Advances in Genetically Modified Food and Ingredient Authentication Testing

Transcription

1 Advances in Genetically Modified Food and Ingredient Authentication Testing Hemanth Shenoi Global Commercialization Marketing Manager Applied Markets Promega Corporation

2 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

3 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

4 Labeling Requirements Traceability from production / importation / table Post market monitoring once authorized for sale Sharing of information on GMOs cultivated Promega Corporation 4

5 Interest in Compliance Across the Production Chain Promega Corporation 5 Figure:

6 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

7 GMO Event Testing Importance of DNA Extraction Promega Corporation

8 Current Trends in Global GMO Environment Rise in GMO complexity Proliferation of authorized GMOs Increased number of tests demanded Promega Corporation 8

9 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

10 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

11 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

12 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

13 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 /s y Promega Corporation 13

14 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/A Beef Pork Canola seed Corn Meal Soy Flour Crispy Rice Promega Corporation 14

15 Cycles Concentrated DNA 24.5 Canola: Endogenous Primers PureFood Competitor non-gm Canola sample type RT73 (GM) Promega Corporation 15

16 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 Breaded fish sticks/fish primers Expect a 3.3 Cq difference between dilutions when no inhibitors are present. Promega Corporation 16

17 Cycle Cycle Cycle Inhibition Testing Canola Eluate 1 Canola Slope ΔCq Eluate Eluate Eluate y = x R² = Log Promega Corporation 17 Eluate 2 y = x R² = Log Eluate 3 y = x R² = Log

18 GMO Event Testing Emerging Methods for GMO Detection Promega Corporation

19 New Methods for GMO Detection qpcr dpcr NGS Figure: Promega Corporation 19 Figure:

20 Digital PCR (dpcr): Precise Quantification of Nucleic Acids Figure: Promega Corporation 20

21 Raw Copy Number Data from Certified Reference Material Using dpcr Promega Corporation 21

22 DNA Quantification of Certified Reference Material Using dpcr Promega Corporation 22 Copies per reaction log copies Mean Cq Standard deviation 5, ,

23 Quantification of Unknown GM Content in Maize Samples Promega Corporation 23

24 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

25 Thank You! Questions? Now by chat or later by Promega Technical Services Promega Corporation 25