1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2. Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs P. R. China, Beijing 100025, China
3. Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Abstract：Based on the digital PCR technology for absolute quantification of DNA single molecule, a droplet digital PCR (ddPCR) dual (transformant-specific gene and soybean internal standard gene) quantitative method for the transformant was established. The main contents include:optimize the double ddPCR primer-probe concentration and annealing temperature, examine the specificity, determine the linear kinetic range and critical parameters such as the limit of detection (3copies/μL) and the limit of quantification (15copies/μL). The genetically modified soybean SHZD32-1 has obtained the production and application safety certificate "Nongjianzhengzi (2019)" No. 293. Its ddPCR quantitative detection method, as an absolute quantitative measurement method, is simple, efficient and accurate, and will provide technical support for the calibration of reference materials, quantitative detection and standardization of the establishment of digital PCR methods in the field of biosafety.
Deng H C, Yin C C, Liu G Z , et al. Progress in Nucleic Acid Detection Techniques for Genetically Modified Organisms ［J］. China Biotechnology, 2011, 31 (1): 86-95.
［2］
Hindson C M, Chevillet J R, Briggs H A, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR ［J］. Nature Methods, 2013, 10 (10): 1003-1005.
［3］
Strain M C, Lada S M, Luong T, et al. Highly Precise Measurement of HIV DNA by Droplet Digital PCR ［J］. Plos One, 2013, 8(4): e55943.
Chen X J, Zong K, Sun J J, et al. Establishment of Droplet Digital PCR Quantitative Detection Method of Transgenic Rice Strain‘Bt Shanyou 63 ［J］. Chinese Agricultural Science Bulletin, 2018, (6): 131-136.
［7］
Burke D G, Dong L H, Bhat S, et al. Digital Polymerase Chain Reaction Measured pUC19 Marker as Calibrant for HPLC Measurement of DNA Quantity ［J］. Anal Chem,2013, 85(3):1657-1664.
White H, et al. A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR［J］. Leukemia,2015, 29(2): 369-76.
［8］
Pinheiro L B, Coleman V A, Hindson C M, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification ［J］. Anal Chem, 2012, 84 (2): 1003-1011.
［13］
Pinheiro L B, Coleman V A, Hindson C M, et al. Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification ［J］. Analytical Chemistry, 2012, 84 (2): 1003-1011.
Li D, Wang J, Yang Z, et al. Experimental design analysis of developing quantitative nucleic acid reference materials ［J］. Acta Metrologica Sinica, 2020, 41 (11): 1436-1442.
［15］
Vogelstein B, Kinzler K W. Digital PCR ［J］. Proceedings of the National Academy of ences of the United States of America, 1999, 96 (16): 9236-9241.
Li L. Biometrics and reference materials: Guarantee of effective and consistent analytical results［J］.Current Biotechnology, 2020, 10 (6): 569.
Li L. Bioanalytical testing: Tools for exploring the mysteries of the life sciences ［J］. Biotechnology Bulletin, 2020, 36 (5): 1.
Niu C Y, Zhang Y Z, Yang J Y, et al. Resrach on the Collaborative Value Assignment of Plasmid Nucleic Acid Ｒefernce Materials Based on Digital PCR ［J］. Acta Metrologica Sinica, 2021, 42 (11): 1522-1527.
Pan G, Zhang G M, Huang X, et al. Studies on quantification of transgenic maize ingredient with duplex droplet digital polymerase chain reaction ［J］. Plant Quarantine, 2016, 30 (3): 65-71.
［11］
Bhat S, Emslie K R. Digital polymerase chain reaction for characterisation of DNA reference materials ［J］. Biomolecular Detection and Quantification, 2016, 10: 47-49.
Hayden R T, Gu Z, Ingersoll J, et al. Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus ［J］. Journal of Clinical Microbiology, 2013, 51 (2): 540-546.
Wang D, Wang Z D, Wu X, et al.Development of Reference Material of SARS-CoV-2 Genomic RNA［J］. Acta Metrologica Sinica, 2021, 42(2): 259-264.
Yang S G. Evolution and developing trend of global biotech/GM crops. ［J］. Journal of China Agricultural University, 2020, 25 (9): 13-26.
［9］
Somanath B, Natalie C, Thomas M, et al. Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units ［J］. Analytical Chemistry, 2010, 82 (17): 7185-7192.
Liu F F, Zhang L, Wang J, et al. Quantifi cation of DNA by Digital Polymerase Chain Reaction and Uncertainty Evaluation of Determination Result ［J］. Chemical Analysis And Meterage, 2013, 22 (1): 18-22.
［12］
Sanders R, Huggett J F, Bushell C A, et al. Evaluation of digital PCR for absolute DNA quantification ［J］. Analytical Chemistry, 2011, 83 (17): 6474-6484.
Wang Q, Liu B R. Application of dropplet digital PCR in personalized cancer therapy ［J］. Modern Oncology, 2015(12): 1771-1774.
［18］
Morisset D, tebih D, Milavec M, et al. Quantitative analysis of food and feed samples with droplet digital PCR ［J］. PloS one, 2013,8 (5):e62583.
Savini C.Event-specific Method for the Quantification of Soybean MON87769 Using Real-time PCR ［R］. European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) 2012.
2023, Vol. 44 
