Browse > Article
http://dx.doi.org/10.4014/jmb.2109.09022

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)
Publication Information
Journal of Microbiology and Biotechnology / v.32, no.1, 2022 , pp. 91-98 More about this Journal
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
Recombinase polymerase amplification; TeNT gene; rapid detection;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Dong M, Masuyer G, Stenmark P. 2019. Botulinum and tetanus neurotoxins. Annu. Rev. Biochem. 88: 811-837.   DOI
2 Cardinal PR, Henry SM, Joshi MG, Lauerman MH, Park HS. 2020. Fatal necrotizing soft-tissue infection caused by Clostridium tetani in an injecting drug user: A case report. Surg. Infect. (Larchmt) 21: 457-460.   DOI
3 Thwaites CL, Beeching NJ, Newton CR. 2015. Maternal and neonatal tetanus. Lancet 385: 362-370.   DOI
4 Chen J, Xu Y, Yan H, Zhu Y, Wang L, Zhang Y, et al. 2018. Sensitive and rapid detection of pathogenic bacteria from urine samples using multiplex recombinase polymerase amplification. Lab. Chip. 18: 2441-2452.   DOI
5 Berger SA, Cherubin CE, Nelson S, Levine L. 1978. Tetanus despite preexisting antitetanus antibody. JAMA 240: 769-770.   DOI
6 Zhuang QQ, Chen RT, Zheng YJ, Huang KY, Peng HP, Lin Z, et al. 2021. Detection of tetanus toxoid with fluorescent tetanus human IgG-AuNC-based immunochromatography test strip. Biosens. Bioelectron. 177: 112977.   DOI
7 Akbulut D, Grant KA, McLauchlin J. 2005. Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments. J. Clin. Microbiol. 43: 4342-4348.   DOI
8 Behrensdorf-Nicol HA, Bonifas U, Hanschmann KM, Kramer B, Weisser K. 2013. Binding and cleavage (BINACLE) assay for the functional in vitro detection of tetanus toxin: applicability as alternative method for the safety testing of tetanus toxoids during vaccine production. Vaccine 31: 6247-6253.   DOI
9 Finn CW, Jr., Silver RP, Habig WH, Hardegree MC, Zon G, Garon CF. 1984. The structural gene for tetanus neurotoxin is on a plasmid. Science 224: 881-884.   DOI
10 Jiang D, Pu X, Wu J, Li M, Liu P. 2013. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay. J. Microbiol. Biotechnol. 23: 1-6.   DOI
11 Masuyer G, Conrad J, Stenmark P. 2017. The structure of the tetanus toxin reveals pH-mediated domain dynamics. EMBO Rep. 18: 1306-1317.   DOI
12 Yen LM, Thwaites CL. 2019. Tetanus. Lancet 393: 1657-1668.   DOI
13 Alves M, Canoui E, Deforges L, Garderet L, Guidet B, Offenstadt G, et al. 2012. An unexpected trismus. Lancet 380: 536.   DOI
14 Njuguna HN, Yusuf N, Raza AA, Ahmed B, Tohme RA. 2020. Progress toward maternal and neonatal tetanus elimination - Worldwide, 2000-2018. MMWR Morb. Mortal. Wkly. Rep. 69: 515-520.   DOI
15 Piepenburg O, Williams CH, Stemple DL, Armes NA. 2006. DNA detection using recombination proteins. PLoS Biol. 4: e204.   DOI
16 Zhao Y, Chen F, Li Q, Wang L, Fan C. 2015. Isothermal amplification of nucleic acids. Chem. Rev. 115: 12491-12545.   DOI
17 Kong M, Li Z, Wu J, Hu J, Sheng Y, Wu D, et al. 2019. A wearable microfluidic device for rapid detection of HIV-1 DNA using recombinase polymerase amplification. Talanta 205: 120155.   DOI
18 Qing Z, Xu J, Hu J, Zheng J, He L, Zou Z, et al. 2019. In situ amplification-based imaging of RNA in living cells. Angew. Chem. Int. Ed. Engl. 58: 11574-11585.   DOI
19 Chi YK, Zhao W, Ye MD, Ali F, Wang T, Qi RD. 2020. Evaluation of recombinase polymerase amplification assay for detecting Meloidogyne javanica. Plant Dis. 104: 801-807.   DOI
20 Yang M, Ke Y, Wang X, Ren H, Liu W, Lu H, et al. 2016. Development and evaluation of a rapid and sensitive EBOV-RPA test for rapid diagnosis of Ebola virus disease. Sci. Rep. 6: 26943.   DOI
