Huber et al., Supplementary materials and methods

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1 Reference Materials Reference material (flours) for the following GM and non GM lines were purchased from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium): maize event SYN-BTØ11-1 (Bt11), SYN-EV176-9 (Bt176), DAS , MON-ØØ6Ø3-6 (NK603), MON-ØØØ21-9 (GA21), non-gm maize; soybean MON-Ø4Ø32-6 (GTS ), non-gm soybean; sugar beet: KM-ØØØ71-4 (H7-1), non-gm sugar beet. 100% GM seeds from rapeseed event ACS-BN010-4 (GS40/90) and ACS-BNØØ5-8 (MS8)/ ACS- BNØØ3-6 (RF3) were available from German field trials. Genomic DNA (gdna) from maize event ACS-ZMØØ4-3 (CBH351), ACS-BNØØ8-2 (T45) rape seed, non-gm rape seed, ACS-BNØØ8-2 (LLRice62) and ACS-GMØØ6-4 (A ) soybean and 100% GM seeds from rapeseed MON-ØØØ73-7 (GT73) were received from the German national reference laboratory of the Federal Office of Consumer Protection and Food Safety (BVL). For the experiments performed at NIB, reference materials for the following GM events were purchased from IRMM: soybean events MON-Ø4Ø32-6 (GTS40-3-2), DAS (68416), DP-356Ø43-5 (DP ), DP-3Ø (DP ), maize events SYN- E (3272), DP-Ø9814Ø-6 (98140), SYN-BTØ11-1 (Bt11), SYN-EV176-9 (Bt176), DAS (DAS59122), MON-ØØØ21-9 (GA21), SYN-IR6Ø4-5 (MIR604), MON- ØØ81Ø-6 (MON810), MON-ØØ863-5 (MON863), MON-ØØ6Ø3-6 (NK603), DAS (40278) and DAS-Ø15Ø7-1 (DAS1507), sugar beet event KM-ØØØ71-4 (H7-1), cotton events DAS ( ) x DAS-21Ø23-5 ( ) and potato event BPS-A (AM ). The following GMOs events were purchased from the American Oil Chemists' Society (AOCS, Urbana, USA): soybean events ACS-

2 GMØØ5-3 (A ), ACS-GMØØ6-4 (A ), MST-FGØ72-2 (FG72), BPS-CV (CV127), MON-877Ø1-2 (MON87701) and MON (MON89788), maize events ACS-ZMØØ3-2 (T25), MON-89Ø34-3 (MON89034), MON-88Ø17-3 (MON88017), MON-8746Ø-4 (MON87460) and SYN-IR162-4 (MIR162), oilseed rape events ACS-BNØØ5-8 (MS8), ACS-BNØØ3-6 (RF3), MON-ØØØ73-7 (GT73) and ACS- BNØØ8-2 (T45), rice event ACS-OSØØ2-5 (LL62) and potato event BPS (EH ). A CaMV isolate was provided in inoculated cabbage tissue by Food and Environment Research Agency (FERA; York, UK). Stability tests of the multiplex real-time PCR mixes The mid- and long-term stability of the pentaplex, triplex and duplex PCR reaction mixes was tested after storage at -20 C. For each multiplex real-time PCR assay, all components (without template DNA) were mixed, aliquoted in volumes necessary for one PCR run on a 96-well plate and stored at -20 C. Reference DNAs from the dilution series R7 and R8 (described in the section absolute limit of detection LOD 6 and LOD C.I. ) used for in-house validation were tested immediately without freezing the realtime PCR mix. After intervals of one week, four weeks, two months, three months, four months and six months, the frozen real-time PCR mixes were thawed once and immediately tested with freshly prepared DNA. Stability was estimated by comparing the Cq values of the standard dilution series for each time point with the Cq-values of the reference point (day 1).

3 Determination of the limits of detection Absolute limit of detection LOD 6 and LOD C.I For the determination of the absolute detection limits (LOD 6 and LOD C.I. ), dilution series of the DNA standards were prepared with the following target copies: 20,000; 5,000; 1,250; 250; 50; 20; 10; 5.0; 2.0; 1.0, 0.1 and 0 target copies. The dilution series of the DNA standards were prepared by mixing DNA from the rapeseed events GS40/90, MS8/RF3, and GT73 with non-gm rapeseed DNA respectively. In total two copies of both P-35S and pat are integrated in the hemizygote GS40/90, one copy of ctp2-cp4- epsps in the hemizygote GT73, four copies of TNOS and two copies of bar in the event MS8/RF3. The target copy numbers were calculated on the basis of the genome size of rapeseed (1.225 pg) 1 and the zygosity status under consideration of the actual integrated GM target copy number. Due to the relative content of the different targets, it was necessary to produce two standard dilution series: one for the LOD abs determination of pat, bar, ctp2-cp4-epsps and P-35S targets (standard series R7, these targets being all at equal concentration) and one adjusted to the T-nos target copies (standard series R8). Three independent runs of the optimized duplex, triplex and pentaplex real-time PCR assays were performed using the standard dilution series R7 and R8 analyzed in six replicates of each dilution step. The LOD 6 determination was validated only if the tests at the level 0.1 target copy per well resulted in a maximum of one positive signal (out of six replicates). In case that more than one replicate test resulted in positive signal at this lower level, evaluation of the target concentration of the whole dilution series was revised 2. The LOD 6 was defined as the last concentration at which all six replicates were positive. The LOD determination for each run was performed in parallel on the Mx3005P (Agilent Technologies, USA) and the LC480

4 (Roche Diagnostics, Germany) real-time PCR cyclers. Based on the same dataset, the absolute LOD according to DIN (LOD C.I. ) and the relative confidence intervals (C.I. 95% ), were also calculated using the arithmetic mean ( ) and standard deviation (SD) of the measured target copy numbers (E copies) at each dilution level (m = 6) of the standards dilution series (R7 and R8) with a student factor (dƒ:α) at α = 0.05 and with dƒ = m -1 degrees of freedom. The LOD is achieved by the copy number exceeding C.I. by 100% 3. C.I. (95%) = SDt df ;α 100 x± m x Relative limit of detection (LOD % ) The relative limit of detection (LOD % ) was determined for the pentaplex real-time PCR assay to validate the compliance of the multiplex real-time PCR assay with the legal 0.9% threshold for adventitious or technically unavoidable traces of authorized GMOs in feed and food products according to EC 1830/ For the determination of the LOD %, a dilution series of a mixture of genomic DNA (gdna) from the rapeseed events GS40/90, GT73 and MS8/RF3 containing all five targets was produced. The gdna standard was adjusted to the following five GMO content levels: 0.5%, 0.1%, 0.05%, 0.025%, and 0.010%, corresponding to 100, 20, 10, 5.0 and 2.0 target copies per reaction in a background of 20,000 non GM target copies, respectively. Three independent runs of the pentaplex real-time PCR assays were performed using the

5 gdna standard dilution series analyzed in six replicates of each concentration level. The LOD % was defined as the last dilution step at which all six replicates were positive. Limit of detection with asymmetric target concentrations (LOD asym ) To evaluate the performance of the multiplex real-time PCR assays in asymmetric target scenarios which can lead to competitive effects, mixtures with low copy numbers of each target in a high background of all other targets were prepared. For this, three different standard dilution series were prepared by mixing gdna from GS40/90, GT73 and MS8/RF3 rapeseed, maintaining the P-35S/pat targets, the ctp2-cp4-epsps target or the T-nos/bar targets at low copy number in a high background of all other targets. The three standard dilution series were prepared to reach the following concentration levels: 1,620, 540, 180, 60 and 20 target copies (3,240, 1,080, 360, 120, 40 copies for TNOS) in a background of 54,000 copies of all other multiplex targets, corresponding to a ratio of 2.8%, 0.96%, 0.33%, 0.11% and 0.36% for the targets (5.6%, 1.93%, 0.65%, 0.22%, 0.072% for T-nos). All dilutions have been performed in a background of 100 ng/µl calf thymus DNA (Sigma, Germany). Three independent runs were performed using the different standard dilution series analyzed in six replicates of each concentration level. The LOD asym determined for each target of the three multiplex realtime PCR assays was defined as the last dilution step at which six out of six replicates were positive.

6 References (1) Arumuganathan, K.; Earle, E. Nuclear DNA content of some important plant species. Plant Mol Biol Rep. 1991, 9, (2) AFNOR Standard XP-V : Criteres de validation intra-laboratoire pour les méthodes de détection et quantification de séquences d'acides nucléiques spécifiques; AFNOR: Saint-Denis La Plaine, (3) International Organization for Standardization ISO 21570:2005 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic acid based methods; ISO 21570:2005 ed.; International Organization for Standardization: Geneva, Switzerland, (4) European Commission Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC. Off J Eur Union 2003, L 268,