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Research article

References

  1. Akbulut, D., K. A. Grant, and J. McLauchlin. 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.
    Pubmed PMC CrossRef
  2. Boyanton, B. L., P. Sural, C. R. Loomis, C. Pesta, L. GonzalezKrellwitz, B. Robinson-Dunn, and P. Riska. 2012. Loopmediated isothermal amplification compared to real-time PCR and enzyme immunoassay for toxigenic Clostridium difficile detection. J. Clin. Microbiol. 50: 640-645.
    Pubmed PMC CrossRef
  3. Chon, J. W., J. S. Park, J. Y. Hyeon, C. Park, K. Y. Song, K. W. Hong, et al. 2012. Development of real-time PCR for the detection of Clostridium perfringens in meats and vegetables. J. Microbiol. Biotechnol. 22: 530-534.
    Pubmed CrossRef
  4. Enomoto, Y., T. Yoshikawa, M. Ihira, S. Akimoto, F. Miyake, C. Usui, et al. 2005. Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method. J. Clin. Microbiol. 43: 951-955.
    Pubmed PMC CrossRef
  5. Fall, J., G. Chakraborty, T. Kono, M. Maeda, T. Itami, and M. Sakai. 2008. Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp. FEMS Microbiol. Lett. 288: 171177.
    Pubmed CrossRef
  6. Han, X., C. Ding, L. He, Q. Hu, and S. Yu. 2011. Development of loop-mediated isothermal amplification (LAMP) targeting the GroEL gene for rapid detection of Riemerella anatipestifer. Avian Dis. 55: 379-383.
    Pubmed CrossRef
  7. He, L., Y. Q. Zhou, M. C. Oosthuizen, and J. L. Zhao. 2009. Loop-mediated isothermal amplification (LAMP) detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758) in China. Vet. Parasitol 165: 36-40.
    Pubmed CrossRef
  8. Kaneko, I., K. Miyamoto, K. Mimura, N. Yumine, H. Utsunomiya, S. Akimoto, and B. A. McClane. 2011. Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods. Appl. Environ. Microbiol. 77: 75267532.
    Pubmed PMC CrossRef
  9. Kim, J. D., J. H. Yoon, Y. H. Park, D. W. Lee, K. S. Lee, C. H. Choi, et al. 2003. Isolation and identification of a lactic acid bacterial strain KJ-108 and its capability for deodorizing malodorous gases under anaerobic culture conditions. J. Microbiol. Biotechnol. 13: 207-216.
  10. Langkamp, D. L., S. Hoshaw-Woodard, M. E. Boye, and S. Lemeshow. 2001. Delays in receipt of immunizations in lowbirthweight children: A nationally representative sample. Arch. Pediatr. Adolesc. Med. 155: 167-172.
    Pubmed
  11. Lee, H. C., W. C. Ko, and Y. C. Chuang. 2000. Tetanus of the elderly. J. Microbiol. Immunol. Infect. 33: 191-196.
    Pubmed
  12. Mao, Z., Y. Qiu, L. Zheng, J. Chen, and J. Yang. 2012. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea). J. Microbiol. Methods 89: 179-184.
    Pubmed CrossRef
  13. Mekata, T., T. Kono, R. Savan, M. Sakai, J. Kasornchandra, T. Yoshida, and T. Itami. 2006. Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP). J. Virol. Methods 135: 151-156.
    Pubmed CrossRef
  14. Nagamine, K., T. Hase, and T. Notomi. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probes 16: 223-229.
    Pubmed CrossRef
  15. Njiru, Z. K., A. S. Mikosza, E. Matovu, J. C. Enyaru, J. O. Ouma, S. N. Kibona, et al. 2008. African trypanosomiasis:Sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int. J. Parasitol. 38: 589-599
    Pubmed CrossRef
  16. Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: E63.
    Pubmed PMC CrossRef
  17. Ohtsuka, K., K. Yanagawa, K. Takatori, and Y. Hara-Kudo. 2005. Detection of Salmonella Enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates. Appl. Environ. Microbiol. 71: 6730-6735.
    Pubmed PMC CrossRef
  18. Peng, Y., Z. Xie, J. Liu, Y. Pang, X. Deng, L. Xie, et al. 2011. Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay. Virol. J. 8: 337.
    Pubmed PMC CrossRef
  19. Plutzer, J., A. Torokne, and P. Karanis. 2010. Combination of ARAD microfibre filtration and LAMP methodology for simple, rapid and cost-effective detection of human pathogenic Giardia duodenalis and Cryptosporidium spp. in drinking water. Lett. Appl. Microbiol. 50: 82-88.
    Pubmed CrossRef
  20. Saadatmandzadeh, M., H. Eshghi, M. Rahimizadeh, M. Sankian, and S. A. Jamehdar. 2011. Detection of Clostridium tetani by fluorescent amplification based specific hybridization. Afr. J. Microbiol. Res. 5: 5489-5492.
  21. Sakuma, T., Y. Kurosaki, Y. Fujinami, T. Takizawa, and J. Yasuda. 2009. Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification. J. Appl. Microbiol. 106: 1252-1259.
    Pubmed CrossRef
  22. Sheffield, J. S. and S. M. Ramin. 2004. Tetanus in pregnancy. Am. J. Perinatol. 21: 173-182.
    Pubmed CrossRef
  23. Song, T., C. Toma, N. Nakasone, and M. Iwanaga. 2005. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method. FEMS Microbiol. Lett. 243: 259-263.
    Pubmed CrossRef
  24. Tomita, N., Y. Mori, H. Kanda, and T. Notomi. 2008. Loopmediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat. Protoc. 3: 877882.
    Pubmed CrossRef
  25. Wang, X., Q. Zhang, F. Zhang, F. Ma, W. Zheng, Z. Zhao, Y. Bai, and L. Zheng. 2012. Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye. Virol J. 9: 138.
    Pubmed PMC CrossRef
  26. Xiao, B., Y. H. Zhu, and Q. M. Zou. 2005. A simple and sensitive technique - Loop-mediated isothermal amplification (LAMP). Chin. J. Lab. Med. 28: 761-763.
  27. Yano, A., R. Ishimaru, and R. Hujikata. 2007. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal amplification. J. Microbiol. Methods 68: 414-420.
    Pubmed CrossRef
  28. Zhao, K., W. Shi, F. Han, Y. Xu, L. Zhu, Y. Zou, et al. 2011. Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method. Virol. J. 8: 126.

