Development of a method of sensitively and specifically detecting a Vibrio sp. strain MA3 associated with mass mortalities of the pearl oyster Pinctada fucata martensii using quantitative PCR

International Aquatic Research

مقاله 2، دوره 14، شماره 4، اسفند 2022، صفحه 241-250   

نوع مقاله: Original research

شناسه دیجیتال (DOI): 10.22034/iar.2022.1958494.1275

نویسندگان

Kaito Hatano^{* 1}؛ Akihiro Sakatoku²؛ Daisuke Tanaka²؛ Shoki Tanaka³؛ Tadashi Isshiki⁴؛ Nobuo Suzuki¹

¹Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Noto-cho, Ishikawa 927-0553, Japan

²School of Science, Academic Assembly, University of Toyama, Toyama 930-8555, Japan

³Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan; Mie Prefecture Fisheries Research Institute, Hamajima, Shima, Mie 517-0404, Japan

⁴Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan

چکیده

The mass mortality of the Japanese pearl oyster Pinctada fucata martensii from 2019 to 2021 had a serious impact on Japan's pearl oyster industry. We previously reported that the possible cause of mass mortality was bacterial infection and that a new strain of Vibrio sp. (strain MA3) closely related to Vibrio alginolyticus was isolated from dead pearl oysters. Therefore, the development of a method for specifically detecting only strain MA3 as distinguished from other genus Vibrio species has been eagerly desired. In the present study, a successful quantitative PCR method was established with primers that specifically amplify the intergenic spacer (IGS) region of the 16S–23S rDNA genes of strain MA3. The specificity of this quantitative PCR was high, and it was possible to amplify only IGS genes prepared from strain MA3; however, it could not amplify IGS genes prepared from V. alginolyticus (NBRC15630) of species closely related to strain MA3. Furthermore, this method was highly sensitive and was able to detect up to 101 copies of the IGS gene. Using this newly developed quantitative PCR method, strain MA3 in tissues such as the adductor muscles and gills of dead pearl oysters in infection experiments was detected, although strain MA3 could not be detected in non-infected pearl oysters. Additionally, this PCR method was utilized for detecting strain MA3 in natural seawater because it could be detected in natural seawater to which a bacterial solution of strain MA3 had been added. By using the specific detection methods developed in the present study, it was possible to detect strain MA3 in seawater and diagnose infected pearl oysters, which will lead to preventing the mass mortality of pearl oysters.

کلیدواژه‌ها

Marine bacterium؛ Bivalves؛ Infection experiment؛ Detection method؛ IGS region