Detecting the presence of genetically modified food may be more challenging than legislators expect. KELLYN
S.
BETTS
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espite formidable opposition from the biotechnology industry, the number of countries mandating testing for genetically modified food is growing. But, as the trendsetting Europeans' experience of grappling with implementing such legislation is making clear, devising tests that will reproducibly detect genetically modified organisms (GMOs) can be even more of a challenge. Although European food manufacturers are now required to alert consumers if their wares contain more than 1% GMOs (by weight), many foodstuffs still lack labels because some key testing kinks have yet to be ironed out. Scientists from the European laboratories charged with the challenge of developing the testing methodologies needed to support the standard say the task is daunting. "These tests are very expensive and timeintensive, and we can't find solutions for all cases," laments Hermann Broil, a biologist with the Novel Foods and Genetic Engineering Unit of the Federal
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© 2000 American Chemical Society
Testing Hurdles
Institute for Health Protection of Consumers and Veterinary Medicine (BGW) in Berlin, Germany. The BGW was the first European governmental laboratory to develop methodologies for detecting GMOs in food, beginning work before the first GMObased product was introduced in Europe in 1997. But the quest became significantly harder after legislators decided that instead of merely signaling the presence or absence of modified DNA, tests should determine whether there was more than 1% of a GMO product in a foodstuff. Broil estimates that each test costs $150 in Germany to qualitatively indicate whether GMO material is present or absent in a sample; quantitative tests cost $250—66% more. But cost is just the tip of the iceberg in terms of implementation hurdles. Unfulfilled requirements "We can't fulfill all the requirements of the European Union's (EU's) labeling law," Broil says. The fi-
nal report resulting from the BGVV's efforts to develop GMO testing methodologies, in conjunction with more than 30 other universities, institutes, and laboratories, had yet to be approved in September, eight months after it was completed. Europe's testing problems are likely to be mirrored by other nations as the trend toward mandatory labeling grows. Australia, Japan, New Zealand, Norway, Poland, South Korea, and Switzerland have now joined the 15 EU nations in mandating labeling, according to Craig Winters, executive director of the U.S.-based Campaign to Label Genetically Engineered Foods. Winters' organization maintains a Web site that tracks the worldwide movement toward labeling (www.thecampaign.org); it documents that there is at least a grassroots movement calling for labeling in 15 more countries on five continents. Several of these countries, including Brazil, India, and the United States, have introduced labeling legislation, Winters says. NOVEMBER 1, 2000 / ENVIRONMENTAL SCIENCE AND TECHNOLOGY / NEWS • 473 A
Here today, gone tomorrow. Tomato paste containing GMO tomatoes is no longer available because the EU has not approved it for official sale. When GMO tomato paste was on the market, however, some retailers, like England's Sainsbury supermarket chain, found that a majority of shoppers bought it because they got more for their money.
Broil is also involved in an effort spearheaded by the World Health Organization to develop an international labeling standard, although that group's efforts have been stymied by a failure to agree on what kinds of GMO-derived foods should require labeling. "My feeling is that [the WHO] effort is not going to define the terrain," says John Fagan, founder of Genetic ID, Inc., a U.S.-based company that offers GMO laboratory testing services through labs in Australia, Hong Kong, and Taiwan. The company is also gearing up to announce alliances with two European laboratories and one South American lab, Fagan says. He predicts that GMO labeling will continue to be defined by the efforts of governments such as the EU Japan and Australia In general, countries are following the European lead in defining a specific threshold—usually 1%, although the Australians have agreed on 0.1%—for GMO content, Winters says. He predicts that this trend may portend additional trouble if the nations that have planted the largest acreage in GMOs— Argentina, Canada, China, and the United States, all of which have at least some movement toward GMO labeling—adopt the same kind of legislation. Testing is showing that wind-borne pollen contamina4 7 4 A • NOVEMBER 1, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
tion of non-GMO organic crops in those countries can result in a threshold above 1%, he says. The brouhaha in the United States in September over the presence of an unauthorized variety of GMO corn in tacos can only complicate the situation. All of the methods BGW is perfecting for use by the EU's food industry are based on polymerase chain reactions (PCRs), specifically the technique that employs heat-stable polymerase, the most popular of which is derived from the Thermus aquaiicus bacterium, native to the hot springs of Yellowstone, WY. Devised in 1983, PCR analyses entail splitting DNA genetic material into two component strands, locating a target section, then adding base pairs of the DNA component building blocks of adenine, thymine, cytosine, and guanine to create multiple copies potentially billions of the targeted DNA.
