Study On Detection Of Transgenic Ingredients In Genetically Modified Cottonseed Meal And Its Degradation During Feed Processing

More and more genetically modified (GM) products entering feed fields has raised increasing public concerning about feed safety. In this paper, the extraction method, qualitative and qualitative analysis and degradation of GM cottonseed meal during feed processing were studied to provide scientific basis for the biosafety evaluation system of GM ingredients.CTAB and SDS were used respectively to explore the right way in genomic extraction for genetically modified cottonseed or meal. It was proved that SDS-based method was more suitable for cottonseed genomic extraction, and an extraction kit based on centrifugal columnar was improved. Experiments showed that the DNA of GM cottonseed meal could be extracted with high purity in a short time, which could fully meet requirements in the downstream experiments.Single PCR, multi PCR and loop mediated isothermal amplification (LAMP) technology were employed for qualitative detection of GM cottonseed with high specificity. The detection limit of single PCR was 100 copies. For the Cry1Ab/c gene and Sad1 genes, annealing temperature in the range of 55.5℃-58.3℃could be suitable for amplification. The primer pairs of Cry1Ab/c gene and Sad1 gene were added in a reaction system in the same time. Both Cry1Ab/c gene and Sad1 gene could be detected with annealing temperature in 55.5℃-58.3℃range, while Cry1Ab/c gene could not be detected with annealing temperature above 59.1℃. The detection sensitivity of the optimized LAMP reaction was 10 copies, which was 10 times of single PCR.A standard plasmid for real time PCR detection of GM cotton was constructed, and the detection method of GM cotton was established on the basis of gene specificity with the calculation method of copies ratio. This method was proved to be an accurate detection method that could be used in quantitative detection of GM cottonseed meal and its by-product. The tested samples in several regions all contain GM ingredient, except for Tacheng and Zhumadian regions. The copies ratio of Cry1Ab/c gene and Sad1 gene were greater than 50%.The GM cottonseed meal was dry heated; wet heated and extruded, and 5 pairs of primers were designed to study the degradation of GM cottonseed meal during feed processing. The results showed that large fragments were degraded gradually, while the magnitudes were different with temperature increasing. After different times of dry heat and wet heat treatment at 105℃, the degradation of the fragments was similar. In extrusion, the temperature was higher; the degradation of large fragment was more drastic. Different water percentage during extrusion had little effect on degradation. The quantitative analysis of small fragments showed that the copies ratio in different conditions of dry treatment heat had no significant tendency. After heat treatment at 105℃in different times, the copies ratio had no significant tendency, either. However, the copies ratio of Cry1Ab/c gene and Sad1 gene decreased when the temperature increased. There is no significant tendency in copies ratio when extruded in different percentage of water, while the copies ratio of Cry1Ab/c gene and Sad1 gene decreased when the temperature increased.