抗逆大豆IND-ØØ41Ø-5转化体特异性定量PCR检测方法的建立及其标准化  

Establishment and Standardization of Event-Specific Real-Time Quantitative PCR Detection Method of Stress-Resistant Soybean IND-ØØ41Ø-5

作　　者：李允静[1] 肖芳 武玉花[1] 李俊[1] 高鸿飞 翟杉杉 梁晋刚 吴刚[1] LI YunJing;XIAO Fang;WU YuHua;LI Jun;GAO HongFei;ZHAI ShanShan;LIANG JinGang;WU Gang(Oil Crops Research Institute of Chinese Academy of Agricultural Science/Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture and Rural Affairs/Supervision and Test Center(Wuhan)for Plant Ecological Environment Safety,Ministry of Agriculture and Rural Affairs/Key Laboratory of Agricultural Genetically Modified Organisms Traceability,Ministry of Agriculture and Rural Affairs,Wuhan 430062;Development Center of Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176)

机构地区：[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/农业农村部植物生态环境安全监督检验测试中心(武汉)/农业农村部农业转基因生物溯源重点实验室,武汉430062 [2]农业农村部科技发展中心,北京100176

出　　处：《中国农业科学》2023年第13期2443-2460,共18页Scientia Agricultura Sinica

基　　金：农业生物育种重大项目(2022ZD04019);中国农业科学院科技创新工程(CAAS-ASYIP-2021-OCRI)。

摘　　要：【目的】抗逆大豆IND-ØØ41Ø-5已获得中国进口加工原料安全证书,建立一种特异、准确、灵敏的抗逆大豆IND-ØØ41Ø-5转化体特异性实时荧光定量PCR(qPCR)检测方法,为抗逆大豆IND-ØØ41Ø-5在中国的安全监管提供精准测量技术。【方法】首先利用Beacon designer 8.0软件对抗逆大豆IND-ØØ41Ø-5转化体5′端旁侧序列设计18对引物探针,结合1对罗萨里奥农业生物技术学院公司提供的引物探针,随后采用qPCR技术对引物探针进行特异性筛选,遴选出1对候选引物探针;设置实时荧光定量PCR的引物/探针浓度梯度,优化qPCR方法的反应体系;设置不同类型的特异性测试样品测试方法的特异性;以纯合抗逆大豆IND-ØØ41Ø-5基因组DNA作为标准样品,梯度稀释成标准溶液模板,绘制标准曲线,考察qPCR方法的线性动态范围,配置拷贝数比值为5%、1%和0.1%的测试样品,评价定量方法的准确性;配置拷贝数比值为0.05%和0.025%的样品,测试方法的检出限;拷贝数比值为0.1%的样品经16次定量测试,确定方法的定量限;最终确定抗逆大豆IND-ØØ41Ø-5转化体特异性实时荧光定量PCR检测方法的关键技术参数。8家有资质单位对该定量PCR方法的特异性、检出限、定量限、准确性等进行验证,采用柯克伦法和格拉布斯法评估qPCR方法的重复性和再现性,利用线性最小二乘法对准确性样品测试结果的测量不确定度进行预评定。【结果】通过特异性筛查确定RBORD-F1/RBORD-R1/RBORD-P1引物探针为候选组合,建立了抗逆大豆IND-ØØ41Ø-5转化体特异性qPCR方法,扩增片段为138 bp,优化的反应体系引物终浓度为0.4μmol·L-1、探针终浓度为0.2μmol·L-1;IND-ØØ41Ø-5和Lectin标准曲线的线性动力学范围为33—83190 copies的基因组DNA,该方法可准确定量5%、1%和0.1%含量的IND-ØØ41Ø-5测试样品,偏差和相对标准偏差(RSD)均小于25%;确定检出限为0.05%、定量限为0.1%。8家【Objective】Stress-resistant soybean IND-ØØ41Ø-5 has been authorized as a safety certificate for imported processed raw materials in China.This study aims to develop a specific,accurate,and sensitive real-time quantitative PCR(qPCR)assay for the quantification of the stress-resistant soybean IND-ØØ41Ø-5 event,providing a precise measurement technique for regulating its safety in China.【Method】18 pairs of primers and probes were designed using Beacon designer 8.0 software for the 5′end flanking sequence of the stress-resistant soybean IND-ØØ41Ø-5 event,in combination with one pair of primer and probe providing by Instituto de Agrobiotecnologia Rosario(INDEAR)S.A.Subsequently,the specific screening of the primers and probes was performed using real-time PCR technology,and one pair of candidate primer and probe was selected.The reaction system parameters,such as primer and probe concentration,were optimized during the establishing the qPCR method.A specificity test was performed using different test samples.The pure stress-resistant soybean IND-ØØ41Ø-5 genomic DNA was serially diluted into standard solution templates,and standard curves were plotted to investigate the linear dynamic range of the qPCR method.Test samples with copy number ratios of 5%,1%and 0.1%were prepared by mixing IND-ØØ41Ø-5 genomic DNA with non-GM counterpart to evaluate the accuracy of the qPCR method.The limit of detection(LOD)was detected by using test samples with copy number ratios of 0.05%and 0.025%.The limit of quantification(LOQ)was determined after 16 tests on samples with a copy number ratio of 0.1%.Finally,the technical parameters of qPCR assay for the stress-resistant soybean IND-ØØ41Ø-5 event were determined.The specificity,LOD,LOQ and accuracy of the qPCR method were validated by eight qualified testing laboratories.The repeatability and reproducibility of the qPCR were evaluated by Cochran′s test and Grubbs'test,and the measurement uncertainty of the accuracy samples were pre-evaluated by linear least-

关 键 词：转基因 抗逆大豆IND-ØØ41Ø-5 转化体特异性 实时荧光定量PCR 

分 类 号：S565.1[农业科学—作物学]