Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Rapid Detection of Clostridium tetani by Recombinase Polymerase Amplification Using an Exo Probe

  • Guo, Mingjing (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University) ;
  • Feng, Pan (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University) ;
  • Zhang, Liqun (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University) ;
  • Feng, Chunfeng (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University) ;
  • Fu, Jie (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University) ;
  • Pu, Xiaoyun (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University) ;
  • Liu, Fei (Department of clinical laboratory, Xinqiao Hospital and The Second Affiliated Hospital, Army Medical University)
  • Received : 2021.09.09
  • Accepted : 2021.11.22
  • Published : 2022.01.28
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Abstract

Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to identify the fragments of the neurotoxin gene of C. tetani. Primers and the exo probe targeting the conserved region were designed, and the resulting amplicons could be detected in less than 20 min, with a detection limit of 20 copies/reaction. The RPA assay displayed good selectivity, and there were no cross-reactions with other infectious bacteria common in penetrating wounds. Tests of target-spiked serum and pus extract revealed that RPA is robust to interfering factors and has great potential for further development for biological sample analysis. This method has been confirmed to be reliable for discriminating between toxic and nontoxic C. tetani strains. The RPA assay dramatically improves the diagnostic efficacy with simplified device architecture and is a promising alternative to real-time PCR for tetanus detection.

Keywords

Acknowledgement

This study was supported by the National Natural Science Foundation of China (grant numbers 81772284), Youth Cultivation Project of the Army Medical University (2018XQN12), Military Medical Science and Technology Youth Training Project (20QNPY029), and General Projects of Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0799). We are grateful to A. Abd El Wahed in Georg-Aaugust-University Goettingen, Goettingen, Germany for his guidance on the experiment. We also thank Meng Li for his great suggestions for our experiments.

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