转基因水稻品系‘Bt汕优63’微滴式数字PCR定量检测方法的建立  

Establishment of Droplet Digital PCR Quantitative Detection Method of Transgenic Rice Strain‘Bt Shanyou 63'

作　　者：陈雪娇[1] 宗凯[1] 孙娟娟[1] 李云飞[1] 郑海松[1] 姚剑[1] Chen Xuejiao, Zong Kai, Sun Juanjuan, Li Yunfei, Zheng Haisong, Yao Jian(Technology Center of Anhui Entry-exit Inspection and Quarantine Bureau, Hefei 23002)

机构地区：[1]安徽出入境检验检疫局技术中心,合肥230022

出　　处：《中国农学通报》2018年第6期131-136,共6页Chinese Agricultural Science Bulletin

基　　金：国家质量监督检验检疫总局科技计划项目"水稻转基因成分定量检测方法研究(数字微滴PCR法)"(2015IK0003);质检公益性行业科研专项"花卉;水稻;棉花等重大疫病检疫关键技术研究"(201410076)

摘　　要：为进一步提升转基因水稻的定量检测效率,推动转基因定量标识制度的实施,对转基因水稻品系‘Bt汕优63’成分进行微滴式数字PCR法定量检测研究。基于转基因水稻品系‘Bt汕优63’的外源插入片段和水稻蔗糖磷酸盐合成酶基因SPS,选择、设计和合成高效特异性的内外源基因引物探针,用于微滴式数字PCR法扩增‘Bt汕优63’的内源基因和品系特异序列。引物探针优化实验表明,SPS60-F/R/P和‘Bt汕优63’品系基因-F/R/P适用于dd PCR双通道法定量检测‘Bt汕优63’成分。准确度验证试验表明,测得值与标准值相近,偏差分别为0.03、0.15、0.15、0.03。测得值的SD介于0.08~0.48之间,RSD介于4.86%~15.10%之间,小于欧盟要求25%,准确度较好。本研究建立的转基因水稻品系‘Bt汕优63’微滴数字PCR定量检测方法简便高效、准确度高,可用于农产品和食品中转基因水稻‘Bt汕优63’成分的定量检测。To improve the quantitative detection efficiency of transgenic rice,and promote the implementationof transgenic food quantitative identification system,the components of transgenic rice‘Bt shanyou 63＇wereanalyzed with droplet digital PCR quantitative detection method.Based on the exogenous inserting sequence oftransgenic rice‘Bt shanyou 63＇and the rice sucrose phosphate synthase（SPS） gene,high-efficiency primerprobes were selected,designed and synthesized to amplify the endogenous gene and the strain specificsequence of‘Bt shanyou 63＇with droplet digital PCR.The result of primer probes optimizing experimentdemonstrated that SPS60-F/R/P and‘Bt shanyou 63＇-F/R/P was suitable for quantitatively detecting thecomponents of‘Bt shanyou 63＇with dual channel dd PCR method.The accuracy verification experimentshowed that the measured value was similar to the standard value,and the biases were 0.03,0.15,0.15 and0.03,respectively.SD of measured value was 0.08-0.48,RSD was 4.86%-15.10%,and they were less than therequirement of European Union,indicating that the accuracy was good.The established detection system ofdroplet dd PCR method for transgenic rice line‘Bt 63＇quantification is simple,high efficient and accurate,and it can be used for quantitative analysis of transgenic rice line‘Bt 63＇in agricultural products and foods.

关 键 词：微滴式数字PCR 双通道法 定量检测 转基因水稻 'Bt汕优63’ 

分 类 号：S184[农业科学—农业基础科学]