Familiarity breeds repeatability Although this technique can produce good results when followed by scientists who use it regularly, it is far more difficult to obtain accurate, repeatable results when the testers are less familiar with trie equipment, according to the German scientists (see Figure 1). The process cannot begin until the DI\A
required for PCR testing has been extracted, which takes about four hours, according to Broil. The consortium evaluated more than a dozen methods and kits and found that all produced acceptable DNA for use with PCR, he says. Even so, DNA extraction techniques can be the source of spuriously high test results, according to Fagan. If the DNA is not extracted properly, it may contain some of the plant's inhibitor molecules, which provide important functions like repelling insects and resisting freezing, and can interfere with the PCR process. "Some spices like cayenne are deadly to the PCR process," he adds. Conducting the PCR tests themselves was even trickier. To date, the German scientists have found that the laboratories taking part in their ring trials of different GMO testing modalities can obtain reasonably repeatable results—with about a 20% deviation—in "real time" by using commercial DNA sequencers, which can cost upward of $100,000. A less expensive option called "dual competitive PCR" provided less optimal results for the European government testers, even though it is based on an older and well-known technology, DNA gel electrophoresis. The semi-quantitative method requires scientists to complete two sets of tests, including one with a known amount of GMO against which to compare the unknown in the sample, and it takes two-and-a-half days to complete, Broil says. But the scientists developing the EU's testing methodologies have yet to find a way to overcome its main disadvantage of requiring subjective visual inspection of the results. "We have some ideas about how to resolve the problem, but it's too early to talk about them " Broil says. To guarantee a positive identification, PCR test results should be confirmed by at least three primer sets, or individual segments, that identify the genetic material that has been introduced into the ag-
FIGURE 1
PCR ring-test results for Bt-176 Maize In trying to develop testing methodologies, European scientists have learned that lack of familiarity with the PCR method of analyzing DNA causes laboratories to generate inconsistent results (19 participants in an interlaboratory trial).
A semi-quantitative method of detecting GMOs in food involves amplification, PCR, and gel electrophoresis to compare the amount of GMO DNA in an unknown sample with the amount in a series of known standards. (Courtesy of Genetic ID, Inc..
ricultural product, stresses John Beeby, Genetic ID's laboratory manager. The technique devised by the BGW uses only two primers, Broil says. "We feel that the triple check is a better method," Fagan says. "The redundancy of information increases the certainty of that information."
Amplified aerosols Nonetheless, "the challenge isn't the developing of the methods," argues Jutta Zagon, a biologist with the BGWs Novel Foods and Genetic Engineering Unit who works closely with Broil. The real challenge is to see how well the methods can be used in different labs, Broil adds. Details like the tendency of amplified DNA to form an aerosol mat could generate false positives in other testing samples are just one of the many hurdles that labs must successfully navigate, he says. The European group has tested commonly available genetically modified products like corn, soybeans, and tomatoes, as well as transgenic salmon. Although the salmon, which have been engineered by Canadian researchers to grow three times faster than normal, have yet to be introduced into any market, the scientists tested them to gain experience in evaluating an animal product.
Randomly constructed GMOs
Source: BGW.
For each genetically modified product, scientists must determine how many copies they should expect to find of the primer or primers being used to signify the presence of the GMO, Beeby points out. "When you're constructing GMOs, there is a lot of randomness that happens," he explains. "It may be that the genetic modification was incorporated into the organism many times, or even that a fragment of the modification can be found through your detection method," he says. It is not uncommon for four copies of a genetically modified sequence to be found in a genetically modified product, he says. NOVEMBER 1, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS » 4 7 5 A
Real-time PCR technology allows GMO content to be quantified precisely. The DNA samples will be amplified and quantified after they enter this thermocycler.
