DOI QR코드

DOI QR Code

Species-Specific Duplex PCR for Detecting the Important Fish Pathogens Vibrio anguillarum and Edwardsiella tarda

  • Jo, Geon-A (Department of Biotechnology, Pukyong National University) ;
  • Kwon, Sae-Bom (Department of Biotechnology, Pukyong National University) ;
  • Kim, Na-Kyeong (Department of Biotechnology, Pukyong National University) ;
  • Hossain, Muhammad Tofazzal (Department of Biotechnology, Pukyong National University) ;
  • Kim, Yu-Ri (Department of Biotechnology, Pukyong National University) ;
  • Kim, Eun-Young (Department of Biotechnology, Pukyong National University) ;
  • Kong, In-Soo (Department of Biotechnology, Pukyong National University)
  • Received : 2013.04.25
  • Accepted : 2013.09.16
  • Published : 2013.12.30

Abstract

Vibriosis caused by Vibrio anguillarum and edwardsiellosis caused by Edwardsiella tarda are septicemic diseases of many commercially important freshwater and marine fishes, and threaten the aquaculture industry in Korea. Early diagnosis and accurate identification of these two bacterial species could help to prevent these diseases and minimize the damage to cultured marine species. This study designed a duplex polymerase chain reaction (PCR) method for the simultaneous detection of two major fish pathogens: V. anguillarum and E. tarda. Each pair of oligonucleotide primers exclusively amplified the target groEL gene of the specific microorganism. Twenty-two Vibrio and ten non-Vibrio enteric species were used to check the specificity of the primers, which were found to be highly specific for the target species, even among closely related species. The detection limit was 400 pg for V. anguillarum and 4 ng for E. tarda when mixed purified DNA was used as the template. This assay showed high specificity and sensitivity in the simultaneous detection of V. anguillarum and E. tarda from artificially inoculated seawater and fish.

