荧光定量PCR辐照食品鉴定技术的优化研究  

An Optimization on Identification of Irradiated Food by Fluorescence Quantitative PCR

作　　者：刘绵学[1,2] 伏毅[1,2] 郑宇 王艳[1,2] 潘琳[1] 李华 黄敏[1,2] LIU Mian-xue;FU Yi;ZHENG Yu;WANG Yan;PAN Lin;LI Hua;HUANG Min(Irradiation Preservation Key Laboratory of Sichuan Province,Sichuan Institute of Atomic Energy,Chengdu 610101;Department ofBiotechnology,Sichuan Institute of Atomic Energy,Chengdu 610101)

机构地区：[1]四川省原子能研究院辐照保藏四川省重点实验室,成都610101 [2]四川省原子能研究院生物技术研究所,成都610101

出　　处：《生物技术通报》2018年第12期77-83,共7页Biotechnology Bulletin

基　　金：四川省科技支撑计划项目(2016SZ0075;2016NZ0117);四川省应用基础项目(2017JY0242;2016JY0030;2018JY0531;18YYJC0284);四川省科技计划重点研发项目(2018GZ0003)

摘　　要：为优化荧光定量PCR辐照食品鉴定技术方法,实现对假阳性样本的鉴别能力,降低模板量对鉴定结果的干扰效应。选择马铃薯作为研究材料,分别进行1-12 kGy的60Co辐照、加热和冻融处理,通过转座子引物筛选、检出限分析、DNA损伤特征比较和剂量曲线回归等方法,探究转座子引物的应用对马铃薯辐照鉴定结果的优化效果。结果显示,转座子引物组合LTR2-5\18S-8与LTR2-2\ACT-5可用于假阳性样本的鉴别,引物组合LTR2-5\18S-5可用于辐照鉴定。优化后方法中"初始循环数差值的指数函数-辐照剂量"标准曲线方程为Y=-0.1581X+4.611,线性关系R2为99.24%,优于"初始循环数差值-辐照剂量"的原有模式。通过转座子引物的应用与方法优化,增强了假阳性鉴别能力,消除了模板差异,增强了荧光定量PCR辐照鉴定技术的"剂量-效应关系"。Purpose of this study is to realize the identification ability of false positive samples and reduce template amount interference with real-time PCR results in optimizing irradiation food identification.Potato tuber was selected as material and treated with60-Co in gradient dose(1-12kGy),irradiating,heating and freezing-thawing respectively.Analysis methods,including primer screening of transposon,detection limit analysis,comparison of DNA damage characteristics and dose curve regression,were used to explore the optimized effects of transposon primers in potato irradiation identification.As results,transposon primer combination of LTR2-5\18S-8and LTR2-2\ACT-5can be used for the identification of false positive samples,and the combination of LTR2-5\18S-5can be used for irradiation identification.In the optimized method,the irradiation dose was linear with the exponential function of the initial cycle difference with the standard equation Y=-0.1581X+4.611and determination coefficient R299.24%,which was superior to the original initial cycle difference-irradiation dose model.In conclusion,by the optimized method,the dose-effect relationship with real-time PCR in irradiation identification was improved with less possibility of false positive,and the result interference caused by template amount was eliminated.

关 键 词：辐照鉴定 转座子 马铃薯 DNA损伤 

分 类 号：TS207.3[轻工技术与工程—食品科学]