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Article

Research article

J. Microbiol. Biotechnol. 2013; 23(1): 1-6

Published online January 28, 2013 https://doi.org/10.4014/jmb.1205.05063

Copyright © The Korean Society for Microbiology and Biotechnology.

Rapid, Sensitive, and Specific Detection of Clostridium tetani by Loop-Mediated Isothermal Amplification Assay

Dongneng Jiang 1, Xiaoyun Pu 1*, Jiehong Wu 1, Meng Li 1 and Ping Liu 1

Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China

Received: May 31, 2012; Accepted: August 28, 2012

Abstract

Tetanus is a specific infectious disease, which is often
associated with catastrophic events such as earthquakes,
traumas, and war wounds. The obligate anaerobe Clostridium
tetani is the pathogen that causes tetanus. Once the
infection of tetanus progresses to an advanced stage
within the wounds of limbs, the rates of amputation and
mortality increase manifold. Therefore, it is necessary to
devise a rapid and sensitive point-of-care detection method
for C. tetani so as to ensure an early diagnosis and clinical
treatment of tetanus. In this study, we developed a detection
method for C. tetani using loop-mediated isothermal
amplification (LAMP) assay, wherein the C. tetani tetanus
toxin gene was used as the target gene. The method was
highly specific and sensitive, with a detection limit of 10
colony forming units (CFU)/ml, and allowed quantitative
analysis. While detecting C. tetani in clinical samples, it
was found that the LAMP results completely agreed with
those of the traditional API 20A anaerobic bacteria
identification test. As compared with the traditional API
test and PCR assay, LAMP detection of C. tetani is simple
and rapid, and the results can be identified through
naked-eye observation. Therefore, it is an ideal and rapid
point-of-care testing method for tetanus.

Keywords: loop-mediated isothermal amplification (LAMP), Clostridium tetani, point-of-care testing (POCT)

