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1 TONY FERNANDEZ Downloaded via on July 3, 2018 at 19:38:55 (UTC). See for options on how to legitimately share published articles A A N A LY T I C A L C H E M I S T R Y / J U LY 1,

2 As world trade issues surrounding biotech foods heat up, there is an increasing need to distinguish crops containing genetically modified organisms (GMOs) from non-gmo crops. Worldwide, the major crops affected include corn (maize), soybeans, canola, and cotton. In some countries, specific GMO food-labeling requirements, but also in those wanting to export food products into countries with restrictions. There isn t just one method for determining GMO levels in food, however, and variability among test results is high. The two most common approaches to GMO detection are polymerase chain reaction (PCR)-based methods, which Variability in methods creates a strong need for international standardization of GMO testing. GMOs, or events, are unapproved for food use. In such cases, even trace amounts of a particular GMO are unacceptable in products intended for human consumption. But it s no longer just a question of whether a food contains a specific GMO. Several countries, including Japan and members of the European Union (EU), have food-labeling laws that incorporate threshold levels. Foods containing soy or maize ingredients with GMO material above the threshold level must be labeled genetically modified. To comply with these food-labeling requirements, accurate methods for determining the amount of genetically modified material in food are needed. In the EU, foods containing soy or maize ingredients with >1% GMO content must be labeled genetically modified. Japan has set the level at 5% for soybeans, but it has not set a level for corn because of the potential for crosspollination. Australia and South Korea have also indicated that they will include threshold levels in their GMO foodlabeling laws. In the United States, there is no mandatory requirement for labeling foods that contain GMOs. Despite pressure from consumer advocacy groups and Congress, the U.S. Food and Drug Administration (FDA) maintains that genetically modified foods are substantially equivalent to conventional foods and therefore do not require labeling, unless the nutritional content has been altered or the product contains known allergens. The need to verify GMO levels in food has created a new demand for analytical testing, not only in countries with detect genetically modified DNA sequences, and immunoassays, which measure levels of proteins expressed by transgenic genes. Laboratories around the world are developing new methods for detecting GMOs in food, but there is no agreement regarding the validity of tests. Currently, there are no internationally recognized methods or standards for quantifying GMO levels in food. It s another case of science trying to catch up with policy. Standards and measurement agencies around the globe, however, are working to change that. Toward standardization To support European legislation, the Institute of Health and Consumer Protection (IHCP), which is part of the European Commission s Joint Research Centre (JRC), in Ispra, Italy, has been heavily involved in validating methods for GMO detection through its Food Products Unit. One of its first efforts in this area was to establish a collaborative study for the qualitative detection of genetically modified soybeans and maize. The study involved 41 laboratories, 29 of which actually submitted results, from EU member states, Switzerland, and the United States. Participating laboratories were instructed to follow standard PCR protocols but were allowed to choose any DNA-extraction and PCR procedures that were compatible with the given protocols. The participants, however, were not allowed to alter the primers (35S promoter and NOS terminator) that determine the DNA target sequence. Results from this ring J U LY 1, / A N A LY T I C A L C H E M I S T R Y A

3 study, which was completed in June 1998, showed that GMOs can be consistently detected in soybean and corn flour, provided the sample contains at least 2% genetically modified material (J. AOAC Int. 1999, 82, ). In addition to establishing a validation program for PCR-based methods, IHCP also organized a collaborative study in which 38 laboratories performed qualitative GMO tests using a commercially available diagnostic kit manufactured by Strategic Diagnostics, Inc., (SDI). The test kit uses an enzyme-linked immunosorbent assay (ELISA) to detect the protein in Roundup Ready soybeans that makes the plants resistant to the herbicide Roundup. All but one laboratory submitted results, suggesting the kit is relatively easy to use, and, according to IHCP, the results were promising. For samples containing <2% GMO, negative scores were 99% accurate and positive scores 94%. Although results are generally good for detecting the presence of GMOs on a yes/no basis, quantifying the amount of genetically modified material in food ingredients is another story. Based on results from collaborative studies, the general opinion among the experts seems to be, With regards to quantification, it doesn t look that good. Part of the problem is the lack of internationally recognized GMO standard reference materials. The Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium, which is also part of JRC, has certified three series of GMO reference materials