Development of Loop-mediated Isothermal Amplification (LAMP) for Detection of Vibrio alginolyticus

Loop-mediated isothermal amplification (LAMP)법을 이용한 Vibrio alginolyticus의 신속 진단법 개발

  • Received : 2015.05.30
  • Accepted : 2015.06.26
  • Published : 2015.08.30


Loop-mediated isothermal amplification (LAMP), a PCR-based diagnostic method, is based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal conditions. With the help of four specific primers that recognize six different sequences of a target DNA, LAMP has high specificity in pathogenic identification in a short time. Hence, in the present study, LAMP is used as a diagnostic tool in the identification of the most dreadful aquatic pathogenic species, Vibrio alginolyticus, and to develop species-specific LAMP primers and optimization of LAMP reaction conditions such as annealing temperature, elongation time, and other PCR chemical concentrations, including MgSO4, dNTPs, Betaine, and Bst polymerase. The optimized LAMP primers were also checked for specificity with other Vibrio species, which showed that the designed primers were very specific to V. alginolyticus After the first introduction of a species name like this one, the first part (“Vibrio” in this case) should be abbreviated to only the first letter.only. These are usually the most harmful pathogens of the Vibrio species that appear in shrimp and crabs. The results also revealed that the LAMP assay could be 10-fold more sensitive than conventional PCR in detecting V. alginolyticus. This could be the first report on using a rapid and highly sensitive technique, the LAMP assay, in the effective diagnosis of the pathogenic bacteria V. alginolyticus, which could help in the early detection of diseases, particularly in aquaculture.


LAMP (Loop-mediated Isothermal Amplification);PCR;primer;streptococcus alginolyticus


  1. Yeh, M. S., Liu, C. H., Hung, C. W. and Cheng, W. 2009. cDNA cloning, identification, tissue localisation, and transcription profile of a transglutaminase from white shrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus. Fish Shellfish Immunol. 27, 748-756.
  2. Chunhua, Ren., Chaoqun, Hu., Xiao, Jiang., Hongyan, Sun., Zhe, Zhao., Chang, Chen. and Peng, Luo. 2013. Distribution and pathogenic relationship of virulence associated genes among Vibrio alginolyticus from the mariculture systems. Mol. Cell. Probes. 27, 164-168.
  3. Tseng, I, T. and Chen, J. C. 2004. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress. Fish Shellfish Immunol. 17, 325-333.
  4. Kang, B. J., Im, B. S. and Heo, M. S. 2010. Disease and Treatment of Farmed fish, pp. 73-78, Aqua Info: Yeongdeungpo-gu, Seoul, Korea.
  5. Kim, J., S. 2012. Development of loop-mediated isothermal amplification technique and trivalent vaccine for olive flounder against streptococcusis and edwardsiellosis. Ph. D. dissertation, Jeju National University, Jeju, South Korea.
  6. Lili, Sheng., Yuanzhi, Lv., Qin, Liu., Qiyao, Wang. and Yuanxing, Zhang. 2013. Connecting type VI secretion, quorum sensing, and c-di-GMP production in fish pathogen Vibrio alginolyticus through phosphatase PppA. Vet. Microbiol. 162, 652-662.
  7. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, 63-69.
  8. Shan Liang, Xu., Cheng Gong, Qiu., Wei, Zhou., Dan Li, Wang., Yan, Jia. and Chun Lin, Wang. 2013. Pathological analysis of hemolymphs of Charybdis japonica infected with Vibrio alginolyticus. Fish Shellfish Immunol. 35, 1577-1584.
  9. Xie, Z. Y., Hu, C. Q., Chen, C., Zhang, L. P. and Ren, C. H. 2005. Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China. 2005. Lett. Appl. Microbiol. 41, 202-207.
  10. Austin, B. 2010. Vibrios as causal agents of zoonoses. Vet. Microbiol. 140, 310-317.
  11. Baumann, P., Baumann, L. and Mandel, M. 1971. Taxonomy of marine bacteria: the genus Beneckea. J. Bacteriol. 107, 268-294.
  12. Blake, P. A., Weaver, R. E. and Hollis. D. G. 1980. Diseases of humans (other than cholera) caused by Vibiros. Annu. Rev. Microbiol. 34, 341-367.