The Combination Of Quantitative Pcr And Western Blot Detecting Cp4-Epsps Components In Roundup Ready Soya Plant Tissues And Commercial Soya-Related Foodstuffs

With the widespread of Roundup Ready Soya (event40-3-2)(RRS), the comprehensive detection of genetically modified (GM) component in foodstuffs is of significant interest but few protein-based approaches have been found useful in processed foods. In this report, a modified Biozol-based one step extraction method for isolation of RNA, DN A and protein from a variety of plants is established combined with the usage of CTAB. Further, the combination of quantitative PCR (qPCR) and Western blot was used to detect CP4-EPSPS gene and its protein product in different RRS plant tissues and commercial soya-containing foodstuffs.Combined with the usage of CTAB, a modified Biozol-based one step extraction method for isolation of RNA, DNA and protein from maize, soybean, alfalfa and cucumber plants is established. The results of ultraviolet spectrophotometer and agarose electrophoresis analysis showed that the obtained RNA and DNA display a good purity. Meanwhile, heme oxygenase-1gene (HO-1) and calcium-dependent protein kinase-1gene (CDPK1) exhibited the positive signals checked by RT-PCR. Similar results of protein were confirmed by using Western blot analysis. The whole isolation procedure costs3hours. Overall, in comparison with extracting RNA, DNA and protein separately, this modified method displays characteristics of shorter duration time and higher efficiency when used as the useful tools for one step isolation of RNA, DNA and protein with higher purity from single plant sample. Thus, it provided the benefit for further researches.Furthermore, the combination of quantitative PCR (qPCR) and Western blot was used to detect CP4-EPSPS gene and its protein product in different RRS plant tissues and commercial soya-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the two methods was confirmed firstly. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the Western blot and the qPCR were not consistent. To be specific, at least two degraded fragments of CP4-EPSPS protein (35.5and24.6kD) were observed. For dried bean curd crust and deep fried bean curd, a degraded protein fragment with the size of24.6kD appeared, while CP4-EPSPS gene can not be traced by qPCR. In contrast, we found a signal of CP4-EPSPS DNA in three foodstuffs, including soy-containing ham cutlet product, meat ball and sausage by qPCR, while CP4-EPSPS protein could not be detected by Western blot in such samples. Our study therefore concluded that the combination of DNA-and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels.