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Development of an absolute quantification method for ribosomal RNA gene copy numbers per eukaryotic single cell by digital PCR.
Yarimizu, Kyoko; Sildever, Sirje; Hamamoto, Yoko; Tazawa, Satoshi; Oikawa, Hiroshi; Yamaguchi, Haruo; Basti, Leila; Mardones, Jorge I; Paredes-Mella, Javier; Nagai, Satoshi.
Afiliación
  • Yarimizu K; Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan; Office of Industry-Academia-Government and Community Collaboration, Hiroshima University, 1-3-2 22 Kagamiyama, Higashi-Hir
  • Sildever S; Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan; Department of Marine Systems, Tallinn University of Technology, Akadeemia tee 15A, 12618 Tallinn, Estonia.
  • Hamamoto Y; Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan.
  • Tazawa S; AXIOHELIX Co. Ltd, 12-17 Kandaizumicho, Chiyoda-ku, Tokyo 101-0024, Japan.
  • Oikawa H; Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan.
  • Yamaguchi H; Faculty of Agriculture and Marine Sciences, Kochi University, Nankoku, Kochi 783-8502, Japan.
  • Basti L; Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan.
  • Mardones JI; Instituto de Fomento Pesquero, Centro de Estudios de Algas Nocivas (IFOP-CREAN), Padre Harter 574, Puerto Montt 5501679, Chile; Centro FONDAP de Investigación en Dinámica de Ecosistemas Marinos de Altas Latitudes (IDEAL), Valdivia, Chile.
  • Paredes-Mella J; Instituto de Fomento Pesquero, Centro de Estudios de Algas Nocivas (IFOP-CREAN), Padre Harter 574, Puerto Montt 5501679, Chile.
  • Nagai S; Japan Fisheries Research and Education Agency, Fisheries Resources Institute, Fisheries Stock Assessment Center, 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan. Electronic address: snagai@affrc.go.jp.
Harmful Algae ; 103: 102008, 2021 03.
Article en En | MEDLINE | ID: mdl-33980448
Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Variaciones en el Número de Copia de ADN / Floraciones de Algas Nocivas Tipo de estudio: Screening_studies Idioma: En Revista: Harmful Algae Año: 2021 Tipo del documento: Article Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Variaciones en el Número de Copia de ADN / Floraciones de Algas Nocivas Tipo de estudio: Screening_studies Idioma: En Revista: Harmful Algae Año: 2021 Tipo del documento: Article Pais de publicación: Países Bajos