRMM of the Joint Research Center (JRC).
Please find in the attached files some ISO guidelines used in our laborartory.
with my best wishes

Maher Chaouachi, PhD
posted on 2013-05-30 14:20 UTC by Dr MAHER CHAOUACHI, Institut Superieur de Biotechnologie de Monastir
This is a reply to 5356 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5359]
Dear all,

as an addition to the previous post by Dr Chaouachi we also find as an excellent tool the Compendium of validated methods by JRC which you can find attached to the post.
posted on 2013-05-31 07:16 UTC by Ms Renata Hanzer, Croatian Centre for Agriculture, Food and Rural Affairs
This is a reply to 5359 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5371]
Dear All,

Attached also find additional material on GMO detection protocols from JRC
posted on 2013-06-05 13:12 UTC by Ms Lilian Okiro, Kenya Plant Health Inspectorate Service
This is a reply to 5371 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5372]
Dear All,

Attached also find additional material on GMO detection protocols from JRC
(edited on 2013-06-05 13:15 UTC by Ms Lilian Okiro)
posted on 2013-06-05 13:12 UTC by Ms Lilian Okiro, Kenya Plant Health Inspectorate Service
This is a reply to 5278 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5386]
Dear colleagues,

the availability of GMO detection methods, ISO methods, and the Compendium of detection methods published by the European Joint Research Centre (JRC) have already been mentioned in the on-going discussion. I would like to add a link to validated event-specific methods that are gathered und updated in the course of the European GMO authorisation process (http://gmo-crl.jrc.ec.europa.eu/gmomethods) and the Chinese GMDD database  (http://gmdd.shgmo.org). Furthermore at national level in Germany various detection methods have been developed and have been or are in the process of validation, e.g. for construct or element-specific detection. Due to the fact that the number of GMOs is increasing steadily, screening of samples under investigation is an important issue. This is usually done with element and construct-specific methods for the detection of e.g. p35S, tnos, bar, pat, epsps, pnos-nptII and many others. This is necessary in order to narrow down the number of possible events for subsequent analysis of a given sample. As a result of combined screening analysis a profile of detected and non-detected elements and constructs is obtained. In order to support analysts in interpretation these profiles an Access based database and interpretation tool called the GMOfinder was developed in our institute. Its structure and different features have been published (Gerdes L., Busch U., Pecoraro S., 2012. GMOfinder—A GMO Screening Database. Food Anal. Methods (2012) 5:1368–1376). An author´s copy of this article is available on request (sven.pecoraro@lgl.bayern.de). Selected features of the GMOfinder are:

•MS Access database for collection and interpretation of information related to the screening for GMOs
•Contains 341 GMO events from 29 plant species (June 2013)
•The underlying idea is the matrix-approach where each GMO is assigned to its specific profile of detectable and non-detectable genetic elements and constructs (see also: http://gmo-crl.jrc.ec.europa.eu/doc/2011-12-12%20ENGL%20UGM%20WG%20Publication.pdf).
•Optional display of events with potentially masked genetic elements (mixed profiles due to more than one GMO in a sample)
•Easy incorporation of further information on GMO events when available
•Results tagged with an explanatory comment if experimental findings in  contradiction with theoretical information
•False positive results due to infection with the natural donors CaMV and Agrobacterium are taken into account
•Documentation of information sources helps to integrate the GMOfinder into the QM system

We use this tool in our lab on a daily basis since 2011. Up to now around 30 reference laboratories for GMO analysis in Europe have received the GMOfinder.
The GMOfinder is free of charge and readily available to all non-profit laboratories that do official GMO analysis. It can be received on request (sven.pecoraro@lgl.bayern.de).

