基于重组酶介导核酸等温扩增反应的曼氏血吸虫基因检测方法的建立  

Establishment of the gene detection method of Schistosoma mansoni based on the recombinase-aided isothermal amplification assay

作　　者：赵松[1] 刘燕红 叶钰滢 李伟[1] 张键锋[1] 郭利川 应清界 羊海涛[1] 杨坤[1] ZHAO Song;LIU Yan-Hong;YE Yu-Ying;LI Wei;ZHANG Jian-Feng;GUO Li-Chuan;YING Qing-Jie;YANG Hai-Tao;YANG Kun(Key Laboratory of National Health Commission on Parasitic Disease Control and Prevention,Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology,Jiangsu Institute of Parasitic Diseases,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与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,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335-339,344,共6页Chinese Journal of Schistosomiasis Control

基　　金：江苏省国际科技合作项目(BZ2020003);江苏省医学重点人才项目(ZDRCA2016056);江苏省卫健委科研项目(X201802)。

摘　　要：目的建立一种可用于曼氏血吸虫特异性基因片段检测的重组酶介导的核酸等温扩增方法(Recombinase-aided amplification,RAA)。方法以曼氏血吸虫121 bp高重复基因片段作为靶序列,根据RAA反应原理设计、合成引物及荧光探针,建立并优化荧光RAA法反应体系。分别以不同拷贝数的含121 bp基因片段的重组质粒及不同浓度曼氏血吸虫基因组DNA为模板进行荧光RAA法扩增,评价该方法的敏感性;分别以日本和埃及血吸虫虫卵、十二指肠钩虫虫卵、华支睾吸虫囊蚴基因组DNA为模板进行荧光RAA法检测,评价其特异性。结果建立的荧光RAA法可在39℃、20 min内特异性扩增曼氏血吸虫基因组DNA。以重组质粒为模板,荧光RAA法最低可检出的质粒拷贝数为10拷贝/μL;以基因组DNA为模板,荧光RAA法最低可检测浓度为0.1 fg/μL。以日本血吸虫虫卵、埃及血吸虫虫卵、十二指肠钩虫虫卵、华支睾吸虫囊蚴基因组DNA为模板进行荧光RAA法检测,结果均为阴性。结论成功建立了一种可用于曼氏血吸虫DN A检测的荧光RAA法,该方法反应快捷、操作简便,敏感性和特异性均较好。Objective To establish a recombinase-aided isothermal amplification(RAA) assay for nucleic acid detection of Schistosoma mansoni.Methods The 121 bp highly-repeated sequence of S.mansoni was selected as the target gene fragment to be detected.The primers and fluorescent probes were designed using the Amplfix software,and a fluorescent RAA assay was established and optimized.The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of S.mansoni genomic DNA to determine the sensitivity,and this assay was applied to detect the genomic DNA of S.japonicum,S.haematobium,Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity.Results A fluorescent RAA assay was successfully established,which was effective to amplify the specific gene fragments of S.mansoni within 20 min at 39 ℃.The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using S.mansoni genomic DNA samples as templates.The fluorescent RAA assays were all negative for detecting the genomic DNA from S.japonicum,S.haematobium,A.duodenale and C.sinensis.Conclusion A novel fluorescent RAA assay is successfully established,which is simple,rapid,sensitive and specific to detect genomic DNA of 5.mansoni.

关 键 词：曼氏血吸虫 基因检测 核酸等温扩增 荧光探针 重组酶 

分 类 号：R383.24[医药卫生—医学寄生虫学]