References

  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 1998. Current Protocols in Molecular Biology. John Wiley and Sons, New York, US.
  2. Castro N, Toranzo AE, Nunez S, Osorio CR and Magarinos B. 2010. Evaluation of four polymerase chain reaction primer pairs for the detection of Edwardsiella tarda in turbot. Dis Aquat Org 90, 55-61. http://dx.doi.org/10.3354/dao02203. https://doi.org/10.3354/dao02203
  3. Chizhikov V, Rasooly A, Chumakov K and Levy DD. 2001. Microarray analysis of microbial virulence factors. Appl Environ Microbiol 67, 3258-3263. http://dx.doi.org/10.1128/AEM.67.7.3258-3263.2001. https://doi.org/10.1128/AEM.67.7.3258-3263.2001
  4. Frans I, Michiels CW, Bossier P, Willems KA, Lievens B and Rediers H. 2011. Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. J Fish Dis 34, 643-661. http://dx.doi.org/10.1111/j.1365-2761.2011.01279.x. https://doi.org/10.1111/j.1365-2761.2011.01279.x
  5. Gonzalez SF, Krug MJ, Nielsen ME, Santos Y and Call DR. 2004. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray. J Clin Microbiol 42, 1414-1419. http://dx.doi.org/10.1128/JCM.42.4.1414-1419.2004. https://doi.org/10.1128/JCM.42.4.1414-1419.2004
  6. Hossain MT, Kim EY, Kim YR, Kim DG and Kong IS. 2012. Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR. Lett Appl Microbiol 54, 67-72. http://dx.doi.org/10.1111/j.1472-765X.2011.03174.x. https://doi.org/10.1111/j.1472-765X.2011.03174.x
  7. Hossain MT, Kim EY, Kim YR, Kim DG and Kong IS. 2013. Development of a groELgene- based species-specific multiplex polymerase chain reaction assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. J Appl Microbiol 114, 448-456. http:/dx.doi.org/10.1111/jam.12056. https://doi.org/10.1111/jam.12056
  8. Izumiya H, Matsumoto K, Yahiro S, Lee J, Morita M, Yamamoto S, Arakawa E and Ohnishi M. 2011. Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholera, Vibrio parahaemolyticus, and Vibrio vulnificus. Mol Cell Probes 25, 174-176. http://dx.doi.org/10.1016/j.mcp.2011.04.004. https://doi.org/10.1016/j.mcp.2011.04.004
  9. Jeyasekaran G, Raj KT, Shakila RJ, Thangarani AJ and Sukumar D. 2011. Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products. Appl Microbiol Biotechnol 90, 1111-1118. http://dx.doi.org/10.1007/s00253-011-3175-9. https://doi.org/10.1007/s00253-011-3175-9
  10. Junick J and Blaut M. 2012. Quantification of human fecal Bifidobacterium species by use of quantitative real-time PCR analysis targeting groEL gene. Appl Environ Microbiol 78, 2613-2622. http://dx.doi.org/10.1128/AEM.07749-11. https://doi.org/10.1128/AEM.07749-11
  11. Kim DG, Ahn SH, Kim LH, Park KJ, Hong YK and Kong IS. 2008. Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR. J Microbiol Biotechnol 18, 1841- 1847. https://doi.org/10.4014/jmb.0800.176
  12. Kim DG, Kim YR, Kim EY, Cho HM, Ahn SH and Kong IS. 2010. Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene. Fish Sci 76, 803-810. http://dx.doi.org/10.1007/s12562-010-0266-y.
  13. Kim YR, Kim EY, Kim DG, Kim YO, Hossain MT and Kong IS. 2012. DNA array with the groESL intergenic sequence to detect Vibrio parahaemolyticus and Vibrio vulnificus. Anal Biochem 424, 32-34. http://dx.doi.org/10.1016/j.ab.2012.02.008. https://doi.org/10.1016/j.ab.2012.02.008
  14. Kita-Tsukamoto K, Oyaizu H, Nanba K and Simidu U. 1993. Phylogenetic relationships of marine bacteria, mainly members of the family Vibrionaceae, determined on the basis of 16S rRNA sequences. Int J Syst Bacteriol 43, 8-19. http://dx.doi.org/10.1099/00207713-43-1-8. https://doi.org/10.1099/00207713-43-1-8
  15. Lan J, Zhang XH, Wang Y, Chen J and Han Y. 2008. Isolation of an unusual strain of Edwardsiella tarda from turbot and establish a PCR detection technique with the gyrB gene. J Appl Microbiol 105, 644-651. https://doi.org/10.1111/j.1365-2672.2008.03779.x
  16. Nishibuchi M. 2006. The biology of vibrios. In: Molecular Identification. Thompson FL, Austin B and Swings J, eds. ASM Press, Washington, DC, US, pp. 44-64.
  17. Paillard C, Le Roux F and Borrego JJ. 2004. Bacterial disease in marine bivalves, a review of recent studies: trends and evaluation. Aquat Living Resour 17, 477-498. http://dx.doi.org/10.1051/alr:2004054. https://doi.org/10.1051/alr:2004054
  18. Park SI. 2009. Disease control in Korean aquaculture. Fish Pathol 44, 19-23. http://dx.doi.org/10.3147/jsfp.44.19. https://doi.org/10.3147/jsfp.44.19
  19. Rao PSS, Lim TM and Leung KY. 2001. Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates. Infect Immun 69, 5689-5697. http://dx.doi.org/10.1128/ IAI.69.9.5689-5697.2001. https://doi.org/10.1128/IAI.69.9.5689-5697.2001
  20. Rodkhum C, Hirono I, Crosa JH and Aoki T. 2006. Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum. J Microbiol Methods 65, 612-618. http://dx.doi.org/10.1016/j.mimet.2005.09.009. https://doi.org/10.1016/j.mimet.2005.09.009
  21. Toranzo AE, Magariños B and Romalde JL. 2005. A review of the main bacterial fish diseases in mariculture systems. Aquaculture 246, 37-61. http://dx.doi.org/10.1016/j.aquaculture.2005.01.002. https://doi.org/10.1016/j.aquaculture.2005.01.002
  22. Yushan H, Lei L, Weijia L and Xiaoguang C. 2010. Sequence analysis of the groEL gene and its potential application in identification of pathogenic bacteria. Afr J Microbiol Res 4, 1733-1741.

Cited by

  1. species vol.48, pp.6, 2016, https://doi.org/10.1111/are.13128