Testing GM crops. Mitesh Shrestha

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1 Testing GM crops Mitesh Shrestha

2 GMO food/feed testing is based on some fundamental principles of genetic engineering and cellular physiology: DNA: The introduction of foreign DNA into a recipient plant s DNA (genetic engineering) Protein: The information coded in DNA is translated into protein that performs the function specified by the DNA instructions (cellular physiology) Two methods have been developed based on these fundamentals: genetic analysis (DNA analysis) and Immunological analysis (Protein analysis). Three tests are currently available using these methods.

3 Detection technology of GMOs Real-time PCR DNA Microarray Captured PCR-ELISA Quicktest strip

4 Genetic Analysis (DNA analysis) Genetic analysis is a GMO testing method that detects the presence of a transgene in a plant cell s genome. The specific GMO test used in this method is called the polymerase chain reaction (PCR) test. Polymerase Chain Reaction (PCR Test): Copies a specific section of a plant s DNA billions of times in order to detect and quantitate foreign DNA (GMO) inserted into the plant s genome. Uses short pieces of DNA (primers) that are complementary to the GMO sequence to vastly amplify and quantitate GMOs. Performed in a laboratory setting. Appropriate for qualitative or quantitative testing. Highly sensitive and specific.

5 How to Detect a GMO Isolate DNA from plant tissue and food products. (PCR) is used to assay for evidence of the 35S promoter that drives expression of the glyphosate resistance gene and many other plant transgenes.

6 Real-time PCR Screening Kits Target genes: CaMV 35S Promotor, Nos teminator, NptII, Bar, FMV promotor, Pat Reference gene: 18S rrna Quantitative Kits Roundup Ready soybean Bt176 Maize Event-specific detection kits GTS40-3-2, Bt176, Mon810, Bt11, GA21, T25, RT73

7 35S PROMOTER INDICATES GMO Herbicide resistance correlates with an insertion allele the 35S promoter that is readily identified by electrophoresis on an agarose mini-gel. Amplification of tubulin, a protein found in all plants, provides evidence of amplifiable DNA in the preparation, while tissue from wild-type and Roundup Ready soy plants are positive controls for the 35S promoter.

8 Two PCR reactions are performed for each plant or food sample. One primer set amplifies the 35S promoter from cauliflower mosaic virus. The presence of a 35S product is diagnostic for the presence of a transgene. The 35S promoter is used to drive expression of the glyphosate (Roundup) resistance gene or Bt gene in edible crops. A second primer set amplifies a fragment of a tubulin gene and controls for the presence of plant template DNA. Since the tubulin gene is found in all plant genomes, the presence of a tubulin product indicates amplifiable DNA in the sample isolated. Tubulin is a housekeeping gene

9 Immuno-analysis (Protein analysis) Immunological analysis, or immuno-analysis for short, is a GMO test method that detects proteins. Currently, there are two types of GMO tests that use this method: the Strip Test and ELISA Method. Strip test (Lateral Flow Device or Dipstick): A rapid antibody-based method used for measuring GMO protein in unprocessed material such as seed, grain, or leaves Uses a detection surface comprised of immobilized GMO protein-specific antibodies on a solid strip Appropriate for qualitative or semi-quantitative testing Suitable for field testing ELISA Test (Enzyme-Linked Immunosorbent Assay) An antibody-based method for measuring GMO protein in unprocessed material such as seed, grain, or leaves Uses a detection surface comprised of immobilized GMO protein-specific antibodies in a multi-well solid plate format Appropriate for qualitative or quantitative testing Performed in a laboratory setting

10 Determining which type of GMO test is most appropriate depends on several factors, including but not limited to: Nature of the sample GMO(s) to be analyzed Required test sensitivity Whether qualitative or quantitative analysis is required Product s intended market