Authors: I M J Scholtens E J Kok L Hougs B Molenaar J T N M Thissen H van der Voet
Publish Date: 2009/12/09
Volume: 396, Issue: 6, Pages: 2213-2227
Abstract
To improve the efficacy of the inhouse validation of GMO detection methods DNA isolation and realtime PCR polymerase chain reaction a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and inhouse reproducibility In the present study 19 methods for GM soy maize canola and potato were validated inhouse of which 14 on the basis of an 8day validation scheme using eight different samples and five on the basis of a more concise validation protocol In this way data was obtained with respect to the detection limit accuracy and precision Also decision limits were calculated for declaring nonconformance 09 with 95 reliability In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML residual maximum likelihood method From these components relative standard deviations for repeatability and reproducibility RSDr and RSDR were calculated The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are important factors for the total variance and should therefore be included in the inhouse validation It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future only the PCR step needs to be validated The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methodsWithin the European Union products that are genetically modified organisms or derived thereof in whole or in part should be labelled as such European regulation 1829/2003 The exception to this rule is when the presence of the GMO component is adventitious and unavoidable In the latter case the GMO component is allowed up to the level of 09 per ingredient If the GMO component exceeds this threshold value then the product needs to be labelled as containing GMO materials It is also laid down in Regulation1829/2003 that producers have to supply an eventspecific detection method for each new GMO variety This detection method as well as the associated reference materials is an integral part of the approval dossier The Joint Research Centre JRC of the European Commission in Ispra Italy being the Community Reference Laboratory for GMO detection methods will subsequently test the method and in a first phase compare the results with the agreed Method Acceptance Criteria for Analytical Methods of GMO Testing as formulated by the European Network of GMO Laboratories 4 If the method meets these requirements it will in a second phase be validated in a European ring trial and the results are evaluated for reproducibility and trueness Method Performance Requirements To this end the JRC will be assisted by the ENGL as is stipulated in EU Regulation 1981/2006 6 To date over 45 methods for GMO analysis have been tested in a EU ring trial http//gmocrljrceceuropaeu/ These ring trials were in some cases carried out on the basis of plant materials then the DNA isolation was included in the ring trial but in most cases with purified DNAThe EU validation studies include methods for maize soy cotton potato and rapeseed The results of the EU ring trials of the eventspecific methods have been published by the JRC in individual reports In these reports the bias repeatability and reproducibility of the methods are presented for DNA from reference materials of different percentages These reports of EU validation studies provide important data on the performance of the respective methods in different laboratories within the European Union
Keywords: