Certified Reference Materials in GMO analysis

Transcription

1 Certified Reference Materials in GMO analysis Sławomir Sowa GMO Controlling Laboratory, Plant Biotechnology and Cytogenetics Dep., Plant Breeding and Acclimatization Institute National Research Institute, Radzikow Poland Funded by the European Union and implemented by a Consortium

2 Reference material (CRM) Reference material (RM) is the generic term for a group of either pure substances or matrix materials, which are used for calibration, method validation, the establishment of traceability, method development, and various quality control purposes (proficiency testing, charting, etc). Certified Reference Material Funded by the European Union and implemented by a Consortium

3 Certified reference material (CRM) CRM is a material which is used either for method validation, in particular to check for method bias, or for calibration. As defined in the ISO Guide a CRM is: characterised by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides: - the value of the specified property - associated uncertainty - the statement of metrological traceability. Traceability is the property of a measurement result whereby the result can be related to a reference through an unbroken chain of calibrations, each contributing to the measurement uncertainty.

4 RM versus CRM REFERENCE MATERIALS CERTFIED REFERENCE MATERIALS homogeneity and stability REQUIREMENTS homogeneity and stability - ADITIONAL REQUIREMENTS proven homogeneity and stability,characterisation of the property values using suitable, well described methods method validation, method development, quality control purposes SPECIFIED PROPERTIES APLICATION specified properties must be characterised by a metrologically valid procedure proven homogeneity and stability, characterisation of the property values using suitable, well described and validated methods this information may be summed up in a certificate the value of the specified property associated uncertainty calibration, method validation, the establishment of traceability, method development, control purposes

5 RM/CRMs Reference materials are used as positive controls for qualitative PCR and quantitative purposes in the Real-Time PCR GMO analysis. requirements for development, production and specific properties are described in Annex II of Regulation (EC) 641/2004 RM/CRM for DNA-based methods contain the DNA powdered material (e.g., flour certified for mass 1%, 5% etc, can be certified for copy number), extracted DNA ( e.g. from leaves) plasmid containing the specific DNA sequence (1:1 GM/reference gene ratio)

6 RM/CRM - considerations Plasmids RM Applied method must target DNA sequence incorporated in the plasmid Biological considerations ofrm made from plant tissue plant zygosity, tissue ploidy, origin of the GM parent s (Holst-Jensen et al ; Zhang et al. 2008a ). seeds are composed of tissues which have different ploidy level (endosperm, embryo, pericarp) have a different ratio of maternal/paternal origin the DNA content of each of these tissues is different (endoreduplication) the relation between mass and DNA copy number can vary from sample to sample..

7 Measurement units Mass, Mass Fraction, or haploid genom equivalent? GMOs content should be expressed in - which units? No specified units of measurement for 0,9% threshold in the Regulation (EC) 1829/2003 Haploid genome equivalent - the percentage of genetically modified DNA copy number in relation to target taxon-specific DNA copy numbers calculated in terms of haploid genomes (Commission Recommendation 2004/787/EC) Mass fraction or haploid genome equivalent In LLP regulations 619/2011 (Article 3) it is stated that the certified value of the GMO content shall be given in mass fraction and, where available, in copy number per haploid genome equivalent ( Mass fraction - Annex II of the LLP regulation further states that when results are primarily expressed as GM-DNA copy numbers in relation to taxon-specifi c DNA copy numbers calculated in terms of haploid genomes, they shall be translated into mass fraction.

8 Measurement units Mass, Mass Fraction, or haploid genom equivalent? CRMs prepared on mass/mass should be used for testing with the expression of the result in mass fraction. CRMs prepared on copy number should be used for testing with the expression of the result in haploid genome equivalent zygosity, tissue ploidy, parental origin of the GMO are sometimes well described for CRMs but not for the samples transformation factor mass/mass to copy number might not work copy number-based quantification could be most consistent regardless of the properties of the sample

9 I. Taverniers, 2005 J. Žel et al., 2012 Endosperm (49% DNA) Seed coat (3% DNA) -GM allele - Wild type allel Embryo (48% DNA) Taking into account that the embryo and endosperm represent 97% of total DNA depending of the GM allele origin we might have 3/5 or 2/5 of the transgene copies in the seed. Endoreduplication in different tissue

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14 IRMM -CRM certificate Application Note 1 i 6, IRMM

15 IRMM -CRM certificate Important information: zygosity of the material instruction for use (mm% or cn%) storage conditions

16 AOCS -CRM certificate Before using the buyer must grind the compleate sample at once

17 Certyfikaty Minimal recommended test portion for DNA extraction

18 CRM- certified value 0%

19 Traces of other GM events in CRMs CRMs are certified for particular event not for absence of other The CRMs should not be used as negative controls for any other GM event than the one certified CRMs are checked for traces of other GM events

20 Traces of GM events in CRMs made from non modified maize

21 Anex 3 JRC 2011 certified reference materials for some events are available only as 0% and 100% Dilutions must be prepared in the laboratory i % (e.g.. 1%; 0,1%, ) Estimation of the reference gene copy number in CRMs should be made using the same standard curve and the same plate. Dilution factor must be calculated

22 Dilution factor for positive reference material prepared from 0% and 100% Verification of analytical methods for GMO testing when implementing interlaboratory validated methods, Anex 3 Method Verification, JRC 2011 Measure the content of the reference gene for the GM positive and a GM negative DNA preparation (the same plate with the same standard curve). Dilution factor X = (A/B)*(Y-1)+1 X = the practical dilution factor (how much the GM material has to be diluted compensated for difference in concentration) A = copy number of reference gene for the GM positive DNA preparation B = copy number of the reference gene for the GM negative DNA preparation Y = the theoretical dilution factor e.g. from 10 % GM to 0.1 % GM = 100x

23 Dilution factor for positive reference material prepared from 0% and 100% Verification of analytical methods for GMO testing when implementing interlaboratory validated methods, Anex 3 Method Verification, JRC 2011 Example preparation of 10% control DNA A = 100% GMO, DNA B = 0% 5 μl DNA is added per PCR well for A and B Quantification as unknown sample on reference gene calibration curve Result: A (from DNA A): 10 copies/5 μl B (from DNA B): 8 copies/5 μl To make 10 % GMO from 100 % GM corresponds to 10 times dilution (theoretically Y = 10). X has to be used like Y in calculating the volumes to be mixed. If X=12.25, then practical dilution factor X: (10/8)*(10-1)+1=((10/8)*9) +1= 90/8 + 1 = = 12.25, so 1 μl A has to be mixed with μl B. the DNA solution has to be mixed thoroughly. The trueness of the mixtures can be analysed using the 100% mixture for standard curve and analysing 3 samples in triplicates on 3 different days.

24 Plasmid CRMs CRMs made from flour or genomic DNA are not available for many GM events Less expensive in use One plasmid dilution can be used as a so-called golden standard (1 µg of plasmid is enough for 10 8 reactions- 5% GMO equivalent)) Usefull in validation, method development and quality control Can be used for LOD estimation and an error in qualitative PCR T.R. Allnut i in, Plasmid standards for real time PCR and GM enforcement testing

25 Plasmid CRMs high risk of cross-contamination the PCR efficiency for sample lower when compared with PCR for plasmid dificulties in concentration measurement (neccessery dilution for calibration vurve) low concentrations used for reaction the reaction efficiency for plasmid and genomic DNA should be compared Funded by the European Union and implemented T.R. Allnut by a i in, Consortium Plasmid standards for real time PCR and GM enforcement testing

26 Funded by the European Union and implemented by a Consortium

27 Application note 5 European Reference Material 2007 Use of Certified Reference Materials for the quantification of GMO in DNA copy number ratio Concentration of CRM ~ 2 x 10 6 of plasmid copies/ µl Dilution buffer 1 mmol/l Tris, 0,01 mmol/l EDTA, ph 8.0 Recommended staring volume for dilutions 50 µl 5 µl in reaction Dilution according to standard ISO 21570:2005 IRMM, P. Corbisier, 2007

28 Application note 5 European Reference Material 2007 Use of Certified Reference Materials for the quantification of GMO in DNA copy number ratio Liczba kopii MON810 Liczba kopii hmg X 100 % = GM % (hge) IRMM, P. Corbisier, 2007

29 Thank you! Funded by the European Union and implemented by a Consortium