蜂蜜中转基因成分启动子和终止子的多重实时荧光PCR法检测  

Detection of Promoters and Terminators of Transgenic Components in Honey by Multiplex Real-Time Fluorescence PCR

作　　者：林碧莲 张雅薇 高宇 何孟杭 邱秀玉 韩涛 张芳 傅德江 陈思琪 宋帆 柯振华 LIN Bilian;ZHANG Yawei;GAO Yu;HE Menghang;QIU Xiuyu;HAN Tao;ZHANG Fang;FU Dejiang;CHEN Siqi;SONG Fan;KE Zhenhua(National Quality Supervision and Testing Center for Processed Food(Fuzhou),Fujian Inspection and Research Institute for Product Quality,Fuzhou 350002,China)

机构地区：[1]福建省产品质量检验研究院,国家加工食品质量监督检验检测中心(福州),福建福州350002

出　　处：《食品与生物技术学报》2024年第1期69-77,共9页Journal of Food Science and Biotechnology

基　　金：福建省市场监督管理局科技项目(FJMS2020003)。

摘　　要：为能高效率定位蜂蜜中的转基因可疑阳性样品,以10种转基因启动子和终止子(花椰菜花叶病毒35S启动子(pCaMV35S)、农杆菌的胭脂碱合成酶基因终止子(tNOS)、玄参花叶病毒的35S启动子(p FMV35S)、农杆菌的胭脂碱合成酶基因启动子(pNOS)、玉米泛素基因的启动子(pUbi)、烟草的花蕊特异性TA29的基因发育调节启动子(pTA29)、豌豆二磷酸核酮糖羧化酶基因的终止子(tE9)、章鱼碱合成酶终止子(t OCS)、谷氨酰胺转移酶7终止子(tg7)、花椰菜花叶病毒终止子(tCaMV35S))为研究靶标,进行10种启动子和终止子的引物和探针组合筛选、反应条件优化、灵敏度测定、质控品测试比对等实验,建立3组多重实时荧光PCR反应体系,并将其应用于市售蜂蜜的检测。结果表明:建立的3组多重实时荧光PCR反应体系能精准检出目的基因,其灵敏度分别为0.01、0.04、0.04 ng/μL;14种转基因质控品的测试显示多重实时荧光PCR和单重实时荧光PCR的检测结果一致;40份市售蜂蜜的筛查中,有两份蜂蜜检出pCaMV35S和tNOS,其余未检出,该方法大大提高了检测效率。该方法为蜂蜜中转基因成分的检测提供了快筛快检技术,同时也适用其它产品转基因成分的检测。In order to efficiently identify suspect transgenic positive samples in honey,ten transgenic promoters and terminators,i.e.,CaMV35S promoter,NOS terminator,FMV35S promoter,NOS promoter,Ubi promoter,TA29 promoter,E9 terminator,OCS terminator,g7 terminator,CaMV35S terminator,were used as research targets.A series of experiments were conducted for these ten promoters and terminators,including primers combination screening,reaction condition optimization,sensitivity determination,and test comparison of quality control products.Three groups of multiple real-time fluorescence PCR reaction systems were established and applied to the detection of commercial honey.The result showed that the established three groups of multiple real-time fluorescence PCR reaction system could accurately detect the target gene,with sensitivities of 0.01,0.04,0.04 ng/μL,respectively.The tests of 14 genetically modified quality control products showed that the detection results of multiplex real-time fluorescence PCR and single-plex real-time fluorescence PCR were consistent.The screening of 40 commercial honeys showed that two honeys contained pCaMV35S and tNOS,and the others were not detected,greatly improving the detection efficiency.This method could provide a fast screening and detection technology for the detection of genetically modified ingredients in honey,and meanwhile,it could also be applicable to the detection of genetically modified ingredients in other products.

关 键 词：蜂蜜 转基因 启动子 终止子 多重实时荧光PCR 

分 类 号：TS207.3[轻工技术与工程—食品科学]