GMO Detection Methods

Transcription

1 GMO Detection Methods by George Ngundo, KEPHIS, Nairobi 22 March 2012

2 Gene construction for plant transformation 1. Selectable Marker 2. Transgene 3. Promoter 4. Terminator

3 New Traits 1. Insect Resistance 2. Herbicide Resistance 3. Virus Resistance 4. Longer Shelf life 5. Modified Oil 6. High Nutrition Value 7. Drought tolerance 8. Aesthetic value 9. Salt and ph tolerance 10.Industrial purposes 11.BioPharming Available now Corn Canola Carnation Cotton Papaya Potato Rice Soybean Squash Sugarbeet Tomato In development Apple Banana Barley Citrus Cassava Eucalyptus Grapes Olive Pineapple Poplar Rubber Sweet Potato Walnut Flowers

4 Frequently tested traits Herbicide tolerance Bt-derived insect resistance Virus resistance Fungal resistance Male sterility/fertility restoration Starch Biosynthesis alteration.

5 GMO Detection GMOs can be identified by detecting the inserted genetic material at several levels DNA level, the mrna transcribed from the newly introduced gene, The resulting protein, Metabolite or phenotype

6 Qualities Of A Good GMO Test Method It must detect all GMOs. It must provide quantitative information on how much GMO content is present. It must deliver informative results with a wide range of foods and agricultural products. It must deliver maximum reliability and reproducibility, and must avoid false positives and false negatives. It must be sensitive and reliable.

7 Analytical Methods For GMO Detection 1. Detection objective is to determine if a product contains GMO or not. The result is a positive/negative statement. 2. Identification purpose is to find out which GMO is present and if they are authorized or not. 3. Quantification determination of amount of the GMO and assess compliance with the threshold regulation

8 Analytical Methods, Cont PCR tests can be designed to detect any of the inserted genetic material: promoter, transgene, terminator or selectable marker gene. The exact design of any particular test depends on the objective. For a general detection of GMOs using primers that recognize commonly used promoters like Cauliflower Mosaic Virus (CaMV) 35S promoter or Agrobacterium tumefasciens nos promoter, or the terminator (nos).

9 Protein-Based GMO Detection This methods give a present / absent result I) Immunoassays a) ELISA b) Lateral Flow Strip II) Western Blot

10 ELISA, Lateral-flow device Protein based Easy to perform, cheap and reliable Test results within few minutes Best for raw agricultural commodities Identification of a specific event not possible

11 Lateral-flow device, Cont Lateral flow sticks can be used to detect GMOs in leaves, seeds and grains. One single step is enough for performing the assay. Antibodies specific to the foreign protein are coupled to a color reagent and incorporated into the lateral flow strip. Lateral flow techniques are qualitative or semiquantitative. By following appropriate sampling procedures, it is possible to obtain a 99% confidence level of less than 0.15% GMO for a given lot.

12 Lateral flow strips cont

13 DNA-Based GMO Detection PCR-Based Detection Qualitative PCR (tests for 35S promoter, nos, etc) This methods give a present / absent result Conventional PCR measures the products of the PCR reaction at a single point in the reaction profile. Relationship between DNA concentration and PCR signal is not linear The precision for quantification using conventional PCR is limited. Confirmatory Assays Restriction, Hybridization, sequencing

14 Qualitative PCR for GMO Detection -results

15 Real Time PCR This method is used to quantify the levels of GMO products Real-time PCR is a system based on the continuous monitoring of PCR products. This is done via fluorometric measurement of an internal probe during the reaction. This probe consists of a short DNA fragment, which contains a fluorophor and a quencher. Concentration of DNA in the real-time PCR reaction is proportional to PCR cycle number during the exponential phase of the PCR reaction.

16 Real Time PCR, Cont This method provides precise quantification of the GMO content of agricultural products. Each series of analyses includes the analysis of a full set of standards, giving rise to a standard curve. The results obtained for individual unknown samples are compared to the standard curve to determine the GMO content of those unknowns. Most real-time systems are automated

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18 Real Time PCR - GMOs Detection Thresholds

19 D) Other Methods of GMO Detection I) Chromatography II) Near Infrared (NIR) Spectroscopy III) Micro fabricated Devices and Microchips New technologies resulting from the merger of chipbased Microsystems such as micro arrays and micro fluidic systems appear to be a promising area for GMO analysis application

20 Other Methods of GMO Detection, Cont E.g. the DualChip GMO Microarray Kit The chip can detect upto 30 GMO target sequences (increased from 14 in the earlier version). The DualChip GMO Microarray technology was successfully validated in 2007 in a ring-trial study coordinated by the Joint Research Centre (JRC) of the European Union (

21 THANK YOU