title: Development of loop-mediated isothermal amplification targeting 18s ribosomal DNA for rapid detection of Azumiobodo hoyamushi (Kinetoplastea).
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.
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Su-Min
authors.initials: SM
authors.lastname: Song
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Dinzouna-Boutamba
authors.initials: DB
authors.lastname: Sylvatrie-Danne
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: So-Young
authors.initials: SY
authors.lastname: Joo
authors.affiliation: Aquaculture Management Division, National Fisheries Research & Development Institute, Busan 619-705, Korea.
authors.forename: Yun Kyung
authors.initials: YK
authors.lastname: Shin
authors.affiliation: Department of Parasitology, School of Medicine, Pusan National University, Yangsan 626-870, Korea.
authors.forename: Hak Sun
authors.initials: HS
authors.lastname: Yu
authors.affiliation: Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, Asan 336-745, Korea.
authors.forename: Yong-Seok
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Department of Life Science and Biotechnology, College of Natural Sciences, Soonchunhyang University, Asan 336-745, Korea.
authors.forename: Ji-Eon
authors.initials: JE
authors.lastname: Jung
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
authors.affiliation: Department of Preventive Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Won Kee
authors.initials: WK
authors.lastname: Lee
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Youn-Kyoung
authors.initials: YK
authors.lastname: Goo
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Dong-Il
authors.initials: DI
authors.lastname: Chung
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Korea.
authors.forename: Yeonchul
authors.initials: Y
authors.lastname: Hong
mesh_terms: D000818:Animals; D056986:Euglenozoa Infections / Q000175:diagnosis / Q000662:veterinary; D016830:Kinetoplastida / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011237:Predictive Value of Tests; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D012680:Sensitivity and Specificity; D014561:Urochordata
chemical_list: D012337:RNA, Ribosomal, 18S
keywords: Azumiobodo hoyamushi; LAMP; ascidian aquaculture; diagnostic method; soft tunic syndrome