转基因棉花COT102品系双重定量检测方法研究  

Duplex Real-time Quantitative PCR Method for Detection of Genetically Modified Cotton(Gossypium hirsutum)Event COT102

作　　者：潘志霖 左翠花 吴启明 钱昌元 尹璐 刘月明 李想 PAN Zhi-Lin;ZUO Cui-Hua;WU Qi-Ming;QIAN Chang-Yuan;YIN Lu;LIU Yue-Ming;LI Xiang(Technical Center for Animal,Plant and Food Inspection and Quarantine of Shanghai Customs,Shanghai 200135,China;College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海关动植物与食品检验检疫技术中心,上海200135 [2]上海海洋大学食品学院,上海201306

出　　处：《农业生物技术学报》2021年第11期2248-2258,共11页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育科技重大专项(2018ZX08012001-007);上海市技术标准专项(18DZ2201300)。

摘　　要：棉花(Gossypium hirsutum)是一种重要的经济作物,截至2019年底,全球转基因棉花的种植面积占棉花总种植面积的76%。瑞士先正达公司研发的具有抗虫性状的转基因棉花COT102品系已获得我国批准,但目前我国相关检测标准中均缺乏该品系的检测方法。为了保障我国棉花产业的顺利发展和进出口贸易的有序进行,针对转基因棉花COT102品系,建立行之有效的检测方法至关重要。本研究基于多重实时荧光PCR技术,根据转基因棉花COT102品系外源插入片段与基因组DNA的两端旁邻序列设计多套引物和探针,经对反应条件、引物探针比例等指标进行筛选和优化后,建立了转基因棉花COT102品系特异性双重实时荧光定量PCR检测方法。对方法的特异性、灵敏度及可重复性进行测定,结果显示,该方法特异于转基因棉花COT102品系检测,无非特异性扩增;方法的灵敏度高,检测下限和定量下限均低至5拷贝棉花基因组DNA;多次PCR扩增重复之间偏差小,可重复性好。对4个含有不同百分比含量转基因棉花COT102品系成分的样品进行定量检测,结果表明,百分比含量测定值与设定的真实值间的偏差(bias)介于-0.42%~4.63%之间,标准偏差(standard deviation,SD)小于0.5,相对标准偏差(relative standard deviation,RSD)小于25%,均在可接受范围内。因此,本研究建立的双重实时荧光定量PCR检测方法,适用于棉花及其制品中转基因棉花COT102品系成分的定性和定量检测。高通量检测方法可有效提升检测效率,降低检测成本,提升通关效率,为进境转基因棉花及其制品的有效监管和我国转基因产品标识制度的顺利实施提供有效的技术支撑,为进出口贸易的顺利有序进行提供必要的技术保障。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 realtime 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

关 键 词：转基因棉花 COT102品系 特异性检测 双重实时荧光定量PCR 高通量检测方法 

分 类 号：S562[农业科学—作物学]