Skip to main content
PMC home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2012 Jul 2;30(4):595–603. doi: 10.1007/s00343-012-1293-6

An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene

Guosi Xie 1,2, Qingli Zhang 1, Nana Han 1, Chengyin Shi 1, Xiuhua Wang 1, Qinghui Liu 1, Jie Huang 1,2,✉
PMCID: PMC7087700  PMID: 32214585

Abstract

Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB — LAMP). In this method, the Mg2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene ( hemolysin -LAMP) for detection of E. tarda. The EsrB -LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.

Keyword: Edwardsiella tarda, LAMP, detection, EsrB

Footnotes

Supported by the Special Fund for Agro-Scientific Research in the Public Interest (No. 201103034), and the Earmarked Fund for Modern Agroindustry Technology Research System (No. nycytx-46)

References

  1. Aryan E., Makvandi M., Farajzadeh A., Huygen K., Bifani P., Mousavi S. L., Fateh A., Jelodar A., Gouya M. M., Romano M. A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex. Microbiol. Res. 2010;165(3):211–220. doi: 10.1016/j.micres.2009.05.001. [DOI] [PubMed] [Google Scholar]
  2. Bernardet J. F., Campbella C., Buswell J. A. Flexibacter maritimus is the agent of ‘black patch necrosis’ in Dover sole in Scotland. Dis. Aquat. Org. 1990;8(3):233–237. doi: 10.3354/dao008233. [DOI] [Google Scholar]
  3. Castro N., Toranzoa E., Nú E. S., Osorio C. R., Magari O. B. Evaluation of four polymerase chain reaction primer pairs for the detection of Edwardsiella tarda in turbot. Dis. Aquat. Org. 2010;90(1):55–61. doi: 10.3354/dao02203. [DOI] [PubMed] [Google Scholar]
  4. Chen J. D., Lai S. Y. PCR for direct detection of Edwardsiella tarda from infected fish and environmental water by application of the hemolysin gene. Zool. Stud. 1998;37(3):169–176. [Google Scholar]
  5. En F. X., Wei X., Jian L., Qin C. Loop-mediated isothermal amplification establishment for detection of pseudorabies virus. J. Virol. Methods. 2008;151(1):35–39. doi: 10.1016/j.jviromet.2008.03.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ewing W. H., Mcwhorter A. C., Escobar M. R., Lubin A. H. Edwardsiella, a new genus of Enterobacteriaceae based on a new species, E. tarda. International Bulletin of Bacteriological Nomenclature and Taxonomy. 1965;15(1):33–38. doi: 10.1099/00207713-15-1-33. [DOI] [Google Scholar]
  7. Hoshina T. On a new bacterium, Paracolobactrum anguillimortiferum n. sp. Bulletin of the Japanese Society of Scientific Fisheries. 1962;28(2):162–164. doi: 10.2331/suisan.28.162. [DOI] [Google Scholar]
  8. Ihira M., Yoshikawa T., Enomoto Y., Akimoto S., Ohashi M., Suga S., Nishimura N., Ozaki T., Nishiyama Y., Notomi T. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification. J. Clin. Microbiol. 2004;42(1):140–145. doi: 10.1128/JCM.42.1.140-145.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Iwamoto T., Sonobe T., Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J. Clin. Microbiol. 2003;41(6):2 616–2 622. doi: 10.1128/JCM.41.6.2616-2622.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kono T., Savan R., Sakai M., Itami T. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods. 2004;115(1):59–65. doi: 10.1016/j.jviromet.2003.09.015. [DOI] [PubMed] [Google Scholar]
  11. Lan J., Zhang X. H., Wang Y., Chen J., Han Y. Isolation of an unusual strain of Edwardsiella tarda from turbot and establish a PCR detection technique with the gyrB gene. J. Appl. Microbiol. 2008;105(3):644–651. doi: 10.1111/j.1365-2672.2008.03779.x. [DOI] [PubMed] [Google Scholar]
  12. Mao X. L., Zhou S., Xu D., Gong J., Cui H. C., Qin Q. W. Rapid and sensitive detection of Singapore grouper iridovirus by loop-mediated isothermal amplification. J. Appl. Microbiol. 2008;105(2):389–397. doi: 10.1111/j.1365-2672.2008.03761.x. [DOI] [PubMed] [Google Scholar]
  13. Michael J., Abbott S. L. Infections associated with the genus Edwardsiella: the role of Edwardsiella tarda in human disease. Clinical Infectious Dis. 1993;17(4):742–748. doi: 10.1093/clinids/17.4.742. [DOI] [PubMed] [Google Scholar]
  14. Mo Z. L., Xiao P., Mao Y. X., Zou Y. X., Wang B., Li J., Xu Y. L., Zhang P. J. Construction and characterization of a live, attenuated EsrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.) Fish Shellfish Immun. 2007;23(3):521–530. doi: 10.1016/j.fsi.2006.11.002. [DOI] [PubMed] [Google Scholar]
  15. Mori Y., Nagamine K., Tomita N., Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Bioph. Res. Co. 2001;289(1):150–154. doi: 10.1006/bbrc.2001.5921. [DOI] [PubMed] [Google Scholar]
  16. Nagamine K., Hase T., Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probes. 2002;16(3):223–229. doi: 10.1006/mcpr.2002.0415. [DOI] [PubMed] [Google Scholar]