one for Roundup Ready in soybean powder, and two for different varieties of Bacillus thuringiensis (Bt) in corn powder. Bt-corn is corn that has been genetically altered to contain genes of Bt, a bacteria that produces a protein that is toxic to some insects, including the European corn borer. Each series of standards contains varying GMO mass fractions. The three GMO-certified reference materials (CRMs) have been used in various GMO methodvalidation programs, including those conducted by JRC. According to Jean Pauwels, head of IRMM s Reference Materials Unit, the degradation of DNA in the CRMs is partly to blame for the variability in results. For this reason, we are developing third-generation CRMs for Roundup Ready soy and Bt-11, Bt-176, and MON-810 in maize with less degradation using dry mixing techniques, says Pauwels. The new CRMs are expected to be available by the end of The U.S. National Institute of Standards and Technology (NIST) has also put standard reference materials for GMOs high on its list of priorities for the next fiscal year, says Willie May, chief of NIST s Analytical Chemistry Division. Although specifics of the project have not yet been established, NIST says it will not duplicate ongoing efforts by other national standards laboratories, such as IRMM. First, we need to see if the IRMM standards meet U.S. needs. In cases where there are already standards that could serve the purpose, we would confirm the accuracy of the certified values and then direct customers to them, says May. Meanwhile, the U.S. Department of Agriculture (USDA), through its Grain Inspection, Packers, and Stockyards Administration (GIPSA), is taking the task of standardizing methods for the detection of genetically modified grains into its own hands. According to Steve Tanner, director of GIPSA s Technical Services Division, GIPSA is planning to open a biotech reference laboratory in Kansas City, MO, later this summer to assist in the standardization efforts. Once the laboratory is set up, GIPSA is going to start a program for accrediting private, independent PHOTODISC A A N A LY T I C A L C H E M I S T R Y / J U LY 1,

4 PHOTODISC laboratories that use PCR testing to detect GMOs in grains, says Tanner. Validating a method is one thing. Accrediting a laboratory based on a set of challenge samples, a statistical analysis, and whether they have the proper expertise and quality control, is another. The technology is changing so fast. We want labs to do the best they can with detecting specific events and quantifying the total amount of genetically modified material present in grain samples. Because the economic stakes are high with regard to international trade, the grain market needs GMO testing now and cannot afford to wait for national standards agencies, such as NIST, to develop reference materials. We have initiated discussions with NIST, and our long-term goal is to work with them to help characterize and standardize reference materials, says Tanner. However, in the beginning, We are going to take executive leadership and establish standards based on samples that are certified by the life sciences companies who actually developed the seeds, he says. Eventually, we want to bring in the Joint Research Centre. I see them as a big player longer term. Another area GIPSA plans to get involved in is sampling. One of the major problems associated with all kinds of analytical testing is the type of sample you get, says Tanner. We are going to publish recommended methods for sampling grain, including what size sample is needed to quantify with some degree of confidence the level of genetically modified material. The sampling methods will be posted on GIPSA s Web site ( In addition, GIPSA is planning to evaluate commercially available antibody-based test kits for detecting GMOs to determine if the kits meet manufacturer s performance claims, says Tanner. Other organizations are also looking ahead to the future and beginning to think about the accreditation of laboratories involved in GMO testing of food products. Traditionally, AOAC International has played a large role in developing proficiency-testing standards for food laboratories. According to Arlene Fox of AOAC, an advisory task force has been developed to explore proficiency-testing options down the road, once GMO-detection methods are ready. Laboratory alliances Because of the lack of international standards for GMO detection, some laboratories have started establishing alliances. We realized early on that there is no uniform industry-wide standard for GMO testing, says John Fagan, founder of Genetic ID (Fairfield, IA), the world s first laboratory to provide GMO foodtesting services. This was a great challenge for people in the agriculture export industry. So we began to establish alliances with other laboratories, either in terms of licensing our technology to those labs or establishing joint ventures with labs in other areas around the world. According to Fagan, Genetic ID is in the process of expanding its Global Laboratory Alliance, which currently includes laboratories in Europe, Australia, Japan, Hong Kong, and Taiwan. All the laboratories in the alliance use the same SOPs [standard operating procedures], which we originated in our laboratory here in the U.S. We develop the methods here and keep them up-to-date with the needs of the market. If a new product needs to be tested, we ll develop DNA-extraction methods and make them available to the laboratories in the alliance, he explains. We also provide a quality assurance program that incorporates all of the alliance laboratories, adds Fagan. The laboratories select a few samples each month and send us subsamples of them. We retest them here and compare our results with theirs. If there is lack of agreement, it is a red flag, he says. The laboratories also participate in round-robin tests. We submit samples of known composition to them; they test them and report the results back to us. In addition, each laboratory in the alliance has its own internal quality assurance program. So there is both an internal quality check and an external alliance- J U LY 1, / A N A LY T I C A L C H E M I S T R Y A

