Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes

Abstract

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65 °C for 30 min, 63 °C for 30 min, and 63 °C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 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. https://doi.org/10.1111/j.1472-765X.2010.02823.x

    CAS  Article  PubMed  Google Scholar 

  2. Dong HJ, Cho AR, Hahn TW, Cho S (2014) Development of a multiplex loop-mediated isothermal amplification assay to detect Shiga toxin-producing Escherichia coli in cattle. J Vet Sci 15:317. https://doi.org/10.4142/jvs.2014.15.2.317

    Article  PubMed  PubMed Central  Google Scholar 

  3. Dou M, Sanjay ST, Dominguez DC, Liu P, Xu F, Li XJ (2017) Multiplexed instrument-free meningitis diagnosis on a polymer/paper hybrid microfluidic biochip. Biosens Bioelectron 87:865–873. https://doi.org/10.1016/j.bios.2016.09.033

    CAS  Article  PubMed  Google Scholar 

  4. Fischbach J, Xander NC, Frohme M, Glökler JF (2015) Shining a light on LAMP assays- a comparison of LAMP visualization methods including the novel use of berberine. Biotechniques 58:189–194. https://doi.org/10.2144/000114275

    CAS  Article  PubMed  Google Scholar 

  5. Frans I, Michiels CW, Bossier P, Willems KA, Lievens B, Rediers H (2011) Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. J Fish Dis 34:643–661

    CAS  Article  Google Scholar 

  6. Fu K, Li J, Wang Y, Liu J, Yan H, Shi L, Zhou L (2016) An innovative method for rapid identification and detection of Vibrio alginolyticus in different infection models. Front Microbiol 7. https://doi.org/10.3389/fmicb.2016.00651

  7. Gao H, Li F, Zhang X, Wang B, Xiang J (2010) Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification. Chin J Oceanol Limnol 28:62–66. https://doi.org/10.1007/s00343-010-9247-3

    CAS  Article  Google Scholar 

  8. Hossain MT, Kim EY, Kim YR, Kim DG, Kong IS (2012) Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR. Lett Appl Microbiol 54:67–72. https://doi.org/10.1111/j.1472-765X.2011.03174.x

    CAS  Article  PubMed  Google Scholar 

  9. Hossain MT, Kim EY, Kim YR, Kim DG, Kong IS (2013) Development of a groEL gene-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. https://doi.org/10.1111/jam.12056

    CAS  Article  PubMed  Google Scholar 

  10. Iseki H, Alhassan A, Ohta N, Thekisoe OMM, Yokoyama N, Inoue N, Nambota A, Yasuda J, Igarashi I (2007) Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J Microbiol Methods 71:281–287. https://doi.org/10.1016/j.mimet.2007.09.019

    CAS  Article  PubMed  Google Scholar 

  11. Jo GA, Lee JM, No G, Kang DS, Kim SH, Ahn SH, Kong IS (2015) Isolation and characterization of a 17-kDa FKBP-type peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum. Protein Expr Purif 110:130–137. https://doi.org/10.1016/j.pep.2015.02.019

    CAS  Article  PubMed  Google Scholar 

  12. Kim H, Lone G (2003) Elucidation of the Vibrio anguillarum genetic response to the potential fish probiont Pseudomonas fluorescens AH2, using RNA-arbitrarily primed PCR. J Bacteriol. https://doi.org/10.1128/JB.185.3.831-842.2003

    CAS  Article  Google Scholar 

  13. Kim DG, Kim YR, Kim EY, Cho HM, Ahn SH, Kong IS (2010) Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene. Fish Sci 76:803–810. https://doi.org/10.1007/s12562-010-0266-y

    CAS  Article  Google Scholar 

  14. Kulkarni A, Caipang CMA, Brinchmann MF et al (2009) Use of loop-mediated isothermal amplification assay for the detection of vibrio anguillarum o2β, the causative agent of vibriosis in Atlantic cod, Gadus morhua. J Rapid Methods Autom Microbiol 17:503–518. https://doi.org/10.1111/j.1745-4581.2009.00186.x

    CAS  Article  Google Scholar 

  15. Li Y, Fan P, Zhou S, Zhang L (2017) Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens. Microb Pathoghttps://doi.org/10.1016/j.micpath.2017.03.016

    CAS  Article  Google Scholar 

  16. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T, Notomi T, Okayama H et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:63e–663e. https://doi.org/10.1093/nar/28.12.e63

