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Research article

Molecular Biology and Omics

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Nocardia salmonicida, the Causative Agent of Nocardiosis in Fish

Liqun Xia 1, 2, 3, Honglian Zhang 1, Yishan Lu 1, 2, 3*, Jia Cai 1, 2, 3, Bei Wang 1, 2, 3 and Jichang Jian 1, 2, 3

1Fisheries College, Guangdong Ocean University, Zhanjiang 524025, P.R. China, 2Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, P.R. China, 3Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions, Zhanjiang 524088, P.R. China

Received: June 17, 2014; Accepted: September 28, 2014

J. Microbiol. Biotechnol. 2015; 25(3): 321-327

Published March 28, 2015 https://doi.org/10.4014/jmb.1406.06052

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 103 CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.

Keywords

loop-mediated isothermal amplification, Nocardia salmonicida, rapid detection

References

  1. Biswas G, Sakai M. 2014. Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives. Appl. Microbiol. Biotechnol. 98: 2881-2895.
    Pubmed CrossRef
  2. Cai SH, Lu YS, Wu ZH, Jian JC, Wang B, Huang YC. 2010. Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish. Lett. Appl. Microbiol. 50: 480-485.
    Pubmed CrossRef
  3. Chen SC, Lee JL, Lai CC, Gu YW, Wang CT, Chang HY, Tsai KH. 2000. Nocardiosis in sea bass, Lateolabrax japonicus, in Taiwan. J. Fish Dis. 23: 299-307.
    CrossRef
  4. Elkesh A, Kantham KPL, Shinn AP, Crumlish M, Richards RH. 2013. Systemic nocardiosis in a Mediterranean population of cultured meagre, Argyrosomus regius Asso (Perciformes:Sciaenidae). J. Fish Dis. 36: 141-149.
    Pubmed CrossRef
  5. Huang YC, Jian JC, Wu ZH, Lu YS, Yu TJ. 2008. Isolation and identification of the pathogen causing sarcoidosis of Trachinotus ovatus. J. Guangdong Ocean Univ. 28: 49-53.
  6. Isik K, Chun J, Hah YC, Goodfellow M. 1999. Nocardia salmonicida nom. rev., a fish pathogen. Int. J. Syst. Bacteriol. 49: 833-837.
    Pubmed CrossRef
  7. Itano T, Kawakami H, Kono T, Sakai M. 2006. Detection of fish nocardiosis by loop-mediated isothermal amplification. J. Appl. Microbiol. 100: 1381-1387.
    Pubmed CrossRef
  8. Itano T, Kawakami H, Kono T, Sakai M. 2006. Live vaccine trials against nocardiosis in yellowtai, Seriola quinqueradiata. Aquaculture 261: 1175-1180.
    CrossRef
  9. Kaneko H, Kawana T, Fukushima E, Suzutani T. 2007. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Biophys. Methods 70: 499-501.
    Pubmed CrossRef
  10. Kono T, Ooyama T, Chen SC, Sakai M. 2002. Sequencing of 16S-23S rRNA internal transcribed spacer and its application in the identification of Nocardia seriolae by polymerase chain reaction. Aquaculture Res. 33: 1195-1197.
    CrossRef
  11. Labrie L, Ng J, Tan Z, Komar C, Ho E, Grisez L. 2008. Nocardial infections in fish: an emerging problem in both freshwater and marine aquaculture systems in Asia, pp. 297-312. In Bondad-Reansato M, Mohan CV, Crumlish M, Subasinghe R (eds.). Diseases in Asian aquaculture VI. Fish Health Section, Asian Fisheries Society, Philippines.
  12. Mori Y, Nagamine K, Tomita N, Notomi T. 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289: 150-154.
    Pubmed CrossRef
  13. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: e63-e63.
    Pubmed PMC CrossRef
  14. Roth A, Andrees S, Kroppenstedt RM, Harmsen D, Mauch H. 2003. Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons. J. Clin. Microbiol. 41: 851-856.
    Pubmed PMC CrossRef
  15. Rucker RR. 1949. A streptomycete pathogenic to fish. J. Bacteriol. 58: 659-664.
    Pubmed PMC
  16. Suebsing R, Oh MJ, Kim JH. 2012. Development of a reverse transcription loop-mediated isothermal amplification assay for detecting nervous necrosis virus in olive flounder Paralichthys olivaceus. J. Microbiol. Biotechnol. 22: 1021-1028.
    Pubmed CrossRef
  17. Tsai MA, Wang PC, Yoshida T, Liaw LL, Chen SC. 2013. Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae. Dis. Aquat. Org. 102: 225-235.
    Pubmed CrossRef
  18. Wang B, Lu YS, Zhang XL, Huang YC, Jian JC, Wu ZH. 2012. A loop-mediated isothermal amplification method targeting the fbsB gene for rapid detection of Streptococcus agalactiae, the causative agent of streptococcosis in farmed fish. Isr. J. Aquacult-bamid 64: 1-7.
  19. Wang GL, Liu L, Li SY. 2012. Development of a SYBR Green I real-time PCR assay for detection of Nocardia seriolae and its application. J. Fisheries China 36: 509-517.
  20. Wang GL, Xu YJ, Jin S, Zhu JL, Zhu WY. 2009. Research on the nocardiasis and pathogen in reared snakehead, Ophiocephalus argus Cantor. Acta Hydrobiol. Sin. 33: 277-283.
    CrossRef
  21. Wang GL, Yuan SP, Jin S. 2005. Nocardiosis in large yellow croaker, Larimichthys crocea (Richardson). J. Fish Dis. 28: 339-345.
    Pubmed CrossRef
  22. Wang PC, Chen SD, Tsai MA, Weng YJ, Chu SY, Chern RS, Chen SC. 2009. Nocardia seriolae infection in the three striped tigerfish, Terapon jarbua (Forsskal). J. Fish Dis. 32: 301-310.
    Pubmed CrossRef
  23. Xia HL, Lu YS, Tang JF, Cai J, Wang M, Xia LQ. 2014. Establishment of a specific PCR assay to detect fish pathogenic Nocardia salmonicida. J. Guangdong Ocean Univ. 34: 56-61.
  24. Yamamura H, Hayakawa M, Iimura Y. 2003. Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil. J. Appl. Microbiol. 95: 677-685.
    Pubmed CrossRef
  25. Yamazaki Y, Oba E, Kashiwagi N, Sugita K, Shiiba K, Baba Y, et al. 2014. Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae. Lett. Appl. Microbiol. 58: 362-369.
    Pubmed CrossRef
  26. Yu LP, Hu YH, Zhang XH, Sun BG. 2013. Development of a triplex loop-mediated isothermal amplification method for rapid on-site detection of three Vibrio species associated with fish diseases. Aquaculture 414: 267-273.
    CrossRef
  27. Yuan SP, Wang GL, Jin S. 2006. Review of pathogenic nocardias in cultured fish. Microbiol. China 33: 137-141.

