Central and Eastern European Workshop on the Detection and Identification of LMOs

POSTED ON BEHALF OF MIROSLAVA FEKETOVA
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Dear Participants of the Forum,

I am pleased to welcome you to the third round of online discussions of CEE workshop on the detection and identification of LMOs.

As agreed at the workshop, and outlined in the report of the meeting (which is available at http://www.cbd.int/doc/meetings/bs/bsdiws-2016-01/official/bsdiws-2016-01-02-en.pdf), the third round of the online discussions is dedicated to inventorying testing methods. The aim of this discussion is to assemble an inventory of commonly used testing methods and experiences involving the adaptation of such methods to specific purposes.

I would like to invite you to post any relevant material on the matter, especially your experiences with different detection methods of GMO/LMO you are using as well as pose questions and comments on specific areas where additional information is needed so that others that may have this information available can share it with the group.

The discussion will take place for the next two weeks. Your views and feedback will be greatly appreciated. I look forward to a fruitful discussion!

Kindest regards,
Miroslava Feketova

Dear Forum Participants,
Third round of online discussion has started this Monday. Thanks Krasimira for discussion about sampling and sample preparation and Mojca for discussion about extraction and purification methods. Topic of my online discussion is „Inventorying testing methods“.
At the beginning of my part of online discussion I would like to summarize important informations about LMO/GMO testing.
As we know after workshop in Slovenia, methods of LMO/GMO detection are divided in:

1. Protein based methods for LMO/GMO detection:
- ELISA – Enzyme-linked immunoabsorbent analysis
- Lateral Flow Strip Test
- Western blot

2. DNA based methods for LMO/GMO detection:
- PCR
- Multiplex PCR
- Real Time PCR – qPCR
- Digital PCR –dPCR

Because most of laboratories use DNA methods for LMO/GMO detection, my discussion will be oriented on this kind of methods.
For LMO/GMO detection we can use step by step (screening, identification and quantification):

1. Screening qPCR methods (P35S, T-nos, CTP2-CP4EPSPS, bar, 35S-pat and others)
2. Gene specific methods
3. Construct specific methods
4. Event specific methods

After sample preparation, isolation and purification of DNA, one of the critical steps is screening of LMO/GMO. Results of screening methods we can use in following GMO/LMO testing.
I prepared tables with the most common GM modifications, but my condition was existence of CRM materials. I separated GM modifications according to plant. 
Please read this table. If you have new informations, please fill them up into the tables.
For its preparation I used GMO Screening Table “German WG „Biochemical and molecularbiological analysis“ in cooperation with Federal Office of Consumer Protection and Food Safety (BVL, Germany).

You can find sources of information about CRM here:

https://ec.europa.eu/jrc/sites/default/files/rm_catalogue_160504.pdf

http://www.aocs.org/LabServices/content.cfm?ItemNumber=19248

http://nippongene.com/english/leaf/list_gmo-related-products-e24.pdf

Interesting informations about the GM constructs you can find here:

http://en.biosafetyscanner.org/index.php

Miroslava

Dear Forum Participants,
After information about screening methods, I would like to continue our discussion about methods of LMO/GMO detection.
Now, when we identified screening elements and we know results of gene specific methods (identification of reference plant genes) we can continue with identification of LMO/GMO.

- In the first place more information about the LMO/GMO methods you can find here:
http://gmo-crl.jrc.ec.europa.eu/gmomethods/
http://gmdd.shgmo.org/index/search 
http://www.detection-methods.com/

- Second: Before starting to use new methods in practice, it is good to know about these documents:
http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm

Selection of methods for identification of LMO/GMO in connection with results of screening elements and gene specific methods is very difficult process. Because now there is a lot of GM modification in the world, it is very expensive and time consuming process. Finding of accurate methods requires experiences.
I had chosen one very good example from our laboratory and with it I would like to describe how I proceed with selection of LMO/GMO methods in practice.

I got sample of feed. After screening and plant reference genes detection I had these positive results:

P35S, tNOS, bar, 35S-pat, CTP2-CP4EPSPS, HMG gene (maize), Lec gene (soybean) and CruA (canola) genes.

1. I used this kind of methods for identification of GM soybean: GTS 40-3-2 (P35S and tNOS positive), MON 89788 (CTP2-CP4EPSPS positive), A 2704-12 (P35S and 35S-pat positive) and A 5547-127 (P35S and 35S-pat positive).

2. I used this kind of methods for identification of GM maize: Bt176 (P35S and bar positive), DAS 59122 (P35S and 35S-pat positive)

3. I used this kind of methods for identification of GM canola: Rf3 (tNOS and bar positive), Ms8 (tNOS and bar positive), Rf1 (tNOS and bar positive), Rf2 (tNOS and bar positive), Ms1 (tNOS and bar positive)

4. Detection of CaMV – false positive P35S – negative results

Finally I had these positive results: 

DAS 59122 maize 43,95 (±14,15)%
MON 89788 soybean 3,17  (±1,31)%
GTS 40-3-2 soybean 49,81 (±16,04)%
A 2704-12 soybean positive
Ms8 canola         positive

In this case, when we have a lot of positive results of screening elements and reference genes it is very helpful to use “prespotted plates”: More information about prespotted plates you can find:
http://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/matrices/prespotted_plates
If you have comments or questions, please join the discussion. I am ready to answer you.

Miroslava

Hallo,
I have a question:
In the compendium of referent methods- there are different referent genes described for one and same matrice. Is it ok to use only one of them ( for instance about Maize- hmg,  soy - lectin and so on)
about potato, tomato, sugar beet- witch will be the best choice for referent Gene
Thank you in advance
Regards
Krasimira

Dear Krasimira and all participants

Please excuse my delayed answer. On Monday and Tuesday I was on a business trip.
Thank you very much for your good question.

Yes, in the Compendium of reference methods there are a lot of methods described for detection of reference genes, but not only for reference genes  but also for Qualitative GMO detection PCR methods and Quantitative GMO detection PCR methods.
If you need to know, which kind of plant material occurs in sample, you can choose any  of reference genes. Result will be identification of  plant  materials in sample and information about good isolation and concentration of DNA. But If you know which plant materials is in sample, you can continue with identification of GMO/LMO in sample. When you will have positive result of qualitative of GMO/LMO methods, you can start with quantification. For quantification is important to choose accurate methods and now, you need to detected target gene and reference gene for calculation of amount of GMO/LMO in sample. But in this case, you choose only one of methods , and you can use  system, which is described in validated methods.

For example.  You can use this validate system for quantificatio of GM MON810 maize:
1. Target gene: (Mail-F1, Mail-R1, Probe Mail-S2)
2. Reference gene: (ZM1-F, ZM1-R, Probe ZM1)
3. CRM of GMO:  (ERM-BF413 MON 810 MAIZE, IRMM Geel, Belgium)

Miroslava