摘要
旨在建立一种基于重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)和成簇规律间隔短回文重复序列/CRISPR相关蛋白12a(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a,CRISPR/Cas12a)系统的MSTN基因编辑猪检测方法,以满足基因编辑动物研发、育种等过程中核酸检测以及基因型鉴定的需求。本研究构建含有猪MSTN基因突变序列和野生型序列的标准质粒并设计靶向标准质粒的crRNA,基于RPA和CRISPR/Cas12a技术建立荧光报告和胶体金试纸条检测体系,筛选特异性识别标准质粒的crRNA、优化其反应条件并评价其检测灵敏度,最后通过检测猪耳组织样品评价本方法的准确性和稳定性。研究结果表明,该方法能够特异性识别MSTN基因2 bp碱基缺失序列和MSTN基因野生型序列。荧光报告体系最低可检测到4.1×10^(1)copies·μL^(-1)的标准质粒,胶体金试纸条体系最低可检测到4.1×10^(3)copies·μL^(-1)的标准质粒。通过对猪耳组织样本的检测,证明了该检测体系的准确性和可靠性。综上所述,本研究建立的MSTN基因编辑猪RPA-CRISPR/Cas12a检测方法具有操作简便、特异性和灵敏度高的特点,为MSTN基因编辑猪检测提供了重要技术支撑,具有广阔的应用前景。
This study aimed to establish a detection method for MSTN gene-edited pigs based on recombinase polymerase amplification(RPA)and the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12(CRISPR/Cas12a)system,to meet the needs of nucleic acid testing and genotype identification in the research and breeding of gene-edited animals.Standard plasmids containing the porcine MSTN gene mutant sequence and wild-type sequence were constructed,with crRNAs targeting these standard plasmids designed.Detection systems integrating fluorescence reporter or colloidal gold test strip were established based on RPA and CRISPR/Cas12a technologies.Through systematic screening of crRNAs specifically recognizing the standard plasmids,optimization of reaction conditions,and evaluation of detection sensitivity,the method′s analytical performance was characterized.Ultimately,the accuracy and stability of this approach were validated by detecting porcine ear tissue samples.The results demonstrated that the method could specifically identify the 2 bp deletion and wild-type sequences of the MSTN gene.The fluorescence reporter system could detect as low as 4.1×10^(1) copies·μL^(-1) of the standard plasmid,while the colloidal gold test strip system could detect as low as 4.1×10^(3) copies·μL^(-1).The accuracy and reliability of the system were validated by testing porcine ear tissue samples.The RPA-CRISPR/Cas12a detection method for MSTN gene-edited pigs established in this study is characterized by its simplicity,high specificity,and high sensitivity.It provides important technical support for the detection of MSTN gene-edited pigs and holds broad application prospects.
作者
迟顺顺
吴丹
王楠
王婉洁
聂雨欣
牟玉莲
刘志国
朱振东
李奎
CHI Shunshun;WU Dan;WANG Nan;WANG Wanjie;NIE Yuxin;MU Yulian;LIU Zhiguo;ZHU Zhendong;LI Kui(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China;Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture,Key Laboratory of Livestock and Poultry Multi-omics of Ministry of Agriculture and Rural Affairs,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518124,China;Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Tianjin Ningheyuan Swine Breeding Farm Co.,Ltd.,Tianjin 301504,China)
出处
《畜牧兽医学报》
北大核心
2025年第8期3734-3748,共15页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
农业生物育种国家科技重大专项(2022ZD04020)
中国农业科学院科技创新工程(ASTIP-IAS05)。
