Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
권호 표지
DOI QR코드

DOI QR Code

Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)

  • Song, Su-Min (Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine) ;
  • Sylvatrie-Danne, Dinzouna-Boutamba (Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine) ;
  • Joo, So-Young (Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine) ;
  • Shin, Yun Kyung (Aquaculture Management Division, National Fisheries Research & Development Institute) ;
  • Yu, Hak Sun (Department of Parasitology, School of Medicine, Pusan National University) ;
  • Lee, Yong-Seok (Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University) ;
  • Jung, Ji-Eon (Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University) ;
  • Inoue, Noboru (National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine) ;
  • Lee, Won Kee (Department of Preventive Medicine, Kyungpook National University School of Medicine) ;
  • Goo, Youn-Kyoung (Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine) ;
  • Chung, Dong-Il (Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine) ;
  • Hong, Yeonchul (Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine)
  • Received : 2014.02.28
  • Accepted : 2014.04.04
  • Published : 2014.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.

Keywords

References

  1. Hirose E, Ohtake SI, Azumi K. Morphological characterization of the tunic in the edible ascidian, Halocynthia roretzi (Drasche), with remarks on 'soft tunic syndrome' in aquaculture. J Fish Dis 2009; 32: 433-445. https://doi.org/10.1111/j.1365-2761.2009.01034.x
  2. Kumagai A, Suto A, Ito H, Tanabe T, Takahashi K, Kamaishi T, Miwa S. Mass mortality of cultured ascidians Halocynthia roretzi associated with softening of the tunic and flagellate-like cells. Dis Aquat Organisms 2010; 90: 223-234. https://doi.org/10.3354/dao02228
  3. Kumagai A, Kamaishi T. Development of polymerase chain reaction assays for detection of the kinetoplastid Azumiobodo hoyamushi, the causative agent for soft tunic syndrome in the ascidian Halocynthia roretzi. Fish Pathol 2013; 48: 42-47. https://doi.org/10.3147/jsfp.48.42
  4. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucl Acids Res 2000; 28: E63. https://doi.org/10.1093/nar/28.12.e63
  5. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 2009; 15: 62-69. https://doi.org/10.1007/s10156-009-0669-9
  6. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 2001; 289: 150-154. https://doi.org/10.1006/bbrc.2001.5921
  7. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 2009; 46: 167-172. https://doi.org/10.2144/000113072
  8. Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 2008; 3: 877-882. https://doi.org/10.1038/nprot.2008.57
  9. Hirose E, Nozawa A, Kumagai A, Kitamura S. Azumiobodo hoyamushi gen. nov. et sp. nov. (Euglenozoa, Kinetoplastea, Neobodonida): a pathogenic kinetoplastid causing the soft tunic syndrome in ascidian aquaculture. Dis Aquat Organisms 2012; 97: 227-235. https://doi.org/10.3354/dao02422
  10. Kim D, Baik KS, Park SC, Kim SJ, Shin TS, Jung SJ, Oh MJ, Seong CN. Cellulase production from Pseudoalteromonas sp. NO3 isolated from the sea squirt Halocynthia rorentzi. J Indust Microbiol Biotechnol 2009; 36: 1375-1382. https://doi.org/10.1007/s10295-009-0623-y
  11. Kim SH, Yang HO, Shin YK, Kwon HC. Hasllibacter halocynthiae gen. nov., sp. nov., a nutriacholic acid-producing bacterium isolated from the marine ascidian Halocynthia roretzi. Int J Syst Evol Microbiol 2012; 62: 624-631. https://doi.org/10.1099/ijs.0.028738-0
  12. Kitamura SI, Ohtake SI, Song JY, Jung SJ, Oh MJ, Choi BD, Azumi K, Hirose E. Tunic morphology and viral surveillance in diseased Korean ascidians: soft tunic syndrome in the edible ascidian, Halocynthia roretzi (Drasche), in aquaculture. J Fish Dis 2010; 33: 153-160. https://doi.org/10.1111/j.1365-2761.2009.01103.x
  13. Kumagai A, Ito H, Sasaki R. Detection of the kinetoplastid Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, in wild ascidians Halocynthia roretzi. Dis Aquat Organisms 2013; 106: 267-271. https://doi.org/10.3354/dao02653
  14. Enomoto Y, Yoshikawa T, Ihira M, Akimoto S, Miyake F, Usui C, Suga S, Suzuki K, Kawana T, Nishiyama Y, Asano Y. Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method. J Clin Microbiol 2005; 43: 951-955. https://doi.org/10.1128/JCM.43.2.951-955.2005
  15. Kaneko H, Kawana T, Fukushima E, Suzutani T. Tolerance of loopmediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 2007; 70: 499-501. https://doi.org/10.1016/j.jbbm.2006.08.008
  16. Sharma S, Srinivasan M, George C. Acanthamoeba keratitis in non-contact lens wearers. Arch Ophthalmol 1990; 108: 676-678. https://doi.org/10.1001/archopht.1990.01070070062035
  17. Srinivasan M, Burman S, George C, Nirmalan PK. Non-contact lens related Acanthamoeba keratitis at a tertiary eye care center in south India: implications for eye care programs in the region. Med Sci Monit 2003; 9: CR125-CR129.

Cited by

  1. Measurement of Tunic Hardness in an Edible Ascidian, Halocynthia roretzi, with Remarks on Soft Tunic Syndrome vol.35, pp.6, 2014, https://doi.org/10.2108/zs180072