绵羊无浆体LAMP-CRISPR/Cas12a检测方法的建立  

Development of LAMP-CRISPR/Cas12a assay for the detection of Anaplasma ovis

作　　者：郭心龙 周勇志[2] 曹杰[2] 王亚楠[2] 张厚双[2] 段德勇[1] 周金林[2] GUO Xinlong;ZHOU Yongzhi;CAO Jie;WANG Ya'nan;ZHANG Houshuang;DUAN Deyong;ZHOU Jinlin(College of Veterinary Medicine,Hunan Agricultural University,Changsha,410128,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2025年第3期330-337,共8页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2022YFE0120100)。

摘　　要：绵羊无浆体(Anaplasma ovis)是一种蜱传、寄生于红细胞内的细菌病原体,引起绵羊、山羊和野生反刍动物等发病,对我国养羊业发展造成严重危害。尽管已有多种绵羊无浆体诊断方法报道,但仍然缺少简易快捷、应用于现场的检测技术。本研究旨在建立一种针对绵羊无浆体msp4基因的LAMP-CRISPR/Cas12a检测方法。该检测方法是在恒定温度下进行的,不需要特殊设备仪器,能够产生荧光呈现可视化的结果。通过优化LAMP-CRISPR/Cas12a检测方法的体系以确定各成分最佳浓度比。该检测方法能够检测到绵羊无浆体msp4基因最低质粒DNA拷贝数为3.90 copies/μL,具有较高的敏感性。检测其他无浆体和其他常见羊病原体时呈阴性,表明特异性良好。对临床样本的应用检测,与常规PCR检测结果符合率为100%。结果表明,建立的LAMP-CRISPR/Cas12a体系适用于绵羊无浆体的检测,具有快捷方便的现场诊断和在资源匮乏的情况下应用的优势。Anaplasma ovis is a tick-borne bacterial pathogen that parasitizes erythrocytes,caus-ing disease in sheep,goats,and wild ruminants.It poses a serious threat to the development of the sheep farming industry in China.Although various diagnostic methods for A.ovis have been reported,there is still a lack of simple and rapid detection techniques for field application.This study aimed to estab-lish a LAMP-CRISPR/Cas12a detection method targeting the msp4 gene of A.ovis.This detection method is conducted at a constant temperature and dose not require special equipment,allowing for the generation of fluorescent results that could be visually interpreted.By optimizing the system for the LAMP-CRISPR/Cas12a method,the optimal concentration ratios of the components were determined.The method could detect a minimum plasmid DNA copy number of 3.90 copies/μL for the msp4 gene of A.ovis,demonstrating high sensitivity.Additionally,the results were negative for other Anaplasma or common sheep pathogens,indicating good specificity.In clinical sample applications,the consistency with conventional PCR results was 100%.The results suggested that the established LAMP-CRISPR/Cas12a system is suitable for the detection of A.ovis,offering advantages for rapid and convenient field di-agnosis and application in resource-scarce situations.

关 键 词：绵羊无浆体 msp4基因 LAMP CRISPR 

分 类 号：S852.64[农业科学—基础兽医学]