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转基因玉米MON87427/zSSIIb 二重微滴数字PCR方法建立及应用
引用本文:肖芳,张秀杰,李俊,王颢潜,李允静,单露英,翟杉杉,高鸿飞,吴刚,武玉花. 转基因玉米MON87427/zSSIIb 二重微滴数字PCR方法建立及应用[J]. 中国油料作物学报, 2021, 43(1): 90. DOI: 10.19802/j.issn.1007-9084.2020322
作者姓名:肖芳  张秀杰  李俊  王颢潜  李允静  单露英  翟杉杉  高鸿飞  吴刚  武玉花
作者单位:1. 中国农业科学院油料作物研究所/农业农村部油料作物生物学与遗传育种重点实验室,湖北武汉,430062;2. 农业农村部科技发展中心,北京,100025
基金项目:转基因生物新品种培育专项(2016ZX08012003)。
摘    要:实施转基因产品定量标识制度需要建立准确可靠的定量检测技术和方法。数字PCR(dPCR)不依赖标准物质,实现对DNA分子的绝对定量,已成功用于转基因含量检测和标准物质定值。为建立可靠的dPCR方法,获得准确测量结果,本研究以耐除草剂玉米MON87427为材料,探索建立二重微滴数字PCR(ddPCR)的策略。以构建的聚合MON87427转化体和5个玉米内标基因的重组质粒pUC57-M为质控样品,将5个不同的玉米内标基因分别与MON87427组合,通过优化退火温度,根据阳性微滴与阴性微滴分辨率、中等信号强度雨滴数量及测量值与理论值的一致性等,确定了二重ddPCR组合为MON87427/zSSIIb,最适退火温度为58.4℃。MON87427/zSSIIb 二重ddPCR的动力学范围为10~60 000拷贝。对盲样进行定量检测,二重ddPCR的定量结果与荧光定量PCR有良好的可比性,表明MON87427/zSSIIb 二重ddPCR可取代qPCR方法进行转基因玉米MON87427的定量检测及标准物质定值。

关 键 词:转基因玉米MON87427  二重微滴数字PCR  荧光定量PCR  内标基因  

Development and application of MON87427/zSSIIb duplex droplet digital PCR method
XIAO Fang,ZHANG Xiu-jie,LI Jun,WANG Hao-qian,LI Yun-jing,SHAN Lu-ying,ZHAI Shan-shan,GAO Hong-fei,WU Gang,WU Yu-hua. Development and application of MON87427/zSSIIb duplex droplet digital PCR method[J]. Chinese Journal of Oil Crop Sciences, 2021, 43(1): 90. DOI: 10.19802/j.issn.1007-9084.2020322
Authors:XIAO Fang  ZHANG Xiu-jie  LI Jun  WANG Hao-qian  LI Yun-jing  SHAN Lu-ying  ZHAI Shan-shan  GAO Hong-fei  WU Gang  WU Yu-hua
Affiliation:1. Oil Crops Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory of Biology and GeneticImprovement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062, China;2. Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025, China
Abstract:Digital PCR(dPCR),an absolute quantitative method of DNA molecules,has been successfully applied for genetically modified organisms(GMO)detection and reference material characterization.In this paper,a duplex ddPCR method was established for quantification of MON87427 content with reference plasmid pUC57-M harboring MON87427-specfic sequence and 5 maize reference gene sequences as quality control.By comparing the 5 reference genes,zSSIIb was determined to combine with MON87427 for duplex ddPCR assay according to the resolution of positive droplets and negative droplets,the number of raindrops with medium signal intensity,and the consistency between measured value and theoretical value.The optimal annealing temperature for MON87427/zSSIIb duplex ddPCR was determined at 58.4℃,and the duplex ddPCR displayed good linearity over the range from 10 to 60000 copies.The quantitative results of blinded samples by duplex ddPCR were well comparable with those by real-time quantitative PCR(qPCR),indicating that the MON87427/zSSIIb duplex ddPCR might replace the qPCR method for quantification of transgenic maize MON87427 and characterization of MON87427 reference materials.
Keywords:transgenic maize MON87427  duplex droplet digital PCR  real-time quantitative PCR  reference gene
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