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

doi:10.12140/j.issn.1000-7423.2020.02.006

Abstract

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

CLC Number:

R383.24

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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