基因编辑外源基因Cas9定性定量检测方法的研究  

Study of Qualitative and Quantitative Detection Methods for Exogenous Gene Cas9 in Gene Editing

作　　者：丁霖 王颢潜 张敏[1] 王雨玲 汪小福[1] 陈笑芸[1] 徐俊锋[1] 张秀杰 彭城 DING Lin;WANG Hao-Qian;ZHANG Min;WANG Yu-Ling;WANG Xiao-Fu;CHEN Xiao-Yun;XU Jun-Feng;ZHANG Xiu-Jie;PENG Cheng(Institute of Agro-product Safety and Nutrition,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Development Center for Science and Technology,Ministry of Agriculture and Rural Affairs of the People's Republic of China,Beijing 100176,China)

机构地区：[1]浙江省农业科学院农产品质量安全与营养研究所,杭州310021 [2]农业农村部科技发展中心,北京100176

出　　处：《农业生物技术学报》2022年第10期1855-1868,共14页Journal of Agricultural Biotechnology

基　　金：浙江省旱粮育种专项(2021C02064-6);浙江省公益项目(LGN22C130015);国家自然科学基金(31471417;31501280)。

摘　　要：对基因编辑产品进行有效监管、消除公众疑虑是基因编辑产品在我国产业化的前提。目前,Cas9核酸内切酶是基因编辑技术中最常用的手段,因此需要建立针对Cas9核酸内切酶的定性定量检测方法。本研究以基因编辑中最常用的Cas9核酸内切酶外源序列为靶位点,设计和筛选引物、探针,进行PCR体系优化,确定最佳的引物浓度与退火温度,通过特异性、灵敏性等测试建立了检测基因编辑外源基因Cas9的普通PCR和qPCR检测方法。在检测方法特异性实验中,普通PCR和qPCR方法均只在含有Cas9等外源DNA的样品中扩增出了阳性结果,而其他不含有Cas9核酸内切酶的样品则均为阴性结果;在方法灵敏性测试中,普通PCR检测方法的检出限为0.1%,q PCR法检测下限(limit of detection,LOD)为16个拷贝;在方法稳定性实验中,普通PCR和qPCR方法在其相应最低检出限浓度下分别进行了67与60次重复实验,结果均为阳性。该定性定量方法适用于不同基因编辑作物,如水稻(Oryza sativa)、大豆(Glycine max)和油菜(Brassica napus)等。本研究建立的Cas9定性及定量检测方法特异性良好、灵敏度较高、稳定性较强。该方法的建立为将来实现对基因编辑产品的有效监测提供了一定的技术支撑。The prerequisites of bringing gene-edited products to market are effective supervision of them and eliminating public doubts in China.At present,Cas9 endonuclease is the most commonly used method of geneediting technology,therefore,it is necessary to establish a qualitative and quantitative detection method for Cas9 endonuclease.In this study,the most commonly used Cas9 endonuclease exogenous sequence in geneediting was used as the target site.The primers and probes of conventional PCR and q PCR were designed,and the most suitable primers and probes were selected through experiments.The system was optimized in the conventional PCR method to determine the optimal primer concentration and annealing temperature for subsequent experiments.By testing specificity and sensitivity,conventional PCR and q PCR detection methods were established for detecting the Cas9 in gene-editing.Meanwhile,the detection limits of qualitative and quantitative detection methods were determined.In the specificity experiment of detection method,the target band was amplified by conventional PCR method in the samples containing Cas9,but the samples without Cas9 endonuclease were negative results.The qPCR method obtained amplification curves in samples containing Cas9,while other samples without Cas9 endonuclease were not amplified;In the method sensitivity test,conventional PCR could detect 0.1%of Cas9 event,and limit of detection(LOD)of q PCR method could reach 16 copies of gene-edited rice genomic DNA;In the method stability experiment,the conventional PCR performed 67 repeated experiments by using the gene-edited samples with a mass fraction of 0.1%,and the experimental results all amplified the target bands.The qPCR method was repeated 60 times at the corresponding minimum detection limit concentration,and the experimental results all obtained the amplification curve.At the same time,this qualitative and quantitative method is applicable to different geneedited crops,such as rice(Oryza sativa),soybean(Glycine max)and rape(Brassica napus).T

关 键 词：基因编辑技术 常规PCR qPCR Cas9 

分 类 号：S41-30[农业科学—植物保护]