核酸斑点杂交技术快速鉴定转基因大豆品系OA北大核心CSTPCD

To establish a high-throughput detection method for genetically modified(GM)soybean varieties,this study focused on GM soybeans DAS-68416-4,DAS-44406-6,DAS-81419,and DP305423.Specific primers were designed based on the unique junctional genomic sequences of each variety and the inserted fragments.DNA was extracted from GM standard materials and used as a template for qualitative PCR amplification.The amplified products were then dot-blotted onto a nylon membrane,and biotinylated probes were prepared for hybridization and colorimetric detection.The concentrations of probes,hybridization temperature and time,and color development duration were optimized.The optimized reaction conditions were applied to detect the GM soybean varieties and a blank control,confirming the specificity of the system.Sensitivity was determined by a tenfold serial dilution of DNA from the four GM soybean varieties using the optimized conditions.Applicability was assessed by testing seven varieties of GM soybean standards and eight proficiency testing samples collected in the laboratory.Furthermore,the reliability of the system was further validated by conducting dot blot hybridization tests on 17 blind samples using a double-blind method.The results indicated that the amplified products from GM soybeans DAS-68416-4,DAS-44406-6,DAS-81419,and DP305423 as templates all exhibited a single clear and bright band,consistent with the expected size.The blank control and internal reference gene results were normal.The specific probes for the four GM soybean varieties showed color reactions only with their corresponding samples and no color reaction with the other three samples,demonstrating the specificity of the method.The lowest detection limits were 20 pg·μL-1 for samples DAS-68416-4,DAS-81419,and DP305423,and 2 pg·μL-1 for sample DAS-44406-6,indicating high sensitivity.The test results for seven standard samples,eight proficiency testing samples,and 17 blind samples were consistent with the current standards SN/T 1204-2016,make it suitable for routine sample testing.The qualitative PCR combined with dot blot hybridization method established in this study enables rapid and accurate identification of four GM soybean varieties.It is of significant importance for the orderly advancement of biobreeding industrial applications,strict inspection of illegal GM seed market circulation and field cultivation,and ensuring food safety.