摘要
以杭州瑞丰生物科技有限公司研发的转Cry1Ab/Vip3Da基因抗虫大豆,即转基因大豆CAL16为材料,建立CAL16转化体特异性定量PCR检测方法,旨在给我国CAL16及其衍生品系的生物育种产业化提供技术支撑。根据5′端和3′端旁侧序列设计引物探针组合,筛选出5′端一对最佳引物组合,PCR产物片段大小为112 bp。经特异性测试、标准曲线的建立、正确度和精密度测试、检出限和定量限测试、稳健性测试、微滴式数字PCR验证,该方法特异性强、灵敏度高、重复性好。检出限达到0.025%,相当于10个拷贝的CAL16特异性片段;定量限达到0.1%,约为40个拷贝。实验室间循环验证显示:该方法符合国家标准方法各项要求,可满足检测需求。最终对CAL16定量方法进行不确定度评定,确保量值科学可靠,满足定量标识的需求。
To provide technical support for biological breeding and commercializing of CAL16 in China,trans-genic soybean CAL16,an insect-resistant soybean with cry1Ab/vip3Da gene developed by Hangzhou RFGENE Co.,Ltd.,is investigated for event-specific quantitative PCR detection method.By combination fuse probes according to 5′-and 3′-end side sequences,a pair of optimal primer combinations is selected at the 5′-end.The PCR product size is 112 bp.After specificity test,standard curve establishment,accuracy and precision test,LOD and LOQ test,robustness test,and micro-drop digital PCR verification,this method turn out to be strong specificity,high sensitivi-ty and good repeatability.The LOD reaches 0.025%,which is equivalent to 10 copies of CAL16 specific fragments;the LOQ reaches 0.1%,about 40 copies.The inter-laboratory cycle verification shows that this method meets the re-quirements of national standard methods and could meet the testing requirements.Finally,the uncertainty evalua-tion of the CAL16 quantitative method is carried out to ensure that the quantitative value is scientific and reliable.Thus this event-specific quantitative PCR detection method of CAL16 could be expected for industrialization of CAL16 and its derivatives in China.
作者
王沛然
陆佳雨
许学
吴爽
李晨
胡笑林
潘伟芹
汪秀峰
WANG Pei-ran;LU Jia-yu;XU Xue;WU Shuang;LI Chen;HU Xiao-lin;PAN Wei-qin;WANG Xiu-feng(Key Laboratory of Rice Genetic Breeding of Anhui Province,Rice Research Institute,Anhui Academy of Agricultural Sciences,Hefei 230031,China)
出处
《中国油料作物学报》
北大核心
2025年第4期1036-1050,共15页
Chinese Journal of Oil Crop Sciences
基金
科技创新2030—重大项目(2022ZD0401815、2022ZD040190902)。
