The US agricultural system is a major exporter of raw materials such as grain and finished foods. A large part of the market consists of commodity crops. Many of these crops are genetically modified (GM) primarily with traits beneficial to their production. Many importing countries regulate which type of GM crops are allowed and often require labeling when the concentration of these materials in the food or grain shipment reaches a specified level. Thus testing is used both prior to export and at the point of import of US products. These tests often involve testing for specific DNA sequences that identify the genetically modified event or for the protein product of introduced genes. Because of the proliferation of testing protocols and reference materials, trade in grain and foods can be blocked due to inconsistencies in the testing and leading to financial loss.
Quantitative real-time PCR is the primary tool for detecting and quantitation of the amount of biotech crop material in food, feed or grain. The target for amplification of the DNA by PCR is the unique DNA sequence that is part of the transgene construct inserted in to the plant cell genome. Investigations were conducted in 2008 to address the quality of measurements and the factors which contribute to that quality.
The goals of this project are to improve measurements and contribute to international harmonization of testing. Laboratory investigations were focused on three projects in 2008 in aspects of testing and measurements:
- Total DNA determination as a prerequisite for PCR assays. It is important to add an appropriate quantity of genomic DNA to a PCR assay since too much or too little each causes a set of problems. The measurement of total nucleic acid in DNA extraction preparations was measured with three different approaches, spectroscopic (UV/VIS), fluorometric (dye intercalation) and phosphorus mass fraction (ICP-OES). Differences between these analyses were investigated as well as the sensitivity of one method to the presence of residual detergent from the extraction process.
- DNA quantitation by PCR when the DNA has been degraded by processes common to food processing. DNA is degraded upon exposure to shearing forces and elevated temperature, which complicates the analysis of biotech crop material in processed food. Investigations were conducted in collaboration with the United States Department of Agriculture Grain Inspection, Packers and Stockyards Administration (USDA-GIPSA) on the effect of degradation on the accurate quantitation of a transgene when compared to an endogenous gene.
- Suitability of various methods targeting the 35S promoter sequence of transgene constructs. One of the common elements found in the transgene constructs used to transform plant genomes in biotech crops is the 35S promoter from Cauliflower mosaic virus. Some testing laboratories utilize this widely used sequence to determine GM crop content for a broad range of biotech events using quantitative PCR directed at the 35S sequences. There are several published assays that have been tested against some but not all the events that carry 35S. NIST will conduct a thorough evaluation of these methods, using the intercalating dye, SYBR Green, as the fluorescent tag to follow the amplification of the target DNA. This will allow the detection of multiple products and therefore the specificity of the target sequence. If the current methods cannot be validated for many or all of the crop events development of a new method will be considered.
- Compared various ways to quantify nucleic acids and determined the potential problems with different methodologies. Developed guidelines for investigators with publication imminent. Demonstrated that a popular detergent (CTAB) used in the lysis of plant cells for the purposes of extracting DNA reduces the fluorescent signal when PicoGreen binds DNA resulting in a serious underestimatation of the quantity of DNA present.
- Demonstrated the difficulty of accurate quantitation of biotech crop material in when the DNA is degraded, as in food processing. Demonstrated the importance of quantitation by comparing the transgene to an endogenous gene (gene present in all plants of the species).
- Tested five different 35S quantitative PCR methods against seven different biotech maize certified reference materials. Determined that only two of the methods show acceptable performance with the panel of reference materials.
January 2, 2006
Lead Organizational Unit:
Biotech crop testing laboratories, US grain and food exporters