Duplex Real-time Quantitative PCR Method for Detection of Genetically Modified Cotton (Gossypium hirsutum) Event COT102
PAN Zhi-Lin1,2, ZUO Cui-Hua1, WU Qi-Ming1, QIAN Chang-Yuan1, YIN Lu1, LIU Yue-Ming1, LI Xiang1,*
1 Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 200135, China; 2 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
Abstract：Cotton (Gossypium hirsutum) is an important cash crop, by the end of 2019, 76% of the total cotton acreage was cultivated on genetically modified (GM) cotton globally. GM cotton event COT102 is a transgenic cotton event with insect resistance developed by Syngenta, Switzerland. This event has been approved by the Ministry of Agriculture of China, but has no detection method in relevant standards in China. In order to ensure the development of cotton industry in China and the orderly conduct of import and export trade, it is very important to establish an effective detection method for GM cotton event COT102. In this study, multiple sets of primers and probes were designed according to the exogenous inserts and adjacent sequences of genomic DNA of GM cotton event COT102 based on duplex real-time PCR technology. After screening and optimizing the reaction conditions, the proportion of primers and probes and other indicators, a duplex real-time quantitative PCR method was developed for the detection of GM cotton event COT102. The specificity, sensitivity and reproducibility of the method were measured. The results showed that the method was specific for the detection of GM cotton event COT102, without any nonspecific amplification. The sensitivity of the method was high, and the lower limit of detection and quantification was as low as 5 copies of cotton genomic DNA. The deviation between repeats of multiple PCR amplification was small and reproducibility was good. The results showed that the bias between the measured value and the expected value was between -0.42% and 4.63%. The standard deviation (SD) was less than 0.5, and the relative standard deviation (RSD) was less than 25%, both were within the acceptable range. Therefore, the duplex real-time quantitative PCR method established in this study was suitable for qualitative and quantitative detection of the components of GM cotton event COT102 in cotton and its products. The high-throughput detection method could effectively improve the detection efficiency, reduce the detection cost, and improve the customs clearance efficiency. This method provides effective technical support for the effective supervision of imported GM cotton and its products, and promotes the implementation of GM product labeling policies in China, meanwhile provides necessary technical support for the smooth and orderly conduct of import and export trade.
[1] C.W.迪芬巴赫, G.S. 德弗克斯勒. 2000. PCR技术实验指南[M]. 科学出版社, 北京. pp. 88-105. (Dieffenbach C. W., Dveksler G. S. 2000. PCR Primer: A Laboratory Manual[M]. Science Press, Beijin, China. pp. 88-105.) [2] 陈翔, 范磊. 2020. 转基因食品检测技术在食品检测工程中的应用[J]. 检测与标准, 45(8): 100-101. (Chen X, Fan L. 2020. Application of genetically modified food testing technology in food testing engineering[J]. Grain Science and Technology and Economy, 45(8): 100-101.) [3] 国际农业生物技术应用服务组织. 2021. 2019年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 41(1): 114-119. (International Service for the Acquisition of Agri-biotech Applications (ISAAA). 2021. The development trend of global biotechnology/genetically modified crops commercialization in 2019[J]. China Biotechnology, 41(1): 114-119. [4] 黄新, 高宏伟, 李想, 等. 2018a. GB/T 19495.4-2018, 转基因产品检测实时荧光定性聚合酶链式反应(PCR)检测方法[S]. 