  17. Noritaka K., Noboru I., Tatsuya S., Francescopaolo D. C., Dennis J. G., Hiroshi S., Chihiro S., Ikuo I. Loop-mediated isothermal amplification for detection of African trypanosomes. J. Clin. Microbiol. 2003;41(12):5 517–5 524. doi: 10.1128/JCM.41.12.5517-5524.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 2000;28(12):e63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Parida M., Posadas G., Inoue S., Hasebe F., Morita K. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J. Clin. Microbiol. 2004;42(1):257–263. doi: 10.1128/JCM.42.1.257-263.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pazos F., Santos Y., Macias A. R., Nú E. S., Toranzo A. E. Evaluation of media for the successful culture of Flexibacter maritimus. J. Fish Dis. 1996;19(2):193–197. doi: 10.1111/j.1365-2761.1996.tb00701.x. [DOI] [Google Scholar]
  21. Pillai D., Bonami J. Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification. J. Fish Dis. 2006;29(5):275–283. doi: 10.1111/j.1365-2761.2006.00718.x. [DOI] [PubMed] [Google Scholar]
  22. Plumb J. A. Edwardsiella Septicaemias. In: Woo P. T. K., Bruno D. W., editors. Fish Diseases and Disorders. New York, USA: CAB International.; 1999. pp. 479–521. [Google Scholar]
  23. Poon L. L. M., Leung C. S. W., Tashiro M., Chan K. H., Wong B. W. Y., Yuen K. Y., Guan Y., Peiris J. S. M. Rapid detection of the severe acute respiratory syndrome (SARS) coronavirus by a loop-mediated isothermal amplification assay. Clin. Chem. 2004;50(6):1 050–1 052. doi: 10.1373/clinchem.2004.032011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sakai T., Iida T., Osatomi K., Kanai K. Detection of type 1 fimbrial genes in fish pathogenic and non-pathogenic Edwardsiella tarda strains by PCR. Fish Pathol. 2007;42(2):115–117. doi: 10.3147/jsfp.42.115. [DOI] [Google Scholar]
  25. Sakai T., Yuasa K., Sano M., Iida T. Identification of Edwardsiella ictaluri and E. tarda by species-specific polymerase chain reaction targeted to the upstream region of the fimbrial gene. J. Aquat. Anim. Health. 2009;21(2):124–132. doi: 10.1577/H08-061.1. [DOI] [PubMed] [Google Scholar]
  26. Savan R., Igarashi A., Matsuoka S., Sakai M. Sensitive and rapid detection of Edwardsiellosis in fish by a loopmediated isothermal amplification method. Appl. Environ. Microb. 2004;70(1):621–624. doi: 10.1128/AEM.70.1.621-624.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sharma V. K., Kaura Y. K., Singh I. P. Frogs as carriers of Salmonella and Edwardsiella. Antonie van Leeuwenhoek. 1974;40(1):171–175. doi: 10.1007/BF00394564. [DOI] [PubMed] [Google Scholar]
  28. Tan Y. P., Zheng J., Tung S. L., Rosenshine I., Leung K. Y. Role of type III secretion in Edwardsiella tarda virulence. Microbiol. 2005;151(7):2 301–2 313. doi: 10.1099/mic.0.28005-0. [DOI] [PubMed] [Google Scholar]
  29. Thune R. L., Stanley L. A., Cooper R. K. Pathogenesis of gram-negative bacterial infections in warmwater fish. Annu. Rev. Fish. Dis. 1993;3:37–68. doi: 10.1016/0959-8030(93)90028-A. [DOI] [Google Scholar]
  30. Tomoko H., Kayoko F., Taketoshi I., Aya N., Alexandre T. O., Tetsuya H., Takahide T., Eiichi H., Yuichi Y., Hideki H. Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification. J. Clin. Microbiol. 2004;42(11):5 349–5 352. doi: 10.1128/JCM.42.11.5349-5352.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang X. P., Lu C. P. Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection. Vaccine. 2009;27(10):1 571–1 578. doi: 10.1016/j.vaccine.2009.01.002. [DOI] [PubMed] [Google Scholar]
  32. White F. H., Simpson C. F., Williams L. E., Jr Isolation of Edwardsiella tarda from aquatic animal species and surface waters in Florida. J. Wildlife Dis. 1973;9(3):204–207. doi: 10.7589/0090-3558-9.3.204. [DOI] [PubMed] [Google Scholar]
  33. Yang C. Z., Wang X. H., Huang J. Identification and phylogenetic analysis of pathogen Ewardsiela tarda from cultured turbot ( Scophthalmus maximus ) J. Shanghai Fisheries Univ. 2008;17(3):280–284. [Google Scholar]
  34. Yano A., Ishimaru R., Hujikata R. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loopmediated isothermal amplification. J. Microbiol. Meth. 2007;68(2):414–420. doi: 10.1016/j.mimet.2006.09.024. [DOI] [PubMed] [Google Scholar]
  35. Yeh H. Y., Shoemaker C. A., Klesius P. H. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri. J. Microbiol. Meth. 2005;63(1):36–44. doi: 10.1016/j.mimet.2005.02.015. [DOI] [PubMed] [Google Scholar]
  36. Zhang Q. L., Shi C. Y., Huang J., Jia K. T., Chen X. H., Liu H. Rapid diagnosis of turbot reddish body iridovirus in turbot using the loop-mediated isothermal amplification method. J. Virol. Methods. 2009;158(1–2):18–23. doi: 10.1016/j.jviromet.2009.01.008. [DOI] [PubMed] [Google Scholar]

Articles from Chinese Journal of Oceanology and Limnology are provided here courtesy of Nature Publishing Group

RESOURCES