Quantitative tests for GMOs rely on molecular factors such as how many sets of chromosomes a crop has (ploidy level) and the number of times the foreign gene is present in the crop’s genome. The ratio of the two is what determines the percentage GM content. This project aims to find out more about these characteristics to enable the development of testing protocols. It also aims to sharpen accuracy, lower the detection limit, and take inventory of materials and equipment to see if there is room for improvement.
To cut back on inaccuracy in PCR tests, researchers are looking into automating certain steps of the process. The project also aims to improve the accuracy of quantitative PCR by pinpointing steps that could introduce uncertainty.
PCR tests for GM content in highly processed samples such as oils typically cannot detect the presence of GMOs below 0.9 percent. The task finds ways to lower the minimum detection limit of PCR tests to be able to conduct tests on highly processed samples.
Finally, this task takes stock of the various reaction solutions and equipment available on the market today. Different types of equipment in combination with different fluorescence based detection chemistries are evaluated in order to be able to make recommendations to maximise accuracy, sensitivity, and cost-effectiveness.
To improve quantification performance, the SIMQUANT approach was developed. The idea is to perform a series of PCR reactions and quantify the target numbers in the sample using the distribution of positive/negative results and most-probable-number statistics. One of the advantages of SIMQUANT is also less sensitivity of qualitative PCR to inhibitors in reactions when compared to quantitative PCR. The SIMQUANT was additionally upgraded to multiplex a version, thus increasing its applicability.
The quality of extracted DNA is known to influence significantly the final result of GM detection and quantification. One approach to control this step is “matrix by matrix” validation of the DNA extraction method. For validation, the quality of DNA solutions should be controlled by testing the presence or not of statistically significant inhibitory effects. This is usually done by adding an exogenous DNA (other taxon genomic DNA than that tested or plasmid, provided they are inhibitor free) containing a specific PCR target into the DNA solutions under study at a concentration close to the limit of quantification or limit of sensitivity (for example, 50-100 copies) and then by amplifying the specific PCR target contained by the exogenous DNA. The most convenient and cheapest way is to use the DNA of other taxon as exogenous DNA. The problem in most routine detection labs is that the matrix is not well defined. Composition of feed and food samples can vary from supplier to supplier and even from batch to batch, making ‘matrix by matrix’ DNA extraction validation not feasible. Modular validation can then be performed, providing appropriate controls of PCR inhibition are applied with every sample.
Similarly, the comparison of different available chemistries was organized to test those most widely used (MGB®, SYBR® Green and Molecular Beacons) in different laboratories and targeting different genes, while the more recently introduced were tested less extensively (Plexor, LNA, lux). The conclusion was that TaqMan®, MGB®, LNA are equal in performance characteristics and they can be used whenever they are better suited for the particular application, e.g. if there is special needs regarding specificity or target regions are problematic for design of longer TaqMan® probes. Molecular Beacons systems were more difficult to design to achieve a robust assay. SYBR® Green chemistry performed well, its drawback being slightly lower sensitivity when compared to probe based assays. The other primer only based system that performed well were Ampliflour and Plexor, while some of the more exotic systems performed significantly below specifications given by the manufacturer.
The costs of analytical procedures in food production chains are significant, therefore, what is needed are more cost-effective, while still reliable, methodologies for GMO detection. One potential solution is the use of a 384 well PCR plate instead of a 96 well format and downsizing total reaction volumes correspondingly. Additional improvement is the use of pipetting robot (automated liquid handling stations). But there are tradeoffs; although this larger through put format may cut down on necessary hands-on work in the lab, certain method performance problems may arise. For this reason, validation and verification of potential new method setups have been performed in order to prove reproducibility of the results and the robustness of the method.
Reliability and costs of GMO detection
Benefits and limits of real‐time PCR setup automation
Different real-time PCR chemistries: suitability for detection and quantification
Public Deliverables of the Co-Extra project
|NAME / ORGANISATION||CONTACT INFORMATION|
GeneScan Analytics GmbH, Germany
Consejo Superior de Investigaciones Cientifíficas (CSIS-IRTA), Spain
Central Science Laboratory Defra (CSL Defra), United Kingdom
Institut National de Recherche Agronomique (INRA), France
National Institute of Biology (NIB), Slovenia
National Veterinary Institute (NVI), Norway
Institute for Agricultural and Fisheries Research (ILVO), Belgium
National Institute of Agricultural Botany (NIAB), United Kingdom
Lumora Ltd., United Kingdom
Joint Research Centre (JRC), Italy
Biolytix AG, Switzerland
Centre Wallon de Recherches Agronomiques (CRA-W), Belgium