While the Starlink corn problem has been a bane for the U.S. grain and food industries, it has been a boon for another industry: GMO testing. Over a dozen U.S. laboratories now offer GMO testing services, with new ones seeming to emerge each month. In addition, several companies manufacture and sell GMO test kits, including ones to detect the Starlink protein, CRY9C.
The three most common methods used to detect GMOs are herbicide bioassay; enzyme linked immunosorbant assay (ELISA), which includes the lateral flow strip tests; and polymerase chain reaction (PCR). Each method has advantages and disadvantages, and each is appropriate for specific applications.
Herbicide bioassay: simplest method
The herbicide bioassay is used to detect genetically modified herbicide resistant traits in Round Up Ready™ and Liberty Link™ soybeans, canola, and corn hybrids. The test, also known as the "sprout-and-spray" test, involves placing seeds in a germination media moistened with a diluted solution containing an herbicide.
The herbicide may also be sprayed on the seedlings. Seeds that sprout with normal roots will be resistant to the herbicide, and thus are GM beans; those that do not develop normal roots or die will be non-GMO. It's that simple.
The test is also inexpensive (U.S.$20 to $30), user friendly, and produces clear-cut results. "Most people can tell the difference," said Tim Gutormson, president, Midwest Seed Services, a seed testing laboratory in Brookings, SD.
On the downside, the test takes up to a week to complete, seeds need to germinate for it to work, and it is limited to herbicide resistant grains only. In addition, the most commonly used sample size, usually a few hundred seeds, may not be large enough to produce results that are statistically significant.
Gutormson said seed and grain companies, particularly those that export, use the test extensively on soybeans. "We receive more samples from exporters than anyone else," he said. Seed companies use the test to check for contamination.
ELISA: fast and inexpensive
Enzyme linked immunosorbant assay (ELISA) methods use antibodies to detect specific proteins produced by genetically modified DNA "events" in soybeans, corn, canola, and cotton.
The test uses a plastic plate containing 96 microwells. Antibodies are coated to the insides of the microwells. A sample is ground and the protein is extracted and added to the microwells.
If the targeted protein is present, it binds to the inside of the wells. The protein is then "sandwiched" by another antibody that has an enzyme attached to it.
A color substrate is added that reacts to the enzyme, creating a color change. The intensity of color indicates the amount of the protein present. The test can take two to four hours.
Another version of the test uses lateral flow strips and delivers results in two to five minutes. The strip tests are commonly used at grain elevators where a fast, "yes or no" answer is needed.
ELISA tests are specific to each GM event. Kit manufacturers Envirologix and Strategic Diagnostics sell both plate and strip tests for a range of genetic events, including Starlink CRY9C.
The main advantages to ELISA tests are speed, user-friendliness, and low cost. Limits of detection vary among the tests, according to Dean Layton, vice- president of marketing at Envirologix, Portland, Maine, U.S. The company's CRY9C plate test can detect the GM protein down to .01%, while the strip tests can detect at .25%.
ELISA tests work best on raw grains, but are limited in detecting proteins in processed foods because heat processes will denature the proteins, making detection difficult.
Some people in the grain industry have questioned the accuracy of strip tests, saying they can produce too many false negative test results. However, Dwight Denham, global business unit director at Strategic Diagnostics, Newark, Delaware, U.S., challenges that view.
"Based on our internal validations, there is less than a 1% chance of false negative and false positive results (using the strip tests)," he said.
PCR: sensitive and precise quantification
PCR is considered the most sensitive and precise GMO test method because it allows direct analysis of the DNA. The method has become the most established testing method worldwide to meet government and industry requirements.
In a PCR analysis, a sample is ground to a powder, and the DNA is purified and extracted. PCR uses biochemical processes to scan through the DNA and locate genetically modified DNA sequences. Short pieces of DNA called primer sets identify the beginning and end of the targeted sequence. When a sequence is located, the PCR equipment multiplies the targeted gene billions of times until it can be measured precisely.
PCR has the ability to detect the presence of GM gene sequences at very low levels, even one or two molecules.
Real-time PCR is a second-generation technology that provides precise quantification over a wide range of GMO concentrations. Real-time uses fluorescent probes to detect and quantify GMOs and operates in a closed system to reduce the chance for cross contamination and human error.
PCR is the only method that can effectively detect GMOs in processed foods. PCR can quantify GM content in a sample to .01%, and provides greater flexibility than ELISA tests, which are specific for one transgenic trait. However, a PCR analysis requires more time, two to three days, and costs from U.S.$75 to over $300 per sample.
