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Fluorescent nucleic acid probe in droplets for bacterial sorting (FNAP-sort) as a high-throughput screening method for environmental bacteria with various growth rates.
Ota, Yuri; Saito, Kanako; Takagi, Taeko; Matsukura, Satoko; Morita, Masamune; Tsuneda, Satoshi; Noda, Naohiro.
Afiliación
  • Ota Y; Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
  • Saito K; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
  • Takagi T; Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
  • Matsukura S; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
  • Morita M; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
  • Tsuneda S; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
  • Noda N; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
PLoS One ; 14(4): e0214533, 2019.
Article en En | MEDLINE | ID: mdl-30995251
We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bacterias / ARN Bacteriano / Sondas de Ácido Nucleico / Transferencia Resonante de Energía de Fluorescencia / Ensayos Analíticos de Alto Rendimiento / Colorantes Fluorescentes Tipo de estudio: Diagnostic_studies / Screening_studies Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2019 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bacterias / ARN Bacteriano / Sondas de Ácido Nucleico / Transferencia Resonante de Energía de Fluorescencia / Ensayos Analíticos de Alto Rendimiento / Colorantes Fluorescentes Tipo de estudio: Diagnostic_studies / Screening_studies Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2019 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Estados Unidos