| | english | español | français |
  Home|The Cartagena Protocol|SD&I|HTPI Portal|Laboratories|2012 - 2014   Printer-friendly version

Past activities 2012 - 2014

Return to the list of threads...
Forum closed. No more comments will be accepted on this forum.
Opening of the discussion: Minimum performance criteria [#5603]
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 compiling information on "Minimum performance criteria for sample handling, extraction, detection and identification methodology".

Further to the process of selecting appropriate methodologies for the detection and identification of LMOs, in the context of each country’s National regulations, considerations of the performance of the selected methodologies is crucial for the production of reliable and reproducible data. In order to assess the methodologies that are appropriate for each national laboratory they must be tested and validated to ensure that they meet minimum performance criteria. The performance of a method encompasses measuring, amongst other things, its precision, accuracy, sensitivity and specificity. Knowledge of these parameters during routine testing would ensure consistent reporting of results within acceptable ranges of variability.

Participants are invited to work towards compiling information that will help National laboratories establish and measure acceptable minimum performance criteria for their choice of detection methods that are applicable to their needs as per the detection and identification of LMOs within their national context. Access to this information will allow each laboratory to develop and establish routine detection methodologies that are based on sound and acceptable criteria.

We are looking forward to your lively participation in this discussion.
Dina
posted on 2014-01-20 00:31 UTC by Dina Abdelhakim, SCBD
This is a reply to 5603 RE: Opening of the discussion: Minimum performance criteria [#5611]
Dear Forum Participants,

The performance of a method encompasses measuring, amongst other things, its precision, accuracy, sensitivity and specificity. Laboratories can select from amongst any number of methods that suit their needs so long as they are validated and are determined to generated data that reaches acceptable minimum performance criteria.

The two attached papers "Criteria for Accreditation of Laboratories Testing Genetically Modified Organisms" and "Detection Methods and Performance Criteria for Genetically Modified Organisms" discuss various criteria against which various methods can be assessed.

Access to this information will allow each laboratory to develop and establish routine detection methodologies that are based on sound and acceptable criteria in line with their national regulatory context.

Are there other criteria that are used in your laboratories? Do you have specific criteria that are unique to different methods that you can contribute to the discussion?

Best regards,
Dina
posted on 2014-02-04 21:28 UTC by Dina Abdelhakim, SCBD
This is a reply to 5611 RE: Opening of the discussion: Minimum performance criteria [#5613]
POSTED ON BEHALF OF BOLDIZSÁR VAJDA
---------------------------------------------------

Dear  Dina,

The link guide you to the website of the European Union Reference Laboratory for GM Food and Feed. You may find a document describing the minimum performance criteria aganst a qPCR method. ( http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf )
That criteria on DNA quality and especially the slope, the R2 in a qPCR run are important in the everyday analysis.

Best regards,

Boldizsár Vajda
posted on 2014-02-05 14:36 UTC by Dina Abdelhakim, SCBD
This is a reply to 5613 RE: Opening of the discussion: Minimum performance criteria [#5614]
For implementing already validated methods in the laboratory there is also a guideline http://gmo-crl.jrc.ec.europa.eu/doc/ENGL%20MV%20WG%20Report%20July%202011.pdf It is based on the Minimum performance requirement document, and are made to enforcement laboratories.
Kind regards Lotte Hougs
posted on 2014-02-05 14:44 UTC by Ph.D. Lotte Hougs, Danish Veterinary and Food Administration
This is a reply to 5614 RE: Opening of the discussion: Minimum performance criteria [#5615]
The method verification document is excellent. We use that in our lab. What we have sometimes problems is with the slope, specially when using Delta Ct, because sometimes the it seems that you have all parameters fulfilled, a almost perfect r2, and it looks like the lower the slope the better the estimate, with the slope reaching -4,0 or -3,9, which are values below the accepted range (-3,1 to -3,6).

Best regards,

Nilson
National Agricultural Laboratory - Brazil
posted on 2014-02-05 15:16 UTC by Nilson César Castanheira Guimarães, Ministry of Agriculture, Livestocks and Supply (MAPA)
This is a reply to 5615 RE: Opening of the discussion: Minimum performance criteria [#5616]
Thank you Nilson for your post.

Would it be possible for you to outline to the group the practical steps your lab took in implementing the recommendations outlined in this document?

Furthermore, would any of the forum members have suggestions for a document that outlines recommendations on how to improve qPCR efficiency/ slope values?

thanks
Dina
posted on 2014-02-05 15:54 UTC by Dina Abdelhakim, SCBD
This is a reply to 5616 RE: Opening of the discussion: Minimum performance criteria [#5617]
Dear Dina and members of the Network,

Greetings!
Thanks Boldizsár and Nilson for bringing up these important issues.
I have never had problems with PCR efficiency, although I had to design my own transgene primers for MGB (Applied) and ZEN (IDT) probes.
I did, however, had problems with multiplexing due to the interactions between endogenous gene controls and transgene targets. Since I have been using one-step pcr kits, the interaction is most probably due to the great difference in the concentration of RNA targets in the sample.
In addition to the discussion on qPCR efficiency and multiplexing, I would like to share with you the adaptations we had to make regarding data normalization. Pfaffl corrections are highly adopted by many research groups and attention should be given for those qPCR equipment softwares that do not consider qPCR efficiency corrections, such as the SteOne software from Applied. Therefore, all of our data is processed afterwards manually in a excel format according to Pfaffl 2001.
Here I present some of the literature that I have been reading regarding these issues. I am sorry if these are not precise recommendations to our discussion-documents.

