Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Determination of Neurotoxin Gene Expression in Clostridium botulinum Type A by Quantitative RT-PCR

  • Shin, Na-Ri (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Shin, Ji-Hun (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Chun, Jeong Hoon (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Yoon, So-Yeon (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Kim, Bong Su (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Oh, Hee-Bok (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health) ;
  • Rhie, Gi-eun (Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health)
  • Received : 2006.09.01
  • Accepted : 2006.09.25
  • Published : 2006.12.31
⟨ Previous Next ⟩

Abstract

Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and $4mg\;ml^{-1}$, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk.

Keywords

Acknowledgement

Supported by : Korean National Institute of Health

References

  1. Baranyi, J. and Roberts, T. A. (1994) A dynamic approach to predicting bacterial growth in food. Int. J. Food Microbiol. 23, 277−294
  2. Center for disease control. (1979) Botulism in the United States, 1899-1977. Handbook for epidemiologists, clinicians, and laboratory workers. DHEW Publ. No. (CDC) 74-8279, plus additional reports by CDC at annual meetings of the Interagency Botulism Research Coordinating Committee (IBRCC), Washington DC
  3. Collins, M. D. and East, A. K. (1998) Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins. J. Appl. Microbiol. 84, 5−17 https://doi.org/10.1046/j.1365-2672.1997.00313.x
  4. Chung, G. T., Kang, D. H., Yoo, C. K., Choi, J. H., and Seong, W. K. (2003) The first outbreak of botulism in Korea. Korean J. Clin. Microbiol. 6, 160−163
  5. Dupuy, B. and Sonenshein, A. L. (1998) Regulated transcription of Clostridium difficile toxin genes. Mol. Microbiol. 27, 107−120
  6. Hatheway, C. L. (1990) Toxigenic clostridia. Clin. Microbiol. Rev. 3, 66−98
  7. Kim, S. H., Song, S. H., Kim, Y. J., and Park, S. Y. (2001) Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody. Mol. Cells 11, 158−163
  8. Klein, D., Janda, P., Steinborn, R., Muller, M., Salmons, B., et al. (1999) Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction : influence of mismatches on quantification. Electrophoresis 20, 291−299 https://doi.org/10.1002/(SICI)1522-2683(19990201)20:2<291::AID-ELPS291>3.0.CO;2-R
  9. Lovenklev, M., Artin, I., Hagberg, O., Borch, E., Holst, E., et al. (2004a) Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B. Appl. Environ. Microbiol. 70, 2928−2934 https://doi.org/10.1128/AEM.70.5.2928-2934.2004
  10. Lovenklev, M., Holst, E., Borch, E., and Radstrom, P. (2004b) Relative neurotoxin gene expression in Clostridium botulinum type B, determined using quantitative reverse transcription- PCR. Appl. Environ. Microbiol. 70, 2919−2927 https://doi.org/10.1128/AEM.70.5.2919-2927.2004
  11. Lund, B. M., George, S. M., and Franklin, J. G. (1987) Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid. Appl. Environ. Microbiol. 53, 935−941
  12. Martin, S. (2003) Clostridium botulinum type D intoxication in a dairy herd in Ontario. Can. Vet. J. 44, 493−495
  13. McGrath, S., Dooley, J. S., and Haylock, R. W. (2000) Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription-PCR. Appl. Environ. Microbiol. 66, 1423−1428 https://doi.org/10.1128/AEM.66.4.1423-1428.2000
  14. Ornella, R., Michela, S., and Paola, C. (2001) Tetanus and botulinum neurotoxins: turning bad guys into good by reseach. Toxicon 39, 27−41
  15. Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2002−2007
  16. Rocke, T. E., Smith, S. R., and Nashold, S. W. (1998) Preliminary evaluation of a simple in vitro test for the diagnosis of type C botulism in wild birds. J. Wild Dis. 34, 744−751
  17. Ronning, I. E. and Frank, H. A. (1987) Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid. Appl. Environ. Microbiol. 53, 1020−1027
  18. Sharkey, F. H., Markos, S. I., and Haylock, R. W. (2004) Quantification of toxin-encoding mRNA from Clostridium botulinum type E in media containing sorbic acid or sodium nitrite by competitive RT-PCR. FEMS Microbiol. Lett. 232, 139−144 https://doi.org/10.1016/S0378-1097(04)00043-6
  19. Sofos, J. N., Busta, F. F., and Allen, C. E. (1979a) Botulism control by nitrite and sorbate in cured meats: a review. J. Food Prot. 42, 739−770
  20. Sofos, J. N., Busta, F. F., and Allen, C. E. (1979b) Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse. Appl. Environ. Microbiol. 37, 1103−1109
  21. Wictome, M., Newton, K. A., and Jameson, K. (1999) Novel assays for the detection of botulinum toxins in foods. Dev. Biol. Stand. 101, 141−145
  22. Woods, L. F., Wood, J. M., and Gibs, B. A. (1981) The involvement of nitric oxide in the inhibition of phosphoroclastic system in Clostiridium sporogenes by sodium nitrite. J. Gen. Microbiol. 125, 399−406
  23. Yoon, S. Y., Chung, G. T., Kang, D. H., Ryu, C. S., Yoo, C. K., et al. (2005) Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food. Microbiol. Immunol. 49, 505−511