收稿日期: 2019-12-31
网络出版日期: 2020-05-11
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
Received date: 2019-12-31
Online published: 2020-05-11
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)
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。
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 . DOI: 10.12140/j.issn.1000-7423.2020.02.006
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.
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