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Article

Research article

J. Microbiol. Biotechnol. 2015; 25(3): 321-327

Published online March 28, 2015 https://doi.org/10.4014/jmb.1406.06052

Copyright © The Korean Society for Microbiology and Biotechnology.

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Nocardia salmonicida, the Causative Agent of Nocardiosis in Fish

Liqun Xia 1, 2, 3, Honglian Zhang 1, Yishan Lu 1, 2, 3*, Jia Cai 1, 2, 3, Bei Wang 1, 2, 3 and Jichang Jian 1, 2, 3

1Fisheries College, Guangdong Ocean University, Zhanjiang 524025, P.R. China, 2Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, P.R. China, 3Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions, Zhanjiang 524088, P.R. China

Received: June 17, 2014; Accepted: September 28, 2014

Abstract

Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study
was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and
sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S
rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as
incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel
electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results
showed the sensitivity of the LAMP assay was 1.68 × 103 CFU/ml (16.8 CFU per reaction) and
10-fold higher than that of PCR. The LAMP method was also effectively applied to detect
N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and
early diagnosis of fish nocardiosis.

Keywords: loop-mediated isothermal amplification, Nocardia salmonicida, rapid detection

References

  1. Biswas G, Sakai M. 2014. Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives. Appl. Microbiol. Biotechnol. 98: 2881-2895.
    Pubmed CrossRef
  2. Cai SH, Lu YS, Wu ZH, Jian JC, Wang B, Huang YC. 2010. Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish. Lett. Appl. Microbiol. 50: 480-485.
    Pubmed CrossRef
  3. Chen SC, Lee JL, Lai CC, Gu YW, Wang CT, Chang HY, Tsai KH. 2000. Nocardiosis in sea bass, Lateolabrax japonicus, in Taiwan. J. Fish Dis. 23: 299-307.
    CrossRef
  4. Elkesh A, Kantham KPL, Shinn AP, Crumlish M, Richards RH. 2013. Systemic nocardiosis in a Mediterranean population of cultured meagre, Argyrosomus regius Asso (Perciformes:Sciaenidae). J. Fish Dis. 36: 141-149.
    Pubmed CrossRef
  5. Huang YC, Jian JC, Wu ZH, Lu YS, Yu TJ. 2008. Isolation and identification of the pathogen causing sarcoidosis of Trachinotus ovatus. J. Guangdong Ocean Univ. 28: 49-53.
  6. Isik K, Chun J, Hah YC, Goodfellow M. 1999. Nocardia salmonicida nom. rev., a fish pathogen. Int. J. Syst. Bacteriol. 49: 833-837.
    Pubmed CrossRef
  7. Itano T, Kawakami H, Kono T, Sakai M. 2006. Detection of fish nocardiosis by loop-mediated isothermal amplification. J. Appl. Microbiol. 100: 1381-1387.
    Pubmed CrossRef
  8. Itano T, Kawakami H, Kono T, Sakai M. 2006. Live vaccine trials against nocardiosis in yellowtai, Seriola quinqueradiata. Aquaculture 261: 1175-1180.
    CrossRef
  9. Kaneko H, Kawana T, Fukushima E, Suzutani T. 2007. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Biophys. Methods 70: 499-501.
    Pubmed CrossRef
  10. Kono T, Ooyama T, Chen SC, Sakai M. 2002. Sequencing of 16S-23S rRNA internal transcribed spacer and its application in the identification of Nocardia seriolae by polymerase chain reaction. Aquaculture Res. 33: 1195-1197.
    CrossRef
  11. Labrie L, Ng J, Tan Z, Komar C, Ho E, Grisez L. 2008. Nocardial infections in fish: an emerging problem in both freshwater and marine aquaculture systems in Asia, pp. 297-312. In Bondad-Reansato M, Mohan CV, Crumlish M, Subasinghe R (eds.). Diseases in Asian aquaculture VI. Fish Health Section, Asian Fisheries Society, Philippines.
  12. Mori Y, Nagamine K, Tomita N, Notomi T. 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289: 150-154.
    Pubmed CrossRef
  13. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: e63-e63.
    Pubmed KoreaMed CrossRef
  14. Roth A, Andrees S, Kroppenstedt RM, Harmsen D, Mauch H. 2003. Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons. J. Clin. Microbiol. 41: 851-856.
    Pubmed KoreaMed CrossRef
  15. Rucker RR. 1949. A streptomycete pathogenic to fish. J. Bacteriol. 58: 659-664.
    Pubmed KoreaMed
  16. Suebsing R, Oh MJ, Kim JH. 2012. Development of a reverse transcription loop-mediated isothermal amplification assay for detecting nervous necrosis virus in olive flounder Paralichthys olivaceus. J. Microbiol. Biotechnol. 22: 1021-1028.
    Pubmed CrossRef
  17. Tsai MA, Wang PC, Yoshida T, Liaw LL, Chen SC. 2013. Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae. Dis. Aquat. Org. 102: 225-235.
    Pubmed CrossRef
  18. Wang B, Lu YS, Zhang XL, Huang YC, Jian JC, Wu ZH. 2012. A loop-mediated isothermal amplification method targeting the fbsB gene for rapid detection of Streptococcus agalactiae, the causative agent of streptococcosis in farmed fish. Isr. J. Aquacult-bamid 64: 1-7.
  19. Wang GL, Liu L, Li SY. 2012. Development of a SYBR Green I real-time PCR assay for detection of Nocardia seriolae and its application. J. Fisheries China 36: 509-517.
  20. Wang GL, Xu YJ, Jin S, Zhu JL, Zhu WY. 2009. Research on the nocardiasis and pathogen in reared snakehead, Ophiocephalus argus Cantor. Acta Hydrobiol. Sin. 33: 277-283.
    CrossRef
  21. Wang GL, Yuan SP, Jin S. 2005. Nocardiosis in large yellow croaker, Larimichthys crocea (Richardson). J. Fish Dis. 28: 339-345.
    Pubmed CrossRef
  22. Wang PC, Chen SD, Tsai MA, Weng YJ, Chu SY, Chern RS, Chen SC. 2009. Nocardia seriolae infection in the three striped tigerfish, Terapon jarbua (Forsskal). J. Fish Dis. 32: 301-310.
    Pubmed CrossRef
  23. Xia HL, Lu YS, Tang JF, Cai J, Wang M, Xia LQ. 2014. Establishment of a specific PCR assay to detect fish pathogenic Nocardia salmonicida. J. Guangdong Ocean Univ. 34: 56-61.
  24. Yamamura H, Hayakawa M, Iimura Y. 2003. Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil. J. Appl. Microbiol. 95: 677-685.
    Pubmed CrossRef
  25. Yamazaki Y, Oba E, Kashiwagi N, Sugita K, Shiiba K, Baba Y, et al. 2014. Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae. Lett. Appl. Microbiol. 58: 362-369.
    Pubmed CrossRef
  26. Yu LP, Hu YH, Zhang XH, Sun BG. 2013. Development of a triplex loop-mediated isothermal amplification method for rapid on-site detection of three Vibrio species associated with fish diseases. Aquaculture 414: 267-273.
    CrossRef
  27. Yuan SP, Wang GL, Jin S. 2006. Review of pathogenic nocardias in cultured fish. Microbiol. China 33: 137-141.