Detection of Genetically Modified Foods
Dr. Suresh Kaushik
IARI, New Delhi
Genetic modification have made improvements in many crop and helped to increase yields, even though genetically modified (GM) foods are often in news as many groups have raised loud protest against transgenic crops. As market restrictions for various transgenic crops, there is increasing interest among growers in determining the presence of genetically modified organisms (GMOs) in crops. GM technology has developed in response to the challenges of feeding an ever-growing global population to create GM crops that are to be pest or virus resistant. The potential risks, benefits and ethical concerns of the presence of GMOs in the food chains are still being debatable. GMO content in food is strictly regulated as consumers seeks to make informed choices about the food they eat. GM foods have not gained worldwide acceptance due to unmollified consumer suspicion resulting from earlier food and environmental concerns, transparent regulatory oversight and mistrust in government bureaucracies, all factors which fueled debate about the environmental and public health safely issues of introduced genes. Growers producing non-GMO grains for markets need to verify that there is no GMO contamination. The default standard for certification as GMO-free has been taken to be zero in many cases or maximum allowable levels in the range of 1 to 3 percent.
Sampling of GM products
Sampling is one of a crucial part of the multifaceted bulk of activities aimed at addressing and managing food issue. Both sample size and sampling procedure are important issues for testing GMOs in raw material and food ingredients. The overall objective of good sampling practice is providing representative sample to be analysed. The sampling plan should be performed in a manner that ensures a statistically representative sample. Sample size must be sufficient to allow adequate sensitivity. In fact, sample is the major source of error in the analysis of GMOs, so the aim of a good sampling plan is to minimize this error.
Reference Materials for GMO testing
Each GMO requires a specific reference material. Grains with altered DNA or expressed proteins have been used a reference material. Reference material should be independent of the analytical methods. Reference materials for positive or negative control provide the basis for the validation of analytical procedure. Both genomic and plasmid DNA can be used a reference material. The availability of reference material is limited due to concern over intellectual property rights (IPR) and costs.
Methods for GMO testing
The detection and identification of GMOs in seeds, grains and other materials are derived by legal requirement and marketing demands. In order to test the amount GMOs in a consistent and reproducible way, suitable analytical methods are required. Processed products such as foods derived from GM crops or raw material such as grains are identified by tests for the presence of introduced DNA or by detecting expressed novels proteins. Both qualitative and quantitative methods are available. Broadly, there are two categories as given below for screening, detecting and identification of GMOs in food articles and raw materials.
A) DNA-based methods
These methods have been developed and applied due to the stability of DNA molecules and their ubiquitous presence in all tissues. Following the appropriate sampling plan, the sample is ground and DNA is extracted from the test portion. The DNA engineering into a crop consists of several elements such as a promoter sequence, structural gene and a stop sequence for the gene. These methods have to cover the widest possible range of GMOs. They target common regulatory genetic elements used in transformation construct such as promoters or terminators from Agrobacterium tumefaciens, cauliflower mosaic virus (CaMV) 35S promoter or the nopalin synthase (NOS) terminator, or the kanamycin-resistance marker gene (nptII) and others. Specificity is given by methods targeting the inserted gene; and targeting the junction region between a common regulatory elements and the gene itself. DNA based method rely on the complementarily of two strands of DNA double helix that hybridize in a sequence specific manner. Several techniques are available but two are commonly used are Southern blot and polymerase chain reaction (PCR) analysis. The Southern blot method involves fixing isolated sample DNA onto nylon membrane or nitrocellulose, probing with double stranded labeled nucleic acid probes specific to the GMO and detecting hybridization radiographically, fluoremetrically or by chemilumiscence. PCR exploits the specifically of DNA polymerase to allow the selective amplification of specific DNA segments occurring at low frequency in a complex mixture of other DNA sequence. In this method, two pairs of primers are used, forward and reverse. These primers are designated to hybridized on opposite strands of the sequence of interest. Amplified segments between the primers are produced millions of times after a series of repetitive cycles. Amplified DNA are separated on a agarose gel electrophoresis according to size. For quantitative PCR analysis of GMOs in food, an internal DNA standard are coamplified with target DNA. In the quantitative competitive (QC)- PCR method, the presence of PCR inhibitors will be noticed immediately because the amplification of both internal standard and target DNA will be simultaneously affected. By this method as little as 0.1% GMO DNA can be detected. In a real time Q-PCR, production of PCR products should proceed exponentially but it reaches a plateau between 30 and 40 cycles because certain reaction components become limiting. The concentration of DNA in real -time PCR reaction is proportional to the PCR cycles number during exponential phase of PCR. If the number of cycle it takes for a sample to reach the same point in its exponential growth curve is known, its precise initial DNA i.e. the GMO content can be determined.
B) Protein - based methods
These methods have the potential to serve as cost-efficient, practical and rapid tools for screening and multi-target analysis. These methods are specially used for GMOs detection by GMOs producer countries for both qualitative and quantitative approaches. Immunoassay technologies with antibodies are ideal for qualitative and quantitative detection of many types of protein in complex matrices when the target analyte is known. Both monoclonal and polyclonal antibodies can be used depending on the amounts needed and the specificity of detection system. The detection limits of protein immunoassay can predict the presence of modified proteins in range of 1 percent GMOs. Both western blot and enzyme-linked immunosorbant assay (ELISA ) techniques have been used for the analyses of protein products. The western blot is a highly specific method that provides qualitative results suitable for determining whether a sample contains the target protein below or above a predetermined threshold level and is useful for the analysis of insoluble protein. ELISA has more than one format - a microwell plate or strip format and a coated tube format. The antibody-coated microwells, with removable strips of 8-12 wells, are quantitative, highly sensitive, and economical. They provide high throughput and ideal for quantitative high-volume lab analysis. The antibody-coated tube format is suitable for field-testing, with typical run times ranging from 20-30 minutes. A variation on ELISA using strips rather than microtitre wells, led to development of lateral flow strip technology. This gives results in 5-10 minutes, is economical and is suitable as an initial screening method.
The detection and the identification of genetically modified organisms (GMOs) in seeds, grains and other food items are driven by legal requirements and marketing demands. If no GMOs are detected with a validated PCR qualitative method, the product (s) would be evaluated for the presence of protein. If no protein is detected, the product is presumed not detectable. If the qualitative PCR shows a positive result, the product is considered as 'non-approved GMO', and a validated Q-PCR is used to detect the level of GMO. Mandatory labeling is required for those products where GMO-derived DNA or proteins are no longer detectable, e.g. as for highly refined oils. Traceability of GMOs has to be guaranteed in all products produced from GMOs in all stages of their placing on the market through the production and distribution chain.
About Author / Additional Info:
Dr. Suresh Kaushik
A biotechnology professional from IARI, India