Best regards,
Sven Pecoraro
posted on 2013-06-13 09:06 UTC by Dr. Sven Pecoraro, Bavarian Health and Food Safety Authority
This is a reply to 5278 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5397]
POSTED ON BEHALF OF GURINDER RANDHAWA
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The methodologies commonly used at various steps of LMO detection are conventional PCR, qPCR, multiplex PCR, Loop mediated isothermal Amplification (LAMP). In all these procedures, starting with preliminary screening of GMOs i.e. detection of most common transgenic elements P-35S /t-nos/ marker genes, we subsequently move on to transgene/construct/event- specific detection. The PCR based assays developed by our GM detection lab. are robust, cost effective and sensitive enough to detect upto 0.1% GMO content. These cost effective assays are being used in the lab. on routine basis.
posted on 2013-06-25 13:31 UTC by Dina Abdelhakim, SCBD
This is a reply to 5397 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5413]
Dear colleagues,
In Lebanon, there are no implemented regulations that define the identity of authorized GM events until now; creating therefore a potential risk for the introduction of various types of GMOs into the country. Accordingly, a cost effective testing strategy that allows for the detection of all the produced GM events is a requirement. In this context, and as we have already mentioned through our previous participation in the online discussion “National regulatory context and current capacity for detecting LMOs” our AUST/ATL laboratory is currently applying ISO and EU-RL GMFF validated methods. Most importantly, in order to face the challenge of the significant increase in both the number and diversification of GM events, we have established our own matrix approach. Our matrix encompasses all GM events in the food and feed related crops and targets the most informative GM elements p35S, T-nos, cp4 epsps, pat, bar and nptII. In the matrix, the events are grouped according to the presence/absence of these screening elements for an easy identification.  Experimentally, the elements are detected using conventional PCR based methods. In this regard, we have optimized the PCR conditions of published primers sequences and the protocols proved to be specific and sensitive to GMOs percentages that are below the lowest worldwide labeling threshold. The validation of these methods is currently in progress.
posted on 2013-07-09 13:14 UTC by Dr Gretta Abou-Sleymane, Lebanon
This is a reply to 5278 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5441]
Good morning,

At the RSS Biosafety Unit, we use the GMO detection methods reported in the following reference:

van den Eede G.,  L. Bonfini, L. Cengia, L. Kluga, M. Mazzara, A. Patak, D. Plan,  M. Querci,  and M. van den Bulcke (2011) Compendium of reference methods for GMO analysis. EUR 24256 EN, EURL-GMFF, ed. (Ispra, Italy, Joint Research Centre, JRC-IHCP).

We found that careful analysis of the results obtained by using the methods of above reference can give new genetic elements not found in the authorized genetic events and not reported in the literatures.

All the best,

Nisreen AL-Hmoud
Royal Scientific Society of Jordan
posted on 2013-07-25 08:53 UTC by Dr. Nisreen AL-Hmoud, Royal Scientific Society
This is a reply to 5441 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5459]
Dear colleagues,

I would like to share a recent publication (not yet assigned to an journal issue; online first) on the applicability of next generation sequencing technologies for molecular characterisation of uncharacterised GMOs.

Wahler D, Schauser L, Bendiek J, Grohmann J (2013) Next-Generation Sequencing as a Tool for Detailed Molecular Characterisation of Genomic Insertions and Flanking Regions in Genetically Modified Plants: a Pilot Study Using a Rice Event Unauthorised in the EU. Food Anal. Methods (online first).

http://link.springer.com/content/pdf/10.1007/s12161-013-9673-x.pdf


Best wishes,

sarah
posted on 2013-08-11 21:46 UTC by Ms. Sarah Agapito-Tenfen, Brazil
This is a reply to 5459 RE: Compilation of laboratory methods for the detection and identification of LMOs [#5460]
Dear Sarah,
thank you very much for the paper. The NGS is nowadays the Highthrouput method used for sequencing and genotyping and adapted for GMO identification. This is considered as a good tool for this purpose. However, the major drawback is the validation of such method and its acceptability in the laboratories and in particular those with a limited budget. i have faced this problem because i have developed few years ago the SNPlex method for the detection of 47 targets and the method worked well (see reference below) but the interlaboratory validation and the transfer of the technique was considered too heavy for the GMO laboratory community.


J Agric Food Chem. 2008 Dec 24;56(24):11596-606. doi: 10.1021/jf801482r.
A high-throughput multiplex method adapted for GMO detection.
Chaouachi M, Chupeau G, Berard A, McKhann H, Romaniuk M, Giancola S, Laval V, Bertheau Y, Brunel D.




With my best wishes,

Maher Chaouachi
posted on 2013-08-11 23:38 UTC by Dr MAHER CHAOUACHI, Institut Superieur de Biotechnologie de Monastir