北京: 国家市场监督管理总局. (Huang X, Gao H W, Li X, et al. 2018-1. GB/T 19495.4-2018, Detection of genetically modified organisms and derived products-Qualitative real-time polymerase chain reaction (PCR) methods[S]. Beijing: State Administration for Market Regulation.) [5] 黄新, 李想, 高宏伟, 等. 2018b. GB/T 19495.5-2018, 转基因产品检测实时荧光定量聚合酶链式反应(PCR)检测方法[S]. 北京: 国际市场监督管理总局. (Huang X, Li X, Gao H W, et al. 2018-2. GB/T 19495.5-2018, Detection of genetically modified organisms and derived products-Quantitative real-time polymerase chain reaction (PCR) methods [S]. Beijing: State Administration for Market Regulation.) [6] 宋明昊. 2018. 转基因食品检测技术的应用与发展[J]. 百科论坛电子杂质, 18: 663. (Song M H. 2018. Application and development of transgenic food detection technology[J]. Encyclopedia Form, 18: 663.) [7] 唐金泉, 王毓蓬, 焦洋. 2009. 我国棉籽油工业发展回顾及思考[J]. 中国棉花加工, 4: 32-34. (Tang J Q, Wang Y P, Jiao Y. Review and reflection on the development of cottonseed oil industry in China[J]. China Cotton Processing, 4: 32-34.) [8] 徐琳杰, 刘培磊, 熊鹂, 等. 2014. 国际上主要国家和地区农业转基因产品的标识制度[J]. 生物安全学报, 23(4): 301-304. (Xu L J, Liu P L, Xiong P, et al. 2014. International labeling policies for genetically modified agricultural products[J]. Journal of Biosafety, 23(4): 301-304.) [9] 杨宏苗, 申孟, 刘杨, 等. 2019. 转基因食品检测技术研究进展[J]. 粮食与油脂,32(12): 8-11. (Yang H M, Shen M, Liu Y, et al. 2019. Research progress on detecting technology of genetically modified food[J]. Cereals & Oils, 32(12): 8-11.) [10] 袁俊杰, 魏霜, 龙阳, 等. 2020. 转基因大豆MON87701和MON87708双重实时荧光PCR检测技术的建立与应用[J]. 农业生物技术学报, 28(2): 342-348. (Yuan J J, Wei S, Long Y, et al. 2020. Development and application of duplex real-time PCR assay for the detection of genetically modified soybean, 28(2): 342-348.) [11] 中华人民共和国农业农村部. 2002. 中华人民共和国农业部令第10号: 农业转基因生物标识管理办法[A]. (Ministry of Agriculture and Rural Affairs of the People's Republic of China. 2002. Order of the Ministry of Agriculture of the People's Republic of China No. 10: Administrative measures for labeling agricultural GMOs [A].) [12] 张帅, 宋贵文, 崔文杰, 等. 2018. 农业部2630号公告-8-2017, 转基因植物及其产品成分检测抗虫棉花COT102及其衍生品种定性PCR方法[S]. 北京: 中华人民共和国农业部. (Zhang S, Song G W, Cui W J, et al. 2018. Bulletin No. 2630-8-2017 of Ministry of Agriculture, Detection of genetically modified plants and derived products-Qualitative PCR method for inset-resistant cotton COT102 and its deliveries[S]. Beijing: The ministry of agriculture of the People's Republic of China.) [13] Bourque S N, Valero J R, Mereier J, et al.1993. Multiple polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis[J]. Applied and Environmental Microbiology, 59: 523-527. [14] EU-RL GMFF (European Union Reference Laboratory for GM Food and Feed). 2012. Event-specific Method for the Quantification of Cotton T304-40 using Real-time PCR [S/OL]. European Union: Joint Research Centre. https://gmo-crl.jrc.ec.europa.eu/summaries/2012-10-29%20EURL%20VL0511%20VP.pdf. [15] European Network of GMO Laboratories (ENGL). 2009. Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing [S/OL]. https://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_ methods.pdf. [16] Li X, Wang X X, Yang J L, et al.2014. A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)[J]. BMC Biotechnology, 14: 43-52. [17] Vandenvelde C, Verstraete M, Van Beers D.1990. Fast multiple polymerase chain reaction on boiled clinical samples for rapid viral diagnosis[J]. Journal of Virological Methods, 30: 215-227. [18] Wang F, Feng J, Ye S, et al.2018. Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms[J]. Biologia, 73(1): 21-29.
