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

  • Jiang, Dongneng (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Pu, Xiaoyun (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Wu, Jiehong (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Li, Meng (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University) ;
  • Liu, Ping (Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University)
  • Received : 2012.05.31
  • Accepted : 2012.08.28
  • Published : 2013.01.28

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

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. https://doi.org/10.1128/JCM.43.9.4342-4348.2005
  2. Boyanton, B. L., P. Sural, C. R. Loomis, C. Pesta, L. Gonzalez- Krellwitz, 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. https://doi.org/10.1128/JCM.01014-11
  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. https://doi.org/10.4014/jmb.1107.07064
  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. https://doi.org/10.1128/JCM.43.2.951-955.2005
  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: 171-177 https://doi.org/10.1111/j.1574-6968.2008.01332.x
  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. https://doi.org/10.1637/9602-112610-Reg.1
  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. https://doi.org/10.1016/j.vetpar.2009.06.036
  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: 7526- 7532. https://doi.org/10.1128/AEM.06216-11
  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 lowbirth- weight children: A nationally representative sample. Arch. Pediatr. Adolesc. Med. 155: 167-172. https://doi.org/10.1001/archpedi.155.2.167
  11. Lee, H. C., W. C. Ko, and Y. C. Chuang. 2000. Tetanus of the elderly. J. Microbiol. Immunol. Infect. 33: 191-196.
  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. https://doi.org/10.1016/j.mimet.2012.03.011
  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. https://doi.org/10.1016/j.jviromet.2006.02.012
  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. https://doi.org/10.1006/mcpr.2002.0415
  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. https://doi.org/10.1016/j.ijpara.2007.09.006
  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. https://doi.org/10.1093/nar/28.12.e63
  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. https://doi.org/10.1128/AEM.71.11.6730-6735.2005
  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. https://doi.org/10.1186/1743-422X-8-337
  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. https://doi.org/10.1111/j.1472-765X.2009.02758.x
  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. https://doi.org/10.1111/j.1365-2672.2008.04084.x
  22. Sheffield, J. S. and S. M. Ramin. 2004. Tetanus in pregnancy. Am. J. Perinatol. 21: 173-182. https://doi.org/10.1055/s-2004-828605
  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. https://doi.org/10.1016/j.femsle.2004.12.014
  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: 877-882 https://doi.org/10.1038/nprot.2008.57
  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. https://doi.org/10.1186/1743-422X-9-138
  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. https://doi.org/10.1016/j.mimet.2006.09.024
  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. https://doi.org/10.1186/1743-422X-8-126

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