For a particular material this must include the methods for DNA extraction and the subsequent quantification in a polymerase chain reaction (PCR) system. In such a case, the whole process from extraction up to the PCR-technique (or equivalent) constitutes a method. The applicant shall provide information about the whole method.
The method shall be event-specific and thus must only be functional with the GMO or GM based product considered and shall not be functional if applied to other events already authorised; otherwise the method cannot be applied for unequivocal detection/identification/quantification. This shall be demonstrated with a selection of non-target transgenic authorised events and conventional counterparts, in the case of GM plants. This testing shall include closely related events, where relevant, and cases where the limits of the detection are truly tested. The same specificity principle must be applied for products that consist of or contain GMOs other than plants.
The method shall be applicable to samples of the food or feed, to the control samples and to the reference material, which is referred to in Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003.
The method shall be developed taking the following documents in consideration as appropriate:
General requirements and definitions: draft European standard prEN ISO 24276:2002,
Nucleic acid extraction prEN ISO 21571:2002,
Quantitative nucleic acid based methods: draft European standard prEN ISO 21570:2002,
Protein based methods: adopted European standard EN ISO 21572:2002,
Qualitative nucleic acid based methods: draft European standard prEN ISO 21569:2002.
in the case of an application for authorisation covering a GMO, products consisting of or containing a GMO or products produced from a GMO, the event-specific quantitative detection method of the GM material;
in addition, in the case of an application for authorisation covering products produced from a GMO where the genetically modified material is detectable, the event-specific quantitative detection method in the foods or feeds produced from the GMO.
Scientific basis: An overview of the principles of how the method works, such as DNA molecular biology based (e.g. for real-time PCR) information must be provided. It is recommended to provide references to relevant scientific publications.
Scope of the method: Indication of the matrix (e.g. processed food, raw materials), the type of samples and the percentage range to which the method can be applied.
Operational characteristics of the method: The required equipment for the application of the method shall be clearly mentioned, with regard to the analysis per se and the sample preparation. Further information of any specific aspects crucial for the application of the method shall also be mentioned here.
Protocol: The applicant shall provide a complete optimised protocol of the method. The protocol shall present all the details as required to transfer and apply the method independently in other laboratories. It is recommended to use a protocol template, which can be obtained from the CRL. The protocol shall include details of:
analyte to be tested,
working conditions, instructions and rules,
all the materials needed, including an estimation of their amounts and storage and handling instructions,
all the equipment needed, including not only the main equipment such as a PCR system or centrifuge but also small items such as micropipettes and reaction tubes with an indication of their appropriate sizes, etc.,
all the steps of the operative protocol, clearly described,
instructions for the data recording (e.g. the programme settings or parameters to be included).
The prediction model (or alike) needed to interpret results and to make inferences must be described in full details. Instructions for the correct application of the model should be provided.
primer pairs tested (in the case of a PCR-based test): justification shall be given of how and why the proposed primer pair has been selected;
stability testing: experimental results from testing the method with different varieties shall be provided;
specificity: the applicant shall submit the full sequence of the insert(s), together with the base pairs of the host flanking sequences needed to establish an event-specific detection method. The CRL shall enter these data in a molecular database. By running homology searches, the CRL will thus be in a position to assess the specificity of the proposed method.
participating laboratories, time of the analysis and outline of the experimental design, including the details about the number of runs, samples, replicates etc.,
description of the laboratory samples (e.g. size, quality, date of sampling), positive and negative controls as well as reference material, plasmids and alike used,
description of the approaches that have been used to analyse the test results and outliers,
any particular points observed during the testing,
references to relevant literature or technical provisions used in the testing.
In view of implementing Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003, the applicant shall, together with the information specified under sections 1, 2 and 3 of this Annex, also provide samples of the food and feed and their control samples of a type and amount to be specified by the CRL for the specific application for authorisation.
The reference material as referred to in Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003 shall be produced in accordance with internationally accepted technical provisions such as ISO Guides 30 to 34 (and more particularly ISO Guide 34, specifying the general requirements for the competence of reference material producers). The reference material shall be preferably certified and, if such is the case, certification shall be done in accordance with ISO Guide 35.
For verification and value assignment, a method that has been properly validated (see ISO/IEC 17025:5.4.5) shall be used. Uncertainties have to be estimated according to GUM (ISO Guide to the Expression of Uncertainty in Measurement: GUM). Major characteristics of these internationally accepted technical provisions are given below.
reference material (RM): material or substance, one or more of whose property values are sufficiently homogenous and well-established to be used for calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials;
Certified reference material (CRM): reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an accurate realisation of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence.
GM RM container (bottles, vials, ampoules, etc.) must be tight and contain not less than the stated amount of material,
samples must have appropriate homogeneity and stability,
the commutability of the GM RM has to be assured,
packaging must be appropriate to the purpose,
labelling must be of good aspect and quality.
between-bottle homogeneity must be examined;
any possible between-bottle heterogeneity must be accounted for in the overall estimated RM uncertainty. This requirement applies even when no statistically significant between-bottle variation is present. In this case, the method variation or the actual calculated between-bottle variation (whichever is larger) must be included in the overall uncertainty;
stability must be positively demonstrated by appropriate statistical extrapolation for the GM RM shelf-life to be within the stated uncertainty; the uncertainty related to this demonstration is normally part of the estimated RM uncertainty;
assigned values are valid only for a limited time and must be subject to a stability monitoring.
the methods used for verification and for certification must:
be applied under metrologically valid conditions,
have been properly technically validated before use,
have precision and accuracy compatible with the target uncertainty;
each set of measurements must:
be traceable to the stated references, and
be accompanied by an uncertainty statement whenever possible;
participating laboratories must:
have the required competence for the execution of the task,
be able to achieve traceability to the required stated references,
be able to estimate its measurement uncertainty,
have in place a sufficient and appropriate quality assurance system.
to avoid a posterior degradation, all samples are best stored under conditions designated for the final storage of the GM RM before measurements are started,
otherwise, they must be transported from door to door keeping them at all times under such storage conditions for which it has been demonstrated that there is no influence on the assigned values.
a certificate complemented by a certification report has to be established, containing all information relevant to and needed by the user. The certificate and report must be made available when the GM CRM is distributed,
certified values must be traceable to stated references and be accompanied by an expanded uncertainty statement valid for the entire shelf-life of the GM CRM.