A more basic problem that the scientists face is obtaining samples of material containing GMOs for use as standards in developing tests. "We're dependent upon the good will of the companies," Zagon explains. Only samples of Monsanto's Bt corn and RoundUp Ready soybeans are commercially available at this time, she says. In other cases, "we must beg to have materials sent to us," she says, although the situation has "relaxed somewhat" in recent months. But Broil points out that the service labs that will probably perform most of the testing are likely to have an even more difficult time obtaining the standards they need. Beeby of Genetic ID says that his company circumvents this obstacle by relying upon samples of known GMO products sent by farmers, but he recognizes that many labs may not have this option. The testing challenge is further complicated by the tendency of some food processing steps to degrade or completely denature the DNA. "It's completely different to extract DNA from processed, as opposed to raw food," Zagon says. The process of refining canola or soy to create a vegetable oil can completely remove the DNA, Broil explains; other processes like the hydrolyzing required to create starch from corn may leave traces of the original introduced DNA. Although the EU's regulation requires a "negative list" of such food processing operations, it has not yet been created. Broil suspects that the regulation will eventually be amended so that the trigger for labeling will not be the detection of a protein or DNA, but the process (such as hydrolyzing) itself, or perhaps the use of the GMO in the raw material. But no amendments are likely to aid the businesses that bear the cost of testing to ensure that their products meet the new law's require4 7 6 A • NOVEMBER 1, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
ments. The law places a "big burden on small- and medium-sized enterprises," Zagon says. lust how much testing these organizations have to pay for is dependent upon a variety of factors, Beeby says. It is determined by where the raw ingredients come from, the serving size of a processed foodstuff, and how the product is shipped, among others. There are no specific requirements for how often testing must be conducted, he says, noting mat the current amount of enforcement in Europe is questionable. However, just as it costs more for the government laboratories that must verify test results to purchase real-time PCR equipment, it costs companies more to obtain the quick results obtainable through them. Genetic ID charges $50 more for realtime results, for example. Yet another potential stumbling block the Europeans are dealing with is how to proceed when a processed food has more than one GMO-containing ingredient, for the law states that food must be labeled if it contains more than 1% of GMOs from any and all sources. This can make testing especially challenging, Zagon explains. To help other laboratories that must implement the testing required to support labeling, the BGW scientists are developing an extensive database documenting key details like the sequence of the introduced DNA and where the GM crops based on that DNA are approved for use. Zagon, Broil, and Beeby are watching developments in DNA chip technology. Companies like GeneScan of Freiburg, Germany, are developing new techniques for GMO testing to meet the needs of what they expect will be a rapidly growing market. "The chip method will be an advance over PCR because it's faster," Beeby says. But Genetic ID is pinning its hopes on another technology that Fagan believes will have superior sensitivity to methods based on DNA chips. The company is developing a technology that is based on hybridization but does not require an amplification step to recognize GMO DNA. It will be able to generate results in under 90 seconds, and those results will be more quantitative than real-time PCR, Fagan says. The German scientists are also looking at proteinbased methods because they are relatively fast and inexpensive, but Zagon stresses that DNA methods are much more powerful because proteins can be so easily denatured. "With PCR, you can synthesize primers for whatever [DNA] you want—you aren't dependent upon expensive antibodies," she says. Perhaps surprisingly, neither Zagon nor Broil say that they believe the benefits of testing for GMOs outweigh the costs. Both are convinced by the extensive laboratory tests conducted by the companies developing GMO-based products that the foods are indeed safe for consumers. "The focus is on health protection; ironically, there is no health concern," Zagon says. But they acknowledge that many consumers around the world appear to see things quite differently. Kellyn S. Betts is an associate editor o/ES&T.
