Workshop and Training Program on Sampling and Detection Methods Applied to Transgenic Crops November 17 19, 2011, NIN, Hyderabad, India

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1 pplied to Transgenic Crops Quantitative PCR for GMO Detection DN-based nalytical Process E. Pearce Smith Eurofins GeneScan ccurate Sampling Sample Prep DN Extraction & Purification NIN, Hyderabad November 7-9, 20 nalyzing the PCR Product(s) PCR Setup: conditions & specificity Run PCR 2 PCR mplification Curve In practice: Efficiency is not 00% Theoretical Target copy number cycle number Depletion of PCR reagents Therefore: Exponential increase is limited Linear increase follows exponential and plateau occurs Log Target DN Experimental PCR Cycle 3 4 What is Real-Time PCR? PCR system in which we can monitor the amplification reaction as it is occurring Proportionality between DN concentration and PCR products is limited Real-Time PCR incorporates the ability to directly measure and quantify the reaction while amplification is taking place Conventional end-point PCR is not suitable for DN quantification 5 6

2 pplied to Transgenic Crops Which detection strategies are available? Intercalating dyes Hybridisation probes 7 ID ID ID Intercalating Dyes - SYR green ID ID λ λ λ ID ID ID λ ID λ ID Repeat inds to doublestranded DN (dsdn). Fluoreseces only when bound to dsdn. s PCR progresses [dsdn] increases, fluorescent signal in sample increases. 8 Reporter (Fluoresceine) Fam R Vic Tet Hybridization Probes Man Fluorogenic Probe Energy Transfer Laser Excitation Quencher (Rhodamine) Q Phosphate Group 9 Man PCR Chemistry R Q R = Reporter Q = Quencher forward primer 0 Man Minor Groove inder (MG) probes Which is the best phase to extract quantitative information? 96 PCR replicates of identical samples have very different individual efficiencies at the end of the reaction. MG are peptides derived from natural antibiotics that bind DN in the minor groove stabilizing duplex DN/Probe 2. No Fluorescent Quenchers (NFQ) lose excitation energy by emission of heat (instead of light as in the case of R). 3. dvantages: increased stability and specificity, lower background 2

3 pplied to Transgenic Crops What is the Threshold Cycle (C T )? The Threshold Cycle (C T ) is the PCR cycle at which the fluorescence signal of the sample rises above the baseline signal Relationship between template, copy number and Threshold Cycle Threshold aseline Sample Relative Fluorescence Copy Number C T Cycle Number The Threshold Cycle, C T Correlates with the starting copy number If you have twice the template, you get to C T one cycle earlier If you have half the template, you reach C T one cycle later GMO quantitation by real-time PCR: what do we need? Standard Curve prepared with material at known GM content, commonly Certified Reference Materials mplification of two targets: Transgene Endogenous species-specific gene The Threshold Cycle, Ct, is a reliable indicator of initial amount of template 5 6 The quantitation of GMOs is a relative measurement The Standard Curve method Two separate Standard Curves: transgene and endogenous gene GM DN QUNTITTION (i.e. RR soybean, maize t-76 etc.) SPECIES-SPECIFIC DN QUNTITTION (i.e lectin, invertase, zein, DH) GMO quantification Endogenous gene quantification % GMO = GM DN/Species DN 7 GM TRGET Copy Number Standard 2000 Standard Standard Standard Standard 5 25 Unknown 853 Unknown Unknown 3 32 ENDOGENOUS Copy Number Standard Standard Standard Standard Standard Unknown Unknown Unknown

4 Workshop and Training Program on Sampling and Detection Methods pplied to Transgenic Crops Percentage of Transgenic Material Plate layout for GMO quantitation: example of simplex assay The percentage of transgenic material (DN) is determined as being the ratio of transgenic to total DN quantities G M O GM DN % = DN Q transgenic system DN Q endogenous system x 00 Sample Transgene Copy Number Endogenous copy number % GMO Unknown Unknown Unknown Positive DN target control Negative DN target control mplification reagent control 20 EN DO GE NO US What and how do we measure? (Some) Critical factors affecting quantitative measurements in GMO analysis. Main Critical performance indicatorsfor quantitative PCR methods in GMO analysis Target copy number Stability of reference gene PCR efficiency/inhibition Discrepancies in GM target copy number between standard and unknown samples may lead to at least 00% over/underestimation of the GM content Reference gene should be tested for stability of amplifiable target copy number across species (i.e. allelic variation) Sub-optimal PCR efficiency due to inhibition may greatly affect linearity of the system and thus correctness of measurements. Specificity >>> Primer design and testing, choice of unique target sequences. Occurance of false positive results Sensitivity >>> PCR design and optimisation, quality of DN template (degree of DN degradation, presence of inhibitors, etc.). LImit of Detection and false negative results. Precision and trueness >>> Method optimisation, inter-laboratory transferibility. Limit of Quantification 2 22 What does % GMO mean? What is the Unit of Measurement? weight : weight? seed : seed? mass : mass? protein : protein? DN : DN?.?? GMO Quantification by PCR - Ratio, not an bsolute mount Corn DN* GMO DN* Corn 80, % Corn gluten 8, % Result on test report Corn starch 800 8? Not quantifiable Maltodextrin ?? Not quantifiable Glucose syrup 0???? Not quantifiable *pproximate relative numbers of target DN molecules that can typically be extracted from a sample and analyzed in quantitative PCR reactions Percentages represent the relative composition of the DN solution extracted from a sample: amount of biotech DN relative to the amount of the plant species DN. This percentages is not a measure of the absolute amount of biotech DN in a sample. 23

5 Workshop and Training Program on Sampling and Detection Methods pplied to Transgenic Crops Testing for % weight / weight? dventitious presence of stacked events Stacked events result in an analytical overestimate of the percentage of biotech corn as compared to the actual w/w percentage of biotech corn in the sample. /20 =5% Overestimate! 2/20 =0% ulk samples Homogenization DN Extraction in 00 NK 603 PCR result = % in 00 MON 863 PCR result = % in 00 NK MON 863 PCR result = 2 % 5% 5% = 0% 5% 5% = 0% PCR tests dapted from Hiroshi kiyama NIHSS, Japan nalysing for combined events No PCR or other molecular approach can distinguish between the presence of a low percentage of stacked events in bulk grain and the presence of a mixture of the two or more individual events that comprise the stack. nalysing for combined events nalysis of Single seeds or sampling approaches have been proposed: kiyama et al. proposed testing individual grains difficult and expensive only feasible for high % GM The International Seed Testing ssociation (IST) has proposed a statistical approach for seed lots Only effective for seed, for limited numbers of stacks and at low concentrations Challenges & Technical Limitations of PCR Is sample representative DN quality and abundance Genetic makeup of the sample (i.e. stacked events)