Best regards,

Sarah

http://www.biomedcentral.com/1471-2199/10/113

http://www.biomedcentral.com/1472-6750/6/37

Pfaffl MW (2001) A new mathematical model for relative quantific-ation in real-time RT-PCR. Nucl. Acids Res. 29: e45.

Pfaffl MW, Hageleit M (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol. Lett. 23: 275–282.
posted on 2014-02-08 11:07 UTC by Ms. Sarah Agapito-Tenfen, Brazil
This is a reply to 5617 RE: Opening of the discussion: Minimum performance criteria [#5618]
Greetings to All,

I would like to thank the moderators for clear designing of what specific information should be discussed under the Minimum performance criteria for laboratory detection and identification.

Thank you to colleagues who provided  here with the useful links of published literature that would help to apply the specific methods for sampling, extraction, testing of LMOs etc.

It would be interesting to see some other available papers to be aware of the countries experiences.

Best,
Angela, MD
posted on 2014-02-11 21:05 UTC by Ms. Angela Lozan, Republic of Moldova
This is a reply to 5603 RE: Opening of the discussion: Minimum performance criteria [#5620]
Dear Forum Participants,
Further to the previous discussions under this topic I would like to further include the following documents on this forum.

The first, "Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection" (http://works.bepress.com/cgi/viewcontent.cgi?article=1031&context=torstein) Provides a review of a number of different PCR chemistries and briefly touches on their performance criteria. This may be useful in the process of selecting specific methodologies that can be used in your laboratories.

Secondly, "Increased efficacy for in-house validation of real-time PCR GMO detection methods" (http://link.springer.com/article/10.1007/s00216-009-3315-6) provides an overview of the process of carrying out in-house validation of PCR methodologies with the view to determine and establish a more efficient and accurate validation procedure.

I hope you find this information useful and look forward to seeing your additional contributions to the forum.

Best regards,
Dina
posted on 2014-02-17 19:21 UTC by Dina Abdelhakim, SCBD
This is a reply to 5603 RE: Opening of the discussion: Minimum performance criteria [#5621]
Dear participants,

I am forwarding the Codex guideline for specific DNA methods (which includes PCR), which also covers detection methods for specific proteins.  It is attached for convenience.  ISO standards are consistent with this Codex guideline.

In addition, a paper by Lipp et al. (also attached) gives a good overview of PCR method criteria, and also mentions the sampling that is inherent in laboratory work.  The approach of using false positiive and false negative rates to characterise qualitative methods shown in this paper is now out of date, and AOAC has published (http://www.eoma.aoac.org/app_n.pdf) a guideline for qualitative methods which, that as well as DNA methods, is also applicable to dipstick type (Lateral flow strip) protein detection methods.

Best regards,

Ray Shillito
(edited on 2014-02-17 21:51 UTC by Dr Raymond Shillito)
posted on 2014-02-17 20:03 UTC by Dr Raymond Shillito, Bayer CropScience
This is a reply to 5603 RE: Opening of the discussion: Minimum performance criteria - protein methods [#5624]
Whilst most of the focus in this forum has been on PCR methods, protein based testing can form an important part of any screening approach, and these tests are used extensively in acceptance testing of grain entering commodity chains.   They are also an effective and important tool in ensuring the purity of seed.  The paper by Grothaus et al. (attached) outlines the use of these technologies.  The document highlights the many areas to which attention must be paid in order to produce reliable test results, Including sample preparation and method validation.
Appropriate use and validation (including criteria) are also described in the Codex Guideline CXG_074 previously posted.

Best regards,

Ray Shillito
posted on 2014-02-17 21:50 UTC by Dr Raymond Shillito, Bayer CropScience
This is a reply to 5620 RE: Opening of the discussion: Minimum performance criteria [#5626]
Dear Forum Participants,
I would like to thank all for providing useful information. I would like to add further documents to the forum.
NATA's Criteria for Accreditation of Laboratories Testing Genetically Modified Organisms from 2006 were already mentioned. Last year NATA published (or updated) two documents on accreditation of laboratories: Biological Testing ISO/IEC 17025 Application
Document and Biological Testing Annex D: Accreditation of facilities testing for genetically modified organisms (GMO).

Publication on experience of a university laboratory in accreditation of methods for GMO detection according to the ISO/IEC 17025 is describing verification studies performed for screening, construct specific and event-specific methods: Verification of Real-Time PCR Methods for Qualitative and Quantitative Testing of Genetically Modified Organisms.