5 wide assessment, which is overseen by Genetic ID. The key is to provide testing to the same standards of performance here in the U.S. and in every other market around the world, emphasizes Fagan. Manufacturers of immunoassay test kits for detecting genetically modified products in food have also started licensing out their technology to other parts of the world. For example, U.S.-based SDI has licensed its GMO Check test kit for Roundup Ready soybeans to the Japan Oilstuff Inspector s Corporation (JOSIC). According to Dwight Denham, SDI s global business unit director, the soybean GMO Check test kit has been evaluated and approved by the National Food Research Institute under Japan s Ministry of Agriculture, Forestry, and Fisheries. In addition to the GMO Check test kit for soybeans, SDI is planning to offer similar test kits for Bt-corn and Liberty Link (an herbicide from Aventis) corn. JOSIC is coming on line with our test kit for Bt-corn, and they want to add the capability for Liberty Link, says Denham. Demand for GMO detection With so many laboratories around the world developing methods for detecting GMOs in food, it should come as no surprise that the demand for GMO testing is quite large. The need for testing goes all the way back to the life sciences companies, who are the ones actually incorporating transgenic material into various seeds. The seed companies need to ensure that, in the end, the GMO trait did indeed show up in the actual seed crop, says SDI s Denham. That is how SDI got its start back in We developed products specifically for the life sciences and seed companies so they can do QA/QC testing of their final products, recalls Denham. It wasn t long, however, before the demand for non-gmo products became an issue. We took the same basic products and redeveloped them for testing low incidents of genetically modified traits in non-gmo products, rather than testing for highpurity GMO traits, he explains. Verification that non-gmo food products really don t contain GMOs will likely continue to drive the demand for GMO testing. A large number of companies around the world have decided to play it safe and not wait for GMO food regulations. Instead, they are eliminating GMO ingredients from their food products, just in case somewhere down the road a regulation banning a particular GMO is passed or a particular GMO is found to be unsafe. Gerber, a leading baby food manufacturer, was one of the first major U.S. companies to voluntarily pull ingredients containing GMOs from its products. Other companies have since followed suit. Frito Lay has told its growers not to supply genetically modified corn, and McDonalds and Procter & Gamble have chosen not to use Monsanto s new genetically modified potato in their products. The situation is even more pronounced in Europe, where the list of non-gmo food producers continues to grow. The U.S. organic market is also extremely interested in GMO testing because of pending organic certification regulations, says Denham. USDA s proposed national organic standards prohibit the use of genetic engineering in the production of all foods and ingredients that carry the organic label. Verification that organically certified foods do not contain GMOs is likely to create more demand for testing. One area, in particular, that is likely to have a dramatic effect on the demand for GMO testing is the growing market for food products that are nutritionally enhanced through genetic engineering. As new attributes that have consumer benefits start getting incorporated into crops, whether it be vaccines in bananas or heart-healthy grains, it is going to call for the need to differentiate crops that are being PHOTODISC A A N A LY T I C A L C H E M I S T R Y / J U LY 1,

6 PHOTODISC Current capabilities for detecting GMOs in food PCR and antibody-based immunoassays have complementary roles in the GMO food-testing market. Most would agree that if time is at a premium and testing has to be done on the spot, then immunoassays are a good way to go. However, when timing is not an issue, PCR is commonly used instead. There is a great deal of controversy surrounding both methods with respect to quantifying GMO levels in food. Although quantitative results have been reported, critics argue that neither method is capable of producing reproducible quantitative results for GMO food applications. The lack of internationally recognized GMO standards is the biggest reason for the variability in results. Nonetheless, they are the best methods currently available. Although there are several ways to approach quantitative PCR (Anal. Chem. 1999,71, 191 A 195 A), real-time or kinetic PCR, which uses hybridization or hydrolysis (Taqman) probes, is becoming the method of choice for determining levels of GMOs in food. Two platforms are widely used for real-time PCR PE Biosystem s 7700 sequencer and Roche s LightCycler although others are becoming available, says David Mc- Dowell of LGC in the United Kingdom. Real-time PCR uses fluorescence to monitor the PCR amplification process. Rather than providing a snapshot at a particular time during the reaction, data is collected in real time during the entire course of the reaction. Additional steps after amplification, such as gel electrophoresis, are not needed, and quantitative results are obtained without opening the reaction tube, thus lowering the risk of contamination. Real-time PCR, however, is quite an investment. Although they have come down in price, real-time PCR machines still cost $36,000 95,000 and require a highly trained technician. Most labs using PCR to detect GMOs in food say their average turn-around time is ~3 5 days. To comply with food-labeling regulations, methods capable of determining the percentage of genetically modified material are needed. PCR provides this information through the use of a reference target and a GMO-specific target. For example, to determine the amount of genetically modified soybeans with respect to the total amount of soybeans, a generic soybean target, which serves as a reference, and a GMO-specific target are used. The challenge is knowing which GMO-specific DNA sequences to target. In order for [GMO] testing to be effective, testing capabilities have to develop in line with new [GMO] targets, says McDowell. Many laboratories, however, are finding it difficult to keep up with the rate at which the life sciences companies are creating new GMOs. The so-called Swiss/German method, validated by the JRC ring study, is a generic test that targets the common 35S promoter and NOS terminator sequences. Although the method screens for most GMOs in the marketplace, it does not cover all of them, says Genetic ID s Fagan. The method is widely used, but it is usually augmented by other tests, he says. Whereas the Swiss/German method targets two primer sets, Genetic ID s Triple Check approach uses three primer sets for soybeans and four primer sets for corn to cover a broader range of GMOs, says Fagan, who claims that his methods can detect all the GMOs in the market. Not only can we detect them all, we can differentiate between them all, he emphasizes. So if a gene is approved in canola but not in corn, Genetic ID s Varietal tests will discern whether the gene is from corn or canola. According to Fagan, in the beginning, the life sciences companies were far from cooperative in sharing GMO target sequence information. In 1996, the only way we could get the information we needed was through FDA, he recalls. We took what we got from FDA and did some molecular biology detective work to develop primer sets, he says. Today, Genetic ID has an ongoing R&D program to develop primers or targets for new GMOs. The story is somewhat different, however, for U.S. immunoassay manufacturers, who have a history of strategic alliances with the life sciences companies. SDI started out in 1996 developing products specifically for several of the leading ag biotech companies to test their final seeds. Envirologix also works closely with the life sciences companies. It signed an agreement with Aventis in February to license the Liberty Link proteins and antibodies for use in test kits, says Envirologix s Layton. Both SDI and Envirologix offer antibodybased lateral flow test strips, which can answer yes/no in about 5 min as to whether a grain product contains a particular GMO. These disposable membrane devices, which sell for < $5 each, are similar to home pregnancy tests, in that they respond to the presence of a particular compound (in the case of GMOs, a particular protein) by producing a color change. Lateral flow test strips are particularly useful for screening grains, so that non-gmo grains remain segregated from those containing GMOs during the grain-handling process, from the fields to the grain elevators to the export terminals, says Layton. The downside is that each test strip responds to only one particular GMO event. SDI currently offers two lateral flow devices one for Bt and one for Monsanto s Roundup Ready, and it plans to introduce one for Aventis Liberty Link. Envirologix offers a test strip for Bt and is developing ones for Roundup Ready and Liberty Link. In addition to lateral flow devices, SDI and Envirologix offer their antibody-based tests in an ELISA 96-well plate format. ELISA tests are relatively easy to use but require a laboratory. A typical ELISA takes about 2 h/plate, at a cost of ~$125/plate or $3/sample (if samples are run in duplicate with controls), says Layton. ELISA test kits are optimized for detecting specific GMOs in food ingredients, such as flours and protein isolates, says SDI s Denham. Because they detect proteins, which are easily denatured during food processing, ELISA tests are not designed to detect GMOs in finished food products. PCR, on the other hand, can detect GMOs in the final food product, not just the ingredients, says Genetic ID s Fagan. Denham disagrees. Both protein and DNA are destroyed through processing, therefore it is best to test the ingredient. Furthermore, he adds, because labeling regulations are based upon the ingredient, it J U LY 1, / A N A LY T I C A L C H E M I S T R Y A