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Extension of bacterial rDNA sequencing for simultaneous methylation detection and its application in microflora analysis.
Nishimura, Motoi; Tanaka, Tomoaki; Murata, Syota; Miyabe, Akiko; Ishige, Takayuki; Kawasaki, Kenji; Yokoyama, Masataka; Hashimoto, Naoko; Yamagata, Kazuyuki; Nagano, Hidekazu; Tojo-Nishimura, Satomi; Matsushita, Kazuyuki.
Afiliación
  • Nishimura M; Division of Laboratory Medicine, Clinical Genetics and Proteomics, Chiba University Hospital, Chiba, Japan. zxa03460@nifty.ne.jp