环形泰勒虫SHERLOCK-LF检测方法的建立与初步应用  

Establishment and preliminary application of SHERLOCK-LF detection method forTheileria annulata

作　　者：苏书晓 赵帅阳[1] 刘军龙[1] 张楚晗 朱浩瀚 关贵全[1] 殷宏[1,2] 罗建勋 SU Shuxiao;ZHAO Shuaiyang;LIU Junlong;ZHANG Chuhan;ZHU Haohan;GUAN Guiquan;YIN Hong;LUO Jianxun(State Key Laboratory for Animal Disease Control and Prevention/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州大学,江苏扬州225009

出　　处：《中国兽医科学》2024年第9期1182-1187,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32202836,31972706);国家重点研发计划项目(2022YFD1302102);农业科技创新工程项目(CAAS-ASTIP-2016-LVRI);国家肉牛牦牛产业技术体系项目(CARS-37);甘肃省基础研究创新群体项目(22JR5RA024);甘肃省科技厅特派团项目(22CX8NA011);国家寄生虫资源库项目(NPRC-2019-194-30)。

摘　　要：为快速、准确、简便地对环形泰勒虫感染进行检测,选取环形泰勒虫子孢子表面抗原基因作为靶标分子,设计特异性RPA引物及crRNA(CRISPR RNA)序列,建立环形泰勒虫的特异性高灵敏酶报告基因解锁(specific high-sensitive enzymatic reporter unlocking,SHERLOCK)-LF检测方法,并分析该方法的特异性、敏感性和符合率。结果显示:该方法可特异性检测环形泰勒虫基因组,与中华泰勒虫、东方泰勒虫、牛巴贝斯虫和双芽巴贝斯虫基因组均无交叉反应,且检测灵敏度达到1.42 copies/μL;与行业标准PCR方法检测结果相比,其符合率达到86.96%。本研究建立的SHERLOCK-LF检测方法可在1.5 h内完成对环形泰勒虫的检测,为该病的现场诊断及防控提供了技术支撑。To detect the infection of Theileria annulata quickly,accurately,and conveniently,T.annulata sporozoite surface antigen gene was selected as the target molecule to design specific RPA primers and crRNA(CRISPR RNA)sequences,and then a specific and highly sensitive enzyme reporter unlocking(SHERLOCK)-LF method was established to detect T.annulata infection.Meanwhile,the specificity,sensitivity and compliance of the method were analyzed.The results showed that the method could specifically detect the genome of T.annulata,and have no cross-reactivity with the genomes of Theileria sinensis,Theileria orientalis,Babesia bovis,or Babesia bigemina.As for its sensitivity,the method could detect 1.42 copies/μL and have 86.96%compliance compared with the PCR method.Furthermore the method could finish the detection of T.annulata in 1.5 h and provide technical support for its on-site diagnosis,prevention and control.

关 键 词：环形泰勒虫 RPA Cas13a crRNA 侧向流试纸条 

分 类 号：S852.723[农业科学—基础兽医学]