Detection Methods Of Genetically Modified Soybean In Feed Products

The largest consumer of genetically modified crops is the feed industry, which needs scientific, effective, simple and rapid methods to detect genetically modified (GM) components in feed. Qualitative and quantitative analysis were developed for the detection of genetically modified soybean, soybean meal and various feed products on the basis of nucleic acid and protein levels in this study. The effects of temperature, moisture and pressure on the variation tendency of endogenous and exogenous genes as well as CP4 - EPSPS protein in genetically modified soybean after dry heat, moist heat and extrusion were tested in addition, which will help to the regulation of processing technology and the safety evaluation of transgenic feed. Results are as follows:1. The high quality and purity genome DNA of GM soybean was obtained with the modified SDS extracted method which was suitable for the rapid and efficient extraction of genome DNA from feed and raw materials through the comparison of modified SDS method, modified CTAB method and DNA extraction kit.2. Event - specific PCR results indicated that transgenic soybean in feed products belonged to GTS 40 - 3 - 2 lines. A loop - mediated isothermal amplification (LAMP) method was introduced, the sensitivity was 100 - fold higher than the conventional PCR. According to soybean Lectin, CaMV 35S promoter and CP4 - EPSPS gene, triplex PCR containing two sets of primers (Lectin 1 / 35S / CP4?Lectin 2 / 35S / CP4) was used to detect feed materials containing soybeans, the sensitivity were 0.25 % and 0.5 % respectively. The quantitative PCR standard curve (R~2 > 0.99) with good precision and repeatability was established according to a kind of plasmid pTLE8 containing eight target genes which could replace positive Certified Reference Material (CRM), the limit of quantitation (LOQ) was 1.57 copies. Therefore, this method could be applied to detect the GM feed products.3. Analysis of SDS - PAGE indicated that the expression level of recombinant CP4-EPSPS protein was higher in the supernatant; the antigen about 50 kDa with high purity was obtained after purification, dialysis and freeze drying, the recombinant protein can be used to production of the high titer ( 1:729000 ) polyclonal antibody.4. Western Blot and ELISA assays were conducted for the detection of transgenic feed products after screening and optimization of parameters, the limits of detection (LOD) were 0.5 % and 0.25 % respectively. The appearance of hybridization signals revealed that the CP4 - EPSPS protein existed in the feed materials by Western Blot method, the results of indirect ELISA showed that the content of transgenic protein in raw materials was higher than that in the processed products.5. Qualitative PCR and quantitative PCR were applied to study the degradation pattern of Lectin and CP4 - EPSPS gene in transgenic soybean meal after dry heat, moist heat and extrusion, four primer pairs were designed to obtain different length amplicons of Lectin and CP4 - EPSPS gene. The results showed that the degradation in various degrees of both genes was caused by diverse processing, Lectin of 118 bp and CP4 - EPSPS of 155 bp were stable under high temperature, high pressure and high moisture; to a certain degree, high moisture could relieve the gene degradation, CP4 - EPSPS gene had a lesser degradation. The results of Western Blot and indirect ELISA demonstrated that CP4 - EPSPS protein existed in the extruded soybean meal and the content decreased gradually with the increasing of the temperature and the moisture. The variation tendency was similar to the degradation of DNA.