Advances in Genetically Modified Food and Ingredient Authentication Testing Hemanth Shenoi Global Commercialization Marketing Manager Applied Markets Promega Corporation
Outline of Presentation 1. Introduction to GMO 2. Importance of DNA extraction to event detection 3. Development of new methods for DNA extraction 4. Introduction to new GMO detection methodologies Promega Corporation 2
Definition: What is a GMO? Regulatory definition Applicability of GMO testing Goals of regulation (EU): Ensure the development of GMOs is conducted and deployed under safe conditions Promega Corporation 3
Labeling Requirements Traceability from production / importation / table Post market monitoring once authorized for sale Sharing of information on GMOs cultivated Promega Corporation 4
Interest in Compliance Across the Production Chain Promega Corporation 5 Figure: http://www.cdc.gov/foodsafety/outbreaks/investigating-outbreaks/production-chain.html
Over 50 Countries Have GMO Labeling Laws Australia Belarus Bolivia Bosnia and Herzegovina Brazil Cameroon China Czech Republic Ecuador El Salvador Estonia Ethiopia Iceland India Indonesia Japan Jordan Kazakhstan Kenya Malaysia Mali Mauritius New Zealand Norway Peru Russia Saudi Arabia Senegal South Africa South Korea Sri Lanka Taiwan Thailand Tunisia Turkey Ukraine Vietnam Scope and rigorousness of laws vary from country to country Promega Corporation 6
GMO Event Testing Importance of DNA Extraction Promega Corporation
Current Trends in Global GMO Environment Rise in GMO complexity Proliferation of authorized GMOs Increased number of tests demanded Promega Corporation 8
Two Primary Methodologies Used for GMO Testing Based on the detection of foreign DNA into a recipient plant s DNA (genetic engineering) Molecular based PCR Detect and quantitate GMO sequences Uses DNA (primers) complementary to target GMO sequence Highly sensitive Capable of detecting all known GMOs (known sequences) Industry standard Downside: Must be performed in a laboratory Skilled technicians needed Protein based ELISA Proteins encoded by GMO sequence are detected Specific and inexpensive Can be performed in the field Low skill Downside: Inability to differentiate between genetic events that produce the same protein The proteins in highly processed food are generally not recognized (denatured) Promega Corporation 9
Why is DNA Extraction Important? Garbage in, garbage out Garbage, poor DNA quality Garbage data Assay High quality DNA Accurate data/actionable Promega Corporation 10
Common DNA Extraction Methodologies Method Description Benefits Home brew Spin columns Magnetic beads Chloroform-based, phase separation extraction Used in combination with centrifugation; spin columns use a special filtering frit Uses magnetic beads and chaotropic conditions to bind DNA, wash, and elute from sample matrix Inexpensive Requires standard lab equipment Well-established method Requires standard lab equipment Highly efficient DNA extraction Amenable to multiple automated instruments Promega Corporation 11
Partnership to Develop the FFS/ PureFood Kit 1. Fast 2. Easy protocol 3. High purity 4. Concentrates target 5. Simplicity with difficult samples 6. Robust amplification minimal inhibition Promega Corporation 12
LGL Technical Requirements 1. DNA from up to 200 mg or 2 g of food or seed GMO * corn, soybeans, canola, ground pork, ground beef, chocolate, honey 2. Automated run time 40 minutes 3. Sufficient amplifiable DNA Findings were published in European Food Research Technology D0I 10.1007/s00217-013-1916-y Promega Corporation 13
PureFood Competitor A Competitor B PureFood Competitor A Competitor B PureFood Competitor A Competitor B PureFood Competitor A Competitor B PureFood Competitor A Competitor B PureFood Competitor A Competitor B Abs ratios Highly Pure DNA 2.5 A260/A280 A260/A230 2 1.5 1 0.5 0 Beef Pork Canola seed Corn Meal Soy Flour Crispy Rice Promega Corporation 14
Cycles Concentrated DNA 24.5 Canola: Endogenous Primers 24.0 23.5 23.0 22.5 22.0 21.5 PureFood Competitor 21.0 20.5 20.0 non-gm Canola sample type RT73 (GM) Promega Corporation 15
Robust Amplification, Minimal Inhibition Neat 1:10 1:100 Efficiency Sample Avg. N= 3 Std. Dev. Avg. N= 3 Std. Dev. Avg. N= 3 Std. Dev. Avg. N= 3 Std. Dev. Honey 21.5 0.2 24.6 0.0 28.0 0.4 103.6 1.3 Breaded fish sticks/fish primers 20.7 0.3 24.0 0.2 31.7 0.2 100.4 1.4 Expect a 3.3 Cq difference between dilutions when no inhibitors are present. Promega Corporation 16
Cycle Cycle Cycle Inhibition Testing Canola Eluate 1 Canola Slope ΔCq Eluate 1-3.404-0.05 Eluate 2-3.369-0.01 Eluate 3-3.341 0.01 34 33 32 31 30 29 28 27 26 y = -3.4041x + 41.128 R² = 0.9996 25 2.000 2.500 3.000 3.500 4.000 4.500 Log 34 33 32 31 30 29 28 27 26 Promega Corporation 17 Eluate 2 y = -3.3693x + 40.792 R² = 0.9992 25 2.000 2.500 3.000 3.500 4.000 4.500 Log 34 33 32 31 30 29 28 27 26 Eluate 3 y = -3.341x + 40.799 R² = 0.9976 25 2.000 2.500 3.000 3.500 4.000 4.500 Log
GMO Event Testing Emerging Methods for GMO Detection Promega Corporation
New Methods for GMO Detection qpcr dpcr NGS Figure: https://en.wikipedia.org/wiki/genomics Promega Corporation 19 Figure: http://biorad.com
Digital PCR (dpcr): Precise Quantification of Nucleic Acids Figure: http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/10/21/digital-pcr-(dpcr)-what-is-itand-why-use-it- Promega Corporation 20
Raw Copy Number Data from Certified Reference Material Using dpcr Promega Corporation 21
DNA Quantification of Certified Reference Material Using dpcr Promega Corporation 22 Copies per reaction log copies Mean Cq Standard deviation 5,000 3.7 24.44 0.1 1,000 3 26.66 0.21 500 2.7 27.82 0.06 100 2 30.32 0.09 50 1.7 31.34 0.21 20 1.3 32.57 0.06
Quantification of Unknown GM Content in Maize Samples Promega Corporation 23
Conclusions GMO is an evolving field development of new crops and new test methods DNA quality is important to assay performance New Maxwell PureFood DNA extraction method provides highly pure, concentrated DNA that contributes to robust amplification New methods for GMO analysis provide additional options to labs Promega Corporation 24
Thank You! Questions? Now by chat or later by email Promega Technical Services techserv@promega.com Promega Corporation 25