四重实时荧光PCR快速检测转基因玉米及其加工产品  

Development of a Quadruplex Fluorescence Quantitative PCR for Detection of Genetically Modified Maize and Processed Products

作　　者：王凤军 叶素丹[1] 凌云[1] 周晓红[1] Wang Fengjun;Ye Sudan;Ling Yun;Zhou Xiaohong(Zhejiang Institute of Economic and Trade,Hangzhou 310012)

机构地区：[1]浙江经贸职业技术学院,杭州310012

出　　处：《中国粮油学报》2020年第10期182-188,共7页Journal of the Chinese Cereals and Oils Association

基　　金：浙江省基础公益研究计划项目(LGC19C200007);浙江省教育厅一般科研项目(Y201840745);浙江经贸职业技术学院省属高校基本科研业务费项目(20SBYB03)。

摘　　要：运用四重实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米及其深加工制品进行筛选检测。选定花椰菜花叶病毒35S启动子(pCaMV35S)、农杆菌的胭脂碱合成酶基因终止子(tNOS)以及根癌农杆菌CP4蛋白基因和5-莽草酸-3-磷酸合成酶基因(EPSPS)为外源基因,选定编码玉米淀粉合成酶异构体zSTSII-2(zSSIIb)基因作为玉米物种的内参照基因。设计和合成靶标基因特异性引物和多重荧光探针,经特异性、重复性、灵敏性和适用性等方法学验证建立了四重实时荧光PCR检测方法。结果表明该方法检测特异性强,重复性好,扩增效率在90%~110%,标准曲线相关系数R^2≥0.99,最低检测限为每20μL反应13个拷贝,最低定量限为每20μL反应1.3个拷贝,其检测结果与SN/T 1204—2016标准方法检测结果一致,由于四个目标基因可以在一个反应管中进行扩增反应,且含有内源基因和外源基因,可降低试剂成本,简化操作程序,缩短检测时间,为玉米及其深加工产品转基因成分的快速检测提供参考。The transgenic maize and its further processed products were screened and detected by quadruple real-time fluorescent PCR.The promoter of cauliflower mosaic virus 35 S(pCaMV35s),the terminator of cochineal synthetase gene(tNOS)of Agrobacterium tumefaciens,the CP4 protein gene and the 5-shikimate-3-phosphate synthetase gene(EPSPS)of Agrobacterium tumefaciens were selected as exogenous genes,and the zSSIIb-2(zSSIIb)gene encoding the isomer of corn starch synthetase was selected as the internal reference gene of corn species.We designed and synthesized target gene specific primers and multiple fluorescent probes,and established a quadruple real-time fluorescent PCR detection method through the methodological verification of specificity,repeatability,sensitivity and applicability.The results showed that the method had high specificity and good repeatability and the amplification efficiency was 90%~110%.The correlation coefficient of standard curve R^2≥0.99 and the minimum detection limit was 13 copies per 20μL reaction,and the minimum quantitative limit was 1.3 copies per 20μL reaction.The detection results were consistent with those of SN/T 1204—2016 standard method.Because four target genes could be amplified in one reaction tube,and contain endogenous genes and exogenous genes,it could reduce the cost of reagents,simplify the operation procedures,shorten the detection time,and provide reference for the rapid detection of transgenic components of corn and its deep-processed products.

关 键 词：转基因玉米 四重实时荧光PCR 快速检测 

分 类 号：S529[农业科学—作物学]