PCR analysis is also a delicate process with many factors influencing its reliability and sensitivity. Laboratories must have the expertise to carefully address all these factors in order to avoid false positive and false negative results.
"Only a few labs are able to do this type of testing," said Anthony Milici, a scientist with GeneThera, Inc., a GMO testing lab in Denver, Colorado, U.S.
STANDARDS NEEDED. A key factor influencing the reliability of PCR and the other methods is obtaining a proper sample for testing, according to Alex Kahler, president of Biogenetic Services, Inc., Brookings, South Dakota, U.S.
"Sampling is the key," he said. "There's a question of how much material needs to be tested to represent the larger lot from where it came."
If the sample is not statistically representative, even the most sophisticated analysis will not yield useful information. Some GMO testing experts recommend large sample sizes to ensure that results reported provide accurate information about the product.
For raw grains, John Fagan, chief scientific officer of Genetic ID, Fairfield, Iowa, U.S., recommends sample sizes of 10,000 soybeans or corn kernels — about 2.5kg. — to achieve statistically valid results. Kahler said there is a need for appropriate sampling methods, especially since the Starlink problem.
Sampling methods are just one of the issues that need to be addressed. Because the GMO issue is so new, there are no internationally standardized protocols or performance standards for GMO testing. As a result, testing labs can vary in their accuracy and reliability, creating risks for grain exporters.
Efforts are under way to remedy this problem. The U.S. Department of Agriculture's Grain Inspection Packers and Stockyard Administration (GIPSA) plans to open a biotechnology accreditation lab in Kansas City, Missouri, this January to help standardize test methods for GM grains. The GIPSA lab will review and accredit GMO testing labs that meet performance standards and evaluate test kits to ensure they are accurate and reliable.
"We want to standardize GMO testing to facilitate the marketing of grain so buyers and sellers feel more confident about their transactions," said Steve Tanner, GIPSA's director of technical services division.
NEW TECHNOLOGIES. What is the best testing method? It will vary according to the application. Fagan recommends that exporters to Europe and Japan use the most rigorous and accurate test before export because their products are likely to face one or more tests after export. Shipments that initially tested negative for GMOs but later tested positive have been rejected by buyers, creating costly problems for exporters.
When a fast "yes or no" answer is required, a lateral flow test may be sufficient. When precise quantification is needed, PCR is the best choice.
Some companies use the strip tests as an initial screen followed by PCR to verify the result. The Illinois Official Grain Inspection follows this practice.
"Grain buyers may not have complete confidence in strip tests, so they will request samples be sent to a lab for testing," said Dallas Stubblefield, an official with the agency.
GMO testing, particularly the lab methods, requires time, a commodity that grain handlers lack when grain arrives at their elevators. In a paper on GMO analysis, Charles Hurburgh, a professor in the Department of Agricultural Biosystems Engineering at Iowa State University states, "In reality there is no practical way to test inbound grain at a country elevator where time and skills are both limiting." He suggests pre-testing samples from producer bins or delaying analysis to a point when more time is available.
Industry experts recognize the limitations of the present methods. "There is no simple test for everything," said Denham of Strategic Diagnostics.
Fagan added, "We need to develop more versatile methods."
New technologies are on the horizon. Europe-based Genescan has developed a "biochip," which is a micro array with hundreds of microscopic wells, each containing GMO primer sets. PCR is used to amplify the DNA, and the material is put on the biochip. A fluorescent reader detects positive or negative events in the wells.
Hundreds of GMO events can be detected using the biochip. Central-Hanse Analytical Laboratory, Belle Chasse, Louisiana, U.S., will offer testing using the biochip technology. GeneThera is developing a real-time PCR method using robotics to streamline the testing process. Genetic ID is developing new proprietary methods that don't involve PCR or other kinds of amplification.
In the meantime, testing experts recommend that grain handlers select GMO testing labs carefully. "It is incumbent on the customer to thoroughly investigate the laboratory they are using, especially the lab's quality control," said Mike Russell, managing director of Central-Hanse Analytical Laboratory.
Fagan recommends that grain handlers ask labs certain questions: How long have they been performing GMO tests? Have they addressed the key steps that influence PCR reliability, such as sample size? Can they screen for all commercially available events? Is the lab accredited by a viable independent organization? Have they developed varietal screening for unapproved GM varieties?
"They should look for a method that has been proven over time," he said.