References

  1. Akbulut, D., K. A. Grant, and J. McLauchlin. 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.
    Pubmed KoreaMed CrossRef
  2. Boyanton, B. L., P. Sural, C. R. Loomis, C. Pesta, L. GonzalezKrellwitz, B. Robinson-Dunn, and P. Riska. 2012. Loopmediated isothermal amplification compared to real-time PCR and enzyme immunoassay for toxigenic Clostridium difficile detection. J. Clin. Microbiol. 50: 640-645.
    Pubmed KoreaMed CrossRef
  3. Chon, J. W., J. S. Park, J. Y. Hyeon, C. Park, K. Y. Song, K. W. Hong, et al. 2012. Development of real-time PCR for the detection of Clostridium perfringens in meats and vegetables. J. Microbiol. Biotechnol. 22: 530-534.
    Pubmed CrossRef
  4. Enomoto, Y., T. Yoshikawa, M. Ihira, S. Akimoto, F. Miyake, C. Usui, et al. 2005. Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method. J. Clin. Microbiol. 43: 951-955.
    Pubmed KoreaMed CrossRef
  5. Fall, J., G. Chakraborty, T. Kono, M. Maeda, T. Itami, and M. Sakai. 2008. Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp. FEMS Microbiol. Lett. 288: 171177.
    Pubmed CrossRef
  6. Han, X., C. Ding, L. He, Q. Hu, and S. Yu. 2011. Development of loop-mediated isothermal amplification (LAMP) targeting the GroEL gene for rapid detection of Riemerella anatipestifer. Avian Dis. 55: 379-383.
    Pubmed CrossRef
  7. He, L., Y. Q. Zhou, M. C. Oosthuizen, and J. L. Zhao. 2009. Loop-mediated isothermal amplification (LAMP) detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758) in China. Vet. Parasitol 165: 36-40.
    Pubmed CrossRef
  8. Kaneko, I., K. Miyamoto, K. Mimura, N. Yumine, H. Utsunomiya, S. Akimoto, and B. A. McClane. 2011. Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods. Appl. Environ. Microbiol. 77: 75267532.
    Pubmed KoreaMed CrossRef
  9. Kim, J. D., J. H. Yoon, Y. H. Park, D. W. Lee, K. S. Lee, C. H. Choi, et al. 2003. Isolation and identification of a lactic acid bacterial strain KJ-108 and its capability for deodorizing malodorous gases under anaerobic culture conditions. J. Microbiol. Biotechnol. 13: 207-216.
  10. Langkamp, D. L., S. Hoshaw-Woodard, M. E. Boye, and S. Lemeshow. 2001. Delays in receipt of immunizations in lowbirthweight children: A nationally representative sample. Arch. Pediatr. Adolesc. Med. 155: 167-172.
    Pubmed
  11. Lee, H. C., W. C. Ko, and Y. C. Chuang. 2000. Tetanus of the elderly. J. Microbiol. Immunol. Infect. 33: 191-196.
    Pubmed
  12. Mao, Z., Y. Qiu, L. Zheng, J. Chen, and J. Yang. 2012. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea). J. Microbiol. Methods 89: 179-184.
    Pubmed CrossRef
  13. Mekata, T., T. Kono, R. Savan, M. Sakai, J. Kasornchandra, T. Yoshida, and T. Itami. 2006. Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP). J. Virol. Methods 135: 151-156.
    Pubmed CrossRef
  14. Nagamine, K., T. Hase, and T. Notomi. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probes 16: 223-229.
    Pubmed CrossRef
  15. Njiru, Z. K., A. S. Mikosza, E. Matovu, J. C. Enyaru, J. O. Ouma, S. N. Kibona, et al. 2008. African trypanosomiasis:Sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int. J. Parasitol. 38: 589-599
    Pubmed CrossRef
  16. Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: E63.
    Pubmed KoreaMed CrossRef
  17. Ohtsuka, K., K. Yanagawa, K. Takatori, and Y. Hara-Kudo. 2005. Detection of Salmonella Enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates. Appl. Environ. Microbiol. 71: 6730-6735.
    Pubmed KoreaMed CrossRef
  18. Peng, Y., Z. Xie, J. Liu, Y. Pang, X. Deng, L. Xie, et al. 2011. Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay. Virol. J. 8: 337.
    Pubmed KoreaMed CrossRef
  19. Plutzer, J., A. Torokne, and P. Karanis. 2010. Combination of ARAD microfibre filtration and LAMP methodology for simple, rapid and cost-effective detection of human pathogenic Giardia duodenalis and Cryptosporidium spp. in drinking water. Lett. Appl. Microbiol. 50: 82-88.
    Pubmed CrossRef
  20. Saadatmandzadeh, M., H. Eshghi, M. Rahimizadeh, M. Sankian, and S. A. Jamehdar. 2011. Detection of Clostridium tetani by fluorescent amplification based specific hybridization. Afr. J. Microbiol. Res. 5: 5489-5492.
  21. Sakuma, T., Y. Kurosaki, Y. Fujinami, T. Takizawa, and J. Yasuda. 2009. Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification. J. Appl. Microbiol. 106: 1252-1259.
    Pubmed CrossRef
  22. Sheffield, J. S. and S. M. Ramin. 2004. Tetanus in pregnancy. Am. J. Perinatol. 21: 173-182.
    Pubmed CrossRef
  23. Song, T., C. Toma, N. Nakasone, and M. Iwanaga. 2005. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method. FEMS Microbiol. Lett. 243: 259-263.
    Pubmed CrossRef
  24. Tomita, N., Y. Mori, H. Kanda, and T. Notomi. 2008. Loopmediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat. Protoc. 3: 877882.
    Pubmed CrossRef
  25. Wang, X., Q. Zhang, F. Zhang, F. Ma, W. Zheng, Z. Zhao, Y. Bai, and L. Zheng. 2012. Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye. Virol J. 9: 138.
    Pubmed KoreaMed CrossRef
  26. Xiao, B., Y. H. Zhu, and Q. M. Zou. 2005. A simple and sensitive technique - Loop-mediated isothermal amplification (LAMP). Chin. J. Lab. Med. 28: 761-763.
  27. Yano, A., R. Ishimaru, and R. Hujikata. 2007. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal amplification. J. Microbiol. Methods 68: 414-420.
    Pubmed CrossRef
  28. Zhao, K., W. Shi, F. Han, Y. Xu, L. Zhu, Y. Zou, et al. 2011. Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method. Virol. J. 8: 126.