Real-time PCR Method for the Detection of Roundup Ready Soybean in Food and Its Application
|Course||Nutrition and Food Hygiene|
|Keywords||Roundup Ready soybean PCR fluorescence dye SYBR GreenⅠ Taqman probe genetically modified soybean oil|
Objective:To establish qualitative and quantitative PCR methods to detect the Roundup Ready soybean in foods and to explore the qualitative method to detect the genetically modified components in soybean oil through improving the DNA extraction method.Methods:Reference materials of Roundup Ready soybean and samples from the supermarkets in Suzhou were analyzed. The primers were designed to amplify the endogenous lectin gene, the 35S promoter and CP4-EPSPS target gene according to Roundup Ready soybean genome. The screening method for Roundup Ready soybean in foods was established based on conventional PCR to detect 35S promoter. The quantitative PCR detection system based on fluorescence dye SYBR GreenⅠwas established to detect CP4-EPSPS target gene, and the samples positive in former screening detection were verified and quantified with it. The parameters such as sensitivity and recovery rate were analyzed by comparing with Taqman quantitative PCR method. The samples obtained from four important stages during soybean oil refining processes were collected and analyzed with four different DNA extraction methods to screen out the most suitable DNA extraction method for soybean oil. The qualitative detection method for genetically modified soybean oil was developed by optimizing Taqman PCR method.Results:1. The detection limit of qualitative screening method for 35S promoter was 0.5ng. Two of the twenty samples contain soybean were proved positive and one of them was confirmed as genetically modified food.2. The detection limit and average recovery rate of SYBR Green real-time PCR was 5pg and 106% respectively. The average relative deviation of the method is 6%, This method had higher sensitivity the same recovery rate and relative deviation compared with Taqman real-time quantitative PCR.3. The modified CTAB method developed in this study was sufficiently useful for extraction DNA from crude oil, neutralisation oil and bleaching oil. The DNA template could be detected by Taqman real-time PCR.Conclusions:A sensitive, accurate and specific method based on SYBR Green real-time quantitative PCR was developed which could be used for the detection of Roundup Ready soybean components in food. The detection of genetically modified soybean oil could be accomplished through using the modified CTAB DNA extraction method and Taqman real-time PCR by analyzing crude oil, neutralisation oil and bleaching oil samples from oil plant.