PCR Methods and Analytical Strategies for GMO Testing. E. Pearce Smith Eurofins GeneScan

Transcription

1 PCR Methods and Analytical Strategies for GMO Testing E. Pearce Smith Eurofins GeneScan 1

2 Genetically Modified Organisms Viruses Bacteria Fungi Plants Animals 2

3 Examples of Tests for Biotech Corn and Soy event trait 35S nos FMV corn MON810 IR + Cry1Ab Bt11 IR + + Cry1Ab TC1507 IR + Cry1F MON863 IR + + Cry3Bb NK603 HT + + EPSPS GA21 HT + no test T25 HT + Pat DAS IR / HT + Cry34/35Ab1 MIR604 IR + Cry3Aa2 MON88017 IR / HT + + EPSPS Cry3Bb1 MON89034 IR Cry1A105/Cry2Ab2 MIR162 IR + Vip3A MON87460 DT + + CspB 5307 IR + ecry3.1ab soybeans PCR LFS detection in grain A HT + Pat MON89788 HT + EPSPS GTS HT + + EPSPS 3

4 Common DNA Testing Strategy for Non-GMO Corn Most accurate: Test each biotech corn event separately, add individual results Cost prohibitive in a commercial situation % + % + % + % + % + % + % + % + % + % + % % + % = % total Event MON DAS MON MON MON T25 Bt11 NK603 TC GA21 MIR604 MIR S % + % + % + % + % % total More cost effective 4

5 Quantitative PCR Tests [biotech DNA] x 100 [species DNA] = % biotech DNA relative quantification 5

6 Sample Size and Detection of Impurities in Trace Amounts For seed and grain, the sensitivity of the overall procedure is typically determined by the test sample size. Example: 3000 seeds One negative sample of 3000 seeds means: <0.1 % with 95% confidence probability of a negative result 0% 20% 40% 60% 80% 100% % true impurity 6

7 How much GMO? 1% 4% 5% corn (100 % biotech) 1%? 100%? wheat flour pea protein rice flour Corn DNA biotech DNA total DNA x x xxxx 90% 1% 4% 5% corn (100 % biotech) no change drastic change dextrose pea protein rice flour Corn DNA biotech DNA total DNA x x xx 90% 7

8 A Case Study Feed additive containing a small amount of corn meal Lab A: Some biotech corn events detected >> 10 % relative to corn DNA Relative quantification Lab B: Some biotech corn events detected >> 10 % relative to corn DNA 35S promoter DNA relative to corn DNA >100 % Relative quantification Lab C: 35S promoter DNA detected, <0.1% Absolute measurement of 35S DNA? 35S DNA relative to total DNA? 8

9 Approximate Sample Sizes and Genome Sizes in Different Crop Plants Crop 3000 Seeds Seeds / 1 kg Genome (bp) Genome (pg) Genomes / 200 ng DNA Wheat 110 g 27, x ,500 Corn 850 g 3, x ,000 Canola 12 g 250, x ,500 Soybeans 600 g 5, x ,500 Flax 20g 170, x ,000 Rice 90 g 33, x ,000 9

10 GMO Quantification by PCR - A Ratio, not an Absolute Amount Corn DNA* GMO DNA* Result LOQ Corn 100,000 1,000 1 % 0.1% * * Corn flour 100,000 1,000 1 % 0.01% Corn gluten 10, % 0.1% Corn starch 1,000 10? Not quantifiable 1% Maltodextrin 100 1?? Not quantifiable 10% Glucose syrup 0???? Not quantifiable 100% *approximate relative numbers of target DNA molecules that can typically be extracted from a sample and analyzed in quantitative PCR reactions ** with a 3000 kernel sample Relative composition of the DNA solution extracted from a sample. This percentage is not a measure of the absolute amount of biotech DNA in a sample. 10

11 DNA content of different matrices 11

12 Which non-gmo soy products are tested? What do PCR results tell us? Sensitivity or LOD Qualitative tests Quantitative tests Whole soybeans Soy flour, meal, flakes protein isolates normal DNA concentrations Typically around 0.1% limited by sample size Can be very sensitive, as low as 0.01% LFS Negative tests results work informative well too Positive results due to low level presence likely Test not necessarily useful ELISA has been Informative, but relatively high uncertainties used too due to sampling More reproducible than sampling & testing grain Crude lecithin or oil limited DNA concentration Refined soy lecithin or oil very little or no DNA Finished products with multiple ingredients sensitivity in terms of % GMO varies with samples GMO poorly or not at all detectable Sensitivity in terms of % GMO varies and is often unknown Test raw materials Interpretation of results limited Interpretation of results very limited Result interpretation limited and challenging Result interpretation requires caution Identity Preservation Systems Traceability Test ingredients separately Test does not work Not Quantifiable Test may not work Interpretation of results especially from screening tests challenging 12

13 Beet sugar production 13

14 Beet sugar production 14

15 Beet sugar production 15

16 Thank you for your attention! E. Pearce Smith Eurofins GeneScan 2219 Lakeshore Drive, Suite 400 New Orleans, LA