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Debate and Policies on Labeling GM Foods
If countries are to require labels on GM foods, one question that must come to mind is how genetic modification can be detected in food products. Prior to testing it is important to ensure three things:
1) That the sample is non homogeneous,
2) The sample size is sufficient enough to achieve statistical significance, and
3) There is reference material that is independent of the sample to serve as a control. Reference material should be raw material, not a finished food product.
Once these things are taken care of, testing can take place. There are two mains methods of testing, protein-based methods and DNA-based methods. Each has several subcategories (Ahmed, 2002).
These tests use antibodies in immunoassays to detect modified proteins in organisms up to a level of 1%. “Immunoassays with antibodies attached to a solid phase have been used in two formats: a competitive assay in which the detector and analyte compete to bind with capture antibodies, or a two site (double antibody sandwich) assay in which the analyte is sandwiched between the capture antibody and the detector antibody.” (Ahmed, 2002). Most scientists prefer the competitive assay. Factors such as quality and sensitivity of kits, threshold limits, work environment, extraction efficiency, and ability to distinguish between close values will affect optimization and validation of the tests. Some GM foods will not express enough protein to be detected, so while protein-based tests are effective, they may not always be useful (Ahmed, 2002).
A) Western Blot: This test is a specific way to determine whether the sample contains the GM protein above or below a preset threshold level. The protein is denatured with detergents and reducing agents, and then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein denaturation eliminates some main problems, including protein solubility and coprecipitation of the target protein with unwanted proteins. The gel is transferred to a nitrocellulose membrane and binding sites on the membrane are blocked. Probing with antibodies occurs, then the bound antibodies are stained or otherwise labeled so they can be detected. A western blot can detect limits of 0.25% for seeds and 1% for toasted meal, and the results are qualitative. This method is more suitable for research than for large scale testing (Ahmed, 2002)
B) ELISA: This method has two formats, a microwell format as well as a coated tube format. Both the wells and the tube are coated with antibodies. The well format is quantitative, economical, and very sensitive making it ideal for large quantities as long as the protein has not been denatured. An ELISA can detect limits of 0.25% for seeds and 1.4% for toasted meal. The tube format returns qualitative results, and no information about the level of GM proteins at the ingredient level can be obtained. ELISA is the preferred method for detecting genetic modification in raw materials, processed ingredients and semiprocessed foods. (Ahmed, 2002).
C) Lateral Flow Strip: This is a variation of ELISA that uses nitrocellulose strips. Antibodies specific for the expressed protein are coupled to a color reagent, and incorporated into the nitrocellulose strip. The strip is then placed in an eppendorf tube that contains a plant tissue with the transgenic protein. A colored antibody sandwich is formed. “This colored sandwich flows to the other end of the strip through a porous membrane that contains two captured zones, one specific for the transgenic protein sandwich and another specific for untreated antibodies coupled to the color reagent.” (Ahmed, 2002). A positive result contains two lines, while a negative result just has one. The lateral flow strip is quick, economical, and is a good method for GM screening “early in the food chain.” (Ahmed, 2002) Strips that are capable of detecting multiple proteins are currently being developed (Ahmed, 2002).
D) Other Immunoassay Formats: Some formats use magnetic particles as a solid support in place of a nitrocellulose membrane. The particles can be coated with antibodies and the reaction can take place in a test tube. A magnet separates bound antibodies from unbound ones. Because the particles are all the same, this method has an advantage of better precision. Techniques using biosensors are also being developed (Ahmed, 2002).
The DNA that has been introduced into a GM crop contains a promoter sequence, a structural gene sequence, and a stop sequence. DNA-based methods exploit this information when detecting genetic modification in foods. As with protein-based methods, some products will not contain enough DNA to be detected (Ahmed, 2002).
A) Southern Blot: Sample DNA is affixed onto a nitrocellulose or nylon membrane and is probed with double stranded nucleic acid probes that are GM specific. The probes are labeled as radiographic, florescent, or chemiluminscent. These probes are as sensitive as radioactive probes, so radioactivity is not employed anymore in many labs. PCR (discussed below) is considered more sensitive than the Southern blot since PCR amplifies the DNA and a single piece can be detected. The Southern blot only uses no amplification (Ahmed, 2002).
B) Qualitative PCR: This is the standard type of PCR. Two pairs of primers are utilized, one sense (5’à 3’) and one antisense (3’à 5’). Through a series of thermal steps, the primers hybridize opposite sides of the sequence of interest and amplify that sequence many times over. The millions of pieces can then be separated by size using gel electrophoresis or other separation methods. DNA extraction and purification are important to this process. The most widely used are the CTAB method (incubates food in the presence of cetyltrimethylammonium bromide) and the Wizard method (uses DNA-binding silica resins). Sometimes sequences also occur naturally in plants and thus PCR gives a false positive. If this happens, a set of primer pairs for adjacent sequences is used (like the promoter or terminator sequence). “Detection limits are in the range of 20 pg to 10 ng of target DNA and 0.0001-1% of the mass fraction of GMOs” (Ahmed, 2002). Studies have shown this method is very reliable (Ahmed, 2002).
C) Quantitative End-point PCR: If labels are required on the basis of particular levels of GM material, a quantitative detection method is necessary. If an internal DNA standard is amplified with the sample DNA it is possible to quantify the sample. Quantitative end-point PCR has four steps: 1) coamplification of sample DNA and the DNA standard; 2) separation of the products by gel electrophoresis; 3) densitometry; 4) estimation of sample and standard DNA levels by regression (Ahmed, 2002)
D) Quantitative Real-time PCR: PCR ought to proceed exponentially with each cycle, but limiting factors prevent this. In real-time PCR, the concentration of DNA is “proportional to PCR cycle number during the exponential phase of PCR. Therefore, if the number of cycles it takes for a sample to reach the same point in its exponential growth curve is known, its precise initial DNA (then GMO) content can be determined.” (Ahmed, 2002).
E) Exhaustive Limiting Dilution PCR method: The basis of this method is twofold: optimization of the PCR so amplification is all or nothing and one or more sequences yielding a positive result. PCR is performed with serial dilutions, and Poisson statistics are used to calculate the number of sample sequences. A disadvantage to this procedure is the fact that contamination can occur with each dilution.
This page was created by Nicole Hesson.
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