21 Xu H, Xia A, Wang D, Zhang Y, Deng S, Lu W, et al. 2020. An ultraportable and versatile point-of-care DNA testing platform. Sci. Adv. 6: eaaz7445.   DOI
22 Kissenkotter J, Bohlken-Fascher S, Forrest MS, Piepenburg O, Czerny CP, Abd El Wahed A. 2020. Recombinase polymerase amplification assays for the identification of pork and horsemeat. Food Chem. 322: 126759.   DOI
23 Ergonul O, Egeli D, Kahyaoglu B, Bahar M, Etienne M, Bleck T. 2016. An unexpected tetanus case. Lancet Infect. Dis. 16: 746-752.   DOI
24 Melkert D, Kahema L, Melkert P. 2014. Reduction of mortality due to tetanus by immunisation and proper wound management of the patients in Sengerema Designated District Hospital, Tanzania. Trop. Doct. 44: 163-165.   DOI
25 Okike CO, Muoneke UV, Uwaezuoke SN, Mbagwu EN, Onyeka-Okite E. 2020. The prevalence and case-fatality rates of post-neonatal tetanus in a population of hospitalized nigerian children: An 8-year retrospective review. J. Trop. Pediatr. 66: 201-209.   DOI
26 Zhang X, Lowe SB, Gooding JJ. 2014. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosens. Bioelectron. 61: 491-499.   DOI
27 Kyu HH, Mumford JE, Stanaway JD, Barber RM, Hancock JR, Vos T, et al. 2017. Mortality from tetanus between 1990 and 2015: findings from the global burden of disease study 2015. BMC Public Health 17: 179.   DOI
28 Lai X, Hosyanto FF, Xu L. 2020. Risk of Clostridium tetani infection in an elderly patient following hemorrhoid ligation. J. Int. Med. Res. 48: 300060520963983.
29 Zhang L, Peng J, Chen J, Xu L, Zhang Y, Li Y, et al. 2021. Highly sensitive detection of low-abundance BRAF V600E mutation in fine-needle aspiration samples by zip recombinase polymerase amplification. Anal. Chem. 93: 5621-5628.   DOI
30 Mori Y, Katasako A, Matsunaga S, Matono T. 2019. Tetanus: remember to vaccinate. Lancet 393: 2331.   DOI
31 Geng Y, Tan K, Liu L, Sun XX, Zhao B, Wang J. 2019. Development and evaluation of a rapid and sensitive RPA assay for specific detection of Vibrio parahaemolyticus in seafood. BMC Microbiol. 19: 186.   DOI
32 Trieu HT, Lubis IN, Qui PT, Yen LM, Wills B, Thwaites CL, et al. 2016. Neonatal tetanus in Vietnam: comprehensive intensive care support improves mortality. J. Pediatric. Infect. Dis. Soc. 5: 227-230.   DOI
33 Pumford EA, Lu J, Spaczai I, Prasetyo ME, Zheng EM, Zhang H, et al. 2020. Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics. Biosens. Bioelectron. 170: 112674.   DOI
34 Yang B, Kong J, Fang X. 2019. Bandage-like wearable flexible microfluidic recombinase polymerase amplification sensor for the rapid visual detection of nucleic acids. Talanta 204: 685-692.   DOI
35 Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. 2019. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat. Protoc. 14: 2986-3012.   DOI
36 Cohen JE, Wang R, Shen RF, Wu WW, Keller JE. 2017. Comparative pathogenomics of Clostridium tetani. PLoS One 12: e0182909.   DOI
37 Higgins M, Ravenhall M, Ward D, Phelan J, Ibrahim A, Forrest MS, et al. 2019. PrimedRPA: primer design for recombinase polymerase amplification assays. Bioinformatics 35: 682-684.   DOI
38 Behrmann O, Bachmann I, Spiegel M, Schramm M, Abd El Wahed A, Dobler G, et al. 2020. Rapid detection of SARS-CoV-2 by low volume real-time single tube reverse transcription recombinase polymerase amplification using an exo probe with an internally linked quencher (Exo-IQ). Clin. Chem. 66: 1047-1054.   DOI
39 Strayer-Scherer A, Jones JB, Paret ML. 2019. Recombinase polymerase amplification assay for field detection of tomato bacterial spot pathogens. Phytopathology 109: 690-700.   DOI