基于重组酶介导等温扩增技术的细粒棘球绦虫核酸检测方法的建立  

Establishment of a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification assay

作　　者：丁昕 刘燕红 倪碧娴 王晓婷[1] 徐祥珍[1] 应清界 戴洋[1] 曹俊[1] DING Xin;LIU Yan-Hong;NI Bi-Xian;WANG Xiao-Ting;XU Xiang-Zhen;YING Qing-Jie;Dai Yang;CAO Jun(National Health Commission Key Laboratory of Parasitic Disease Control and Prevention,Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology,Jiangsu Institute of Parasitic Diseases,Public Health Research Center,Jiangnan Universi-ty,Wuxi 214064,China;Jiangsu Qitian Gene Technology Co.,Ltd.,China)

机构地区：[1]国家卫生健康委员会寄生虫病预防和控制技术重点实验室、江苏省寄生虫与媒介控制技术重点实验室、江苏省血吸虫病防治研究所、江南大学公共卫生研究中心,无锡214064 [2]江苏省奇天生物科技有限公司

出　　处：《中国血吸虫病防治杂志》2020年第4期340-344,共5页Chinese Journal of Schistosomiasis Control

基　　金：江苏省“科教强卫工程”项目(ZDXKA2016016);江苏省属公益类科研院所自主科研项目(BM2018020);江苏省青年医学人才项目(QNRC2016622)。

摘　　要：目的建立一种基于重组酶介导等温扩增技术(RAA)的细粒棘球绦虫核酸检测方法。方法针对细粒棘球绦虫12S rRNA基因序列片段,设计、筛选并合成RAA特异性扩增引物和荧光检测探针,构建细粒棘球绦虫荧光RAA检测方法。分别以含靶序列的不同拷贝数重组质粒和不同浓度细粒棘球绦虫基因组DNA为模板进行荧光RAA扩增,评价其检测灵敏度;分别以细粒棘球绦虫、多房棘球绦虫、日本血吸虫、曼氏血吸虫、十二指肠钩虫、华支睾吸虫、牛带绦虫、曼氏迭宫绦虫、猪带绦虫基因组DNA为模板进行荧光RAA扩增,评价其检测特异性。结果成功建立了细粒棘球绦虫荧光RAA检测法,在39℃条件下20 min内可以实现对细粒棘球绦虫基因组DNA特异性扩增,最低可以检测出10拷贝/μL含靶序列的重组质粒DN A和0.1 ng/μL细粒棘球绦虫基因组DN A样本,具备较高敏感性;对多房棘球绦虫、日本血吸虫、曼氏血吸虫、十二指肠钩虫、华支睾吸虫、牛带绦虫、曼氏迭宫绦虫、猪带绦虫基因组DNA均无阳性扩增,具备较高特异性;且该荧光RAA法可成功检出细粒棘球绦虫包囊中DNA。结论成功建立了一种快速、灵敏、特异的可用于细粒棘球绦虫核酸检测的荧光RAA法。Objective To establish a nucleic acid assay for detection of Echiuococcus granulosus based on recombinase-aided isothermal amplification(RAA) assay.Methods The 12 S rRNA gene of E.granulosus was selected as the target gene,and the specific primers and fluorescent probes for RAA assay were designed,screened and synthesized to establish a fluorescent RAA assay for detection of E.granulosus.The sensitivity of the fluorescent RAA assay was evaluated using different copy numbers of target gene sequence-contained recombinant plasmids and various concentrations of E.granulosus genomic DNA as templates,and the specificity of the fluorescent RAA assay was evaluated using the genomic DNA from E.granulosus,E.multilocularis,Schistosoma japouicum,S.mansoui,Aucylostoma duodeuale,Clonorchis sinensis,Taenia saginata,Spirometra mansoni and Taeuia solium as templates.Results A fluorescent RAA assay was successfully established for detection of E.granulosus,which achieved specific amplification of E.grauulosus genomic DNA within 20 min at 39℃.The lowest detection limit of the fluorescent RAA assay was 10 copies/μL of recombinant plasmids and 0.1 ng/μL E.granulosus genomic DNA,which exhibited a high sensitivity,and the fluorescent RAA assay was all negative for the genomic DNA from E.multilocularis,S.japouicum,S.mansoui,A.duodeuale,C.sinensis,T.saginata,Spirometra mansoni and T.solium,which exhibited a high specificity.In addition,this fluorescent RAA assay successfully detected genomic DNA from E.granulosus cysts.Conclusion A rapid,sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of E.granulosus.

关 键 词：细粒棘球绦虫 重组酶 核酸等温扩增 荧光探针 诊断效能 

分 类 号：R383.33[医药卫生—医学寄生虫学]