The number of new GMOs is increasing rapidly and laboratories that are already accredited for GMO testing need to take up new methods within their scope of accreditation in timely manner, which is possible if the scope of accreditation is flexible. Guidance document to facilitate harmonised flexible scope accreditation related exclusively to quantitative testing of GMOs is addressing different levels of flexibility concerning the products, the events and the analytical. European technical guidance document for the flexible scope accreditation of laboratories quantifying GMOs.

Best regards,

Mojca
(edited on 2014-02-20 14:18 UTC by dr Mojca Milavec)
posted on 2014-02-20 14:06 UTC by dr Mojca Milavec, Slovenia
This is a reply to 5603 RE: Opening of the discussion: Minimum performance criteria [#5724]
Dear members of the Forum,

Attached is an interesting article outlining a comparative study of LFT and PCR detection methods from samples taken from bulk lots in large shipments of soy.

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

Thanks
Dina
posted on 2014-03-28 16:14 UTC by Dina Abdelhakim, SCBD
This is a reply to 5724 RE: Opening of the discussion: Minimum performance criteria [#5736]
Dear members of the forum,

The paper on KeLDA has been used as justification of an impracticable sampling approach as set out in CEN technical specification CEN/TS 15568, and may have been the inspiration behind a proposed international standard that was rejected by the international community at ISO TC34.
Unfortunately the KeLDA paper failed to compare this process with the standard grain sampling approaches that are described in a number of ISO and national standards, in that it failed to compare the results with the composite bulk sample.  Thus the conclusions cannot be fully justified.
It is unlikely (because of cost) that the study will be repeated by the authors while incorporating this important control.
I will will further discuss sampling in the international grain trade in a separate post.

Best regards,

Ray Shillito
posted on 2014-04-04 18:59 UTC by Dr Raymond Shillito, Bayer CropScience
This is a reply to 5724 RE: Opening of the discussion: Minimum performance criteria (sampling) [#5737]
Dear members of the forum,

The existence of a draft International Standard ISO/DIS 21568 (Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Sampling) has been mentioned in some of the documents posted to the forum (specifically those used in EU capacity building posted by Galina).  The proposed standard raised serious concerns as it would have removed flexibility and decision making from the trade, would have been difficult and expensive to implement in practice, and was likely to be in conflict with the work carried out by the Codex Alimentarius Commission and other established sampling protocols (e.g. ISO, GAFTA, FOSFA).

However, the proposed standard was considered unnecessary and impractical and was removed from the work program by vote of the members of TC34.  One major consideration was that grain is already sampled for many characteristics; an additional sampling regime such as proposed in the draft standard was and is not practicable to apply in the real world.
FOFSA and the International Grain Trade Coalition (see attached) have all stated that this apparent restriction should be removed and GAFTA rules (No. 124) include GMO sampling.  In addition ISTA rules for sampling for GMOs do not differ from those for sampling for other characteristics.  Section 19.4.1 states that “Chapter 2: Sampling gives definitions of various sample types, including primary, composite, submitted and working samples, as well as guidelines for obtaining seed lot samples that represent the properties of the seed lot. These definitions and guidelines apply also to GMO testing.”

An ISO international workshop agreement meeting on sampling was convened in May 2008. The issue of sampling of bulk grains, including for the presence of GMO’s, was considered by the international workshop of ISO, in the light of a draft of the international standard ISO DIS 24333.2.  The experts present at the workshop specifically stated that sampling for the presence of GMO should not be dealt with separately as there are many analytes which follow the same pattern and that therefore the existing sampling standard (for other characteristics) is sufficient to sample for their presence.
Indeed existing sampling standards such as ISO 24333 are used extensively for the determination of impurities and contaminants, including those of a particulate nature.  These include such individual seed characteristics such as sprouted seeds, other species, damaged kernels etc.  Evidence for this was presented by jurgen Moller to CEN TC 338 in 2012 (http://www.njf.nu/filebank/files/20130312$203320$fil$ItQTlF5DLYRYA45H87U0.pdf)
Recently experts working on the revision of ISO542 (sampling of Oilseeds) same to the same conclusion and it is expected that any differentiation of sampling for GMO’s from sampling for other characteristics will be removed from the Oilseeds standard.

The draft standard ISO/DIS 21568 was later incorporated into the European Technical Specification CEN/TS 15568.  Despite this the approach has been little used.  At the meeting of CEN/TC 275/WG 11 in Berlin in 2013, only Germany stated that they use the specification “occasionally” while other countries have not or no longer use the specification as it is not practicable.  France specifically stated in 2012 that they “do not use and have no interest in this Technical Specification” (see attached)

Thus it is clear from the opinion of experts in the field that sampling grain (and seed) for the presence of GMO’s is not different than sampling for other characteristics.
posted on 2014-04-04 19:36 UTC by Dr Raymond Shillito, Bayer CropScience
This is a reply to 5603 RE: Opening of the discussion: Minimum performance criteria [#5738]
Dear Forum Participants,
Please find attached a paper on the use of the pJANUS™-02-001 plasmid as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products and its validation for such usage.
Best regards,
Dina
posted on 2014-04-04 19:56 UTC by Dina Abdelhakim, SCBD