Chin J Schisto Control ›› 2019, Vol. 31 ›› Issue (5): 468-.

Previous Articles     Next Articles

Establishment and preliminary evaluation of recombinase aided isothermal amplification (RAA) assay for specific nucleic acid detection of Clonorchis sinensis

ZHANG Qiang1, 2, DING Xin1, 2, WU Xiao-Min1, 2, LIU Yan-Hong3, LIU Jian-Feng1, 2, XU Xiang-Zhen1, 2, YING Qing-Jie3, CAO Jun1, 2, DAI Yang1, 2*   

  1. 1 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, Wuxi 214064, China; 2 Public Health Research Center, Jiangnan University, China; 3 Jiangsu Qitian Gene Technology Co., Ltd., China
  • Online:2019-11-04 Published:2019-11-05

重组酶介导的华支睾吸虫特异性核酸等温扩增方法的建立及初步评价

张强1, 2,丁昕1, 2,吴小珉1, 2,刘燕红3,刘剑峰1, 2,徐祥珍1, 2,应清界3,曹俊1, 2,戴洋1, 2*   

  1. 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 江南大学公共卫生研究中心;3 江苏省奇天生物科技有限公司
  • 作者简介:张强,男,硕士,医师。研究方向:寄生虫病防治
  • 基金资助:
    江苏省“科教强卫工程”项目(ZDXKA2016016);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)

Abstract: Objective To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis. Methods The 18S ribosomal RNA (18S rRNA) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis. Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA assay was evaluated using the genomic DNA of Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis?infected samples was preliminarily assessed in the field. Results A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 ℃ within 20 min. The lowest detection limit was 10 copies/μL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/μL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides, E. granulosus, S. japonicum, A. duodenale, and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis?infected samples in the field, which successfully detected C. sinensis?infected human and rat fecal samples and Pseudorasbora parva samples. Conclusion A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis.

Key words: Clonorchis sinensis, Isothermal nucleic acid amplification, Recombinase, Detection efficiency

摘要: 目的 建立一种可用于华支睾吸虫检测的重组酶介导的等温核酸扩增方法(RAA)。方法 以华支睾吸虫18S rRNA基因序列作为靶序列,设计、合成、筛选特异性引物和探针,建立快速检测华支睾吸虫的荧光RAA检测方法。分别以含不同拷贝数DNA片段的重组质粒和不同浓度华支睾吸虫基因组DNA为模板进行荧光RAA扩增,评价其检测敏感性;分别以似蚓蛔线虫、细粒棘球绦虫、日本血吸虫、十二指肠钩虫、曼氏血吸虫基因组DNA为模板进行荧光RAA扩增,评价其检测特异性;通过抽提含华支睾吸虫虫卵的粪便及含囊蚴的淡水鱼肉样本DNA并进行荧光RAA扩增,初步评价其检测现场样本的能力。结果 成功建立了华支睾吸虫检测荧光RAA法,其可在39 ℃ 20 min内实现对华支睾吸虫DNA的特异性扩增。以含不同拷贝数DNA片段的重组质粒为模板,该方法最低检出限为10 拷贝/μL;以不同浓度华支睾吸虫基因组DNA为模板,该方法最低检出限为3 pg/μL;以似蚓蛔线虫、细粒棘球绦虫、日本血吸虫、十二指肠钩蚴及曼氏血吸虫基因组DNA为模板,检测结果均为阴性。该方法可成功检出感染华支睾吸虫的人体、大鼠粪便样本以及麦穗鱼样本,具备较好的现场样本检测能力。结论 成功建立了一种简便、快速、敏感、特异的可用于华支睾吸虫检测的RAA法。

关键词: 华支睾吸虫, 核酸等温扩增, 重组酶, 检测效能

CLC Number: