重组酶聚合酶扩增结合电化学DNA传感器检测日本血吸虫方法的建立

doi:10.12140/j.issn.1000-7423.2020.02.006

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (2): 168-174.doi: 10.12140/j.issn.1000-7423.2020.02.006

重组酶聚合酶扩增结合电化学DNA传感器检测日本血吸虫方法的建立

邓王平¹, 徐斌¹, 洪清华², 王盛琳¹, 吕超¹, 李银龙¹, 宋世平³, 陈军虎¹, 许静¹, 李石柱¹, 胡薇¹^,², 周晓农¹^,^*()

1 中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025
2 复旦大学生命科学学院微生物学与微生物工程系,上海 200043
3 中国科学院上海应用物理研究所,上海 201800

收稿日期:2019-12-31 出版日期:2020-04-30 发布日期:2020-05-11
通讯作者: 周晓农
作者简介:邓王平（1984-）,女,博士,助理研究员,从事血吸虫病诊断及分子流行病学研究。E-mail： dengwp@nipd.chinacdc.cn
基金资助:
国家科技重大专项(2018ZX10101002-002);国家科技重大专项(No. 2018ZX10004222-004);第60批中国博士后科学基金面上资助(2016M600111)

Establishment of the detection method for Schistosoma japonicum by recombinase polymerase amplification combined with electrochemical DNA biosensor

Wang-ping DENG¹, Bin XU¹, Qing-hua HONG², Sheng-lin WANG¹, Chao LV¹, Yin-long LI¹, Shi-ping SONG³, Jun-hu CHEN¹, Jing XU¹, Shi-zhu LI¹, Wei HU¹^,², Xiao-nong ZHOU¹^,^*()

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research; WHO Collaborating Center of Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
2 Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
3 Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China

Received:2019-12-31 Online:2020-04-30 Published:2020-05-11
Contact: Xiao-nong ZHOU
Supported by:
Supported by the National Science and Technology Major Projects(2018ZX10101002-002);Supported by the National Science and Technology Major Projects(No. 2018ZX10004222-004);the 60th Batch of General Finantial Grant from the China Postdoctoral Science Foundation(2016M600111)

摘要/Abstract

摘要：

目的联合重组酶聚合酶扩增及电化学DNA传感器检测技术,建立一种敏感、特异且简单的日本血吸虫（Schistosoma japonicum）核酸检测方法。方法提取日本血吸虫基因组DNA,以SjR2基因片段为靶序列,设计重组酶聚合酶扩增（RPA）引物与探针,与电化学DNA传感器（EC）检测技术联合,将探针固定于多通道电极芯片进行界面RPA扩增及电化学检测,建立日本血吸虫核酸等温检测方法。优化RPA反应条件,通过检测10 ^-4、10 ^-5、10 ^-6、10 ^-7、10 ^-8 ng日本血吸虫基因组DNA,评价RPA-EC方法的敏感性;通过检测10 ng日本血吸虫、埃及血吸虫、曼氏血吸虫、卫氏并殖吸虫、大片吸虫及华支睾吸虫的基因组DNA,评价RPA-EC方法的特异性。取10只6~8周龄C57成年小鼠,每鼠经腹部贴片感染40条日本血吸虫尾蚴,分别于感染前（0 d）和感染后7、21、35 d收集尾静脉血清,验证RPA-EC方法检测感染动物动态血清DNA中SjR2基因片段的可行性。结果 RPA-EC联合检测方法可在37 ℃、30 min内完成SjR2基因片段的界面快速扩增及检测,成虫基因组DNA的最低检出限可达10 ^-8 ng,与曼氏血吸虫、埃及血吸虫、卫氏并殖吸虫、大片吸虫及华支睾吸虫基因组DNA无交叉反应,具有较好的特异性。日本血吸虫感染小鼠实验结果显示,RPA-EC联合检测方法可高敏感检出感染后7、21、35 d等不同感染期小鼠血清中的SjR2基因片段。结论本研究所建立的RPA-EC联合检测方法敏感性高、特异性好、操作简便,具有一定的应用前景。

关键词: 日本血吸虫, 重组酶聚合酶扩增, 电化学DNA传感器, 早期检测

Abstract:

Objective To establish a sensitive, specific and simple nucleic acid detection method for Schistosoma japonicum by combining recombinase polymerase reaction (RPA) with electrochemical DNA sensor.Methods Genomic DNA of S. japonicum adult worms was extracted. Applying the SjR2 gene fragment as the target sequence, primers and probe were designed for recombinase polymerase amplification (RPA), which was then combined with electrochemical DNA sensor(EC) technique; the probe was immobilized on the multi-channel electrode chips for interfacial RPA amplification and electrochemical detection, thus establishing the isothermal detection method (RPA-EC) for S. japonicum nucleic acid. The RPA reaction conditions were optimized and the sensitivity of the RPA-EC method was tested by detecting 10 ^-4 ng, 10 ^-5 ng, 10 ^-6 ng, 10 ^-7ng and 10 ^-8 ng of S. japonicum genomic DNA. The specificity of the RPA-EC method was evaluated by detecting 10 ng of genomic DNA of S. japonicum, S. haematobium, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis. To verify the feasibility of the RPA-EC method to detect the dynamic changes of the SjR2 gene fragment in sera, 10 adult C57 mice aged 6 to 8 weeks were each infected with 40 S. japonicum cercariae by abdominal challenge, and serum samples were collected on days before infection (0 d), and 7, 21 and 35 after infection. Results The RPA-EC assay could complete the amplification and detection of SjR2 fragment within 30 min at 37 ℃, and the lower detactable limit for SjR2 fragment in adult S. japonicum genomic DNA could be 10 ^-8 ng. In addition, there was no cross-reaction with the genomic DNA of S. mansoni, S. haematobium, P. westermani, F. gigantica and C. sinensis, suggesting a high specificity. It is of note, the results of animal experiments showed that the RPA-EC assay could sensitively detect the SjR2 gene fragment in the sera of mice on day 7, 21 and 35 post infection. Conclusion The RPA-EC combinational detection method established in this study exhibits high sensitivity and good specificity, and is easy to operate, showing the potential for future application.

Key words: Schistosoma japonicum, Recombinase polymerase amplification, Electrochemical DNA sensor, Early detection

中图分类号:

R383.24

邓王平, 徐斌, 洪清华, 王盛琳, 吕超, 李银龙, 宋世平, 陈军虎, 许静, 李石柱, 胡薇, 周晓农. 重组酶聚合酶扩增结合电化学DNA传感器检测日本血吸虫方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 168-174.

Wang-ping DENG, Bin XU, Qing-hua HONG, Sheng-lin WANG, Chao LV, Yin-long LI, Shi-ping SONG, Jun-hu CHEN, Jing XU, Shi-zhu LI, Wei HU, Xiao-nong ZHOU. Establishment of the detection method for Schistosoma japonicum by recombinase polymerase amplification combined with electrochemical DNA biosensor[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(2): 168-174.

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重组酶聚合酶扩增结合电化学DNA传感器检测日本血吸虫方法的建立

Establishment of the detection method for Schistosoma japonicum by recombinase polymerase amplification combined with electrochemical DNA biosensor

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