    Article  Google Scholar 

  17. Notomi T, Mori Y, Tomita N, Kanda H (2015) Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. J Microbiol 53:1–5

    CAS  Article  Google Scholar 

  18. Pachot A, Blond JL, Mougin B, Miossec P (2004) Peptidylpropyl isomerase B (PPIB): a suitable reference gene for mRNA quantification in peripheral whole blood. J Biotechnol 114:121–124. https://doi.org/10.1016/j.jbiotec.2004.07.001

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Plaon S, Longyant S, Sithigorngul P, Chaivisuthangkura P (2015) Rapid and sensitive detection of Vibrio alginolyticus by loop-mediated isothermal amplification combined with a lateral flow dipstick targeted to the rpoX gene. J Aquat Anim Health 27:156–163. https://doi.org/10.1080/08997659.2015.1037468

    CAS  Article  PubMed  Google Scholar 

  20. Ravan H, Amandadi M, Sanadgol N (2016) A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157: H7. Microb Pathog 91:161–165. https://doi.org/10.1016/j.micpath.2015.12.011

    CAS  Article  PubMed  Google Scholar 

  21. Saitou Y, Kobayashi Y, Hirano S, Mochizuki N, Itou T, Ito FH, Sakai T (2010) A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay. J Virol Methods 168:13–17. https://doi.org/10.1016/j.jviromet.2010.04.008

    CAS  Article  PubMed  Google Scholar 

  22. Sajid M, Kawde AN, Daud M (2015) Designs, formats and applications of lateral flow assay: a literature review. J Saudi Chem Soc 19:689–705. https://doi.org/10.1016/j.jscs.2014.09.001

    Article  Google Scholar 

  23. Sambrook J, Russell D W (2001) Commonly used techniques in milecular cloning. In: Molecular Cloning, (Appendix 8, vol 3, 3rd ed) eds

  24. Shao Y, Zhu S, Jin C, Chen F (2011) Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk. Int J Food Microbiol 148:75–79. https://doi.org/10.1016/j.ijfoodmicro.2011.05.004

    CAS  Article  PubMed  Google Scholar 

  25. Siddique MP, Jang WJ, Lee JM, Ahn SH, Suraiya S, Kim CH, Kong IS (2017) groEL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop-mediated isothermal amplification assay. Lett Appl Microbiol 65:106–113. https://doi.org/10.1111/lam.12760

    CAS  Article  PubMed  Google Scholar 

  26. Silvester R, Alexander D, Antony AC, Hatha M (2017) GroEL PCR- RFLP – an efficient tool to discriminate closely related pathogenic Vibrio species. Microb Pathog 105:196–200. https://doi.org/10.1016/j.micpath.2017.02.029

    CAS  Article  PubMed  Google Scholar 

  27. Tanner NA, Zhang Y, Evans TC (2015) Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques. 58. https://doi.org/10.2144/000114253

  28. Xing J, Xu H, Wang Y, Tang X, Sheng X, Zhan W (2017) Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus). Microb Pathog 107:155–163. https://doi.org/10.1016/j.micpath.2017.03.027

    CAS  Article  PubMed  Google Scholar 

  29. Xu HS, He L, Lv SJ, Gong Q, Li S (2012) Establishment of universal loop-mediated isothermal amplification method (LAMP) for rapid detection of pathogenic Vibrio spp. in aquatic organisms. Afr J Microbiol Res. https://doi.org/10.5897/AJMR11.1417

  30. Yan M, Li W, Zhou Z, Peng H, Luo Z, Xu L (2017) Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony. Microb Pathog 102:1–7. https://doi.org/10.1016/j.micpath.2016.10.025

    CAS  Article  PubMed  Google Scholar 

  31. Zhou D, Guo J, Xu L, Gao S, Lin Q, Wu Q, Wu L, Que Y (2014) Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane. Sci Rep 4. https://doi.org/10.1038/srep04912

Download references

Funding

The authors wish to thank for the financial support from the Korea Institute of Marine Science & Technology promotion (Project number 20140418).

Author information

Affiliations

Authors

Corresponding author

Correspondence to In-Soo Kong.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Siddique, M.P., Jang, W.J., Lee, J.M. et al. Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes. Int Microbiol 22, 501–509 (2019). https://doi.org/10.1007/s10123-019-00079-z

Download citation

Keywords

  • LAMP assay
  • Loop-mediated isothermal amplification
  • Duplex LAMP
  • Vibrio anguillarum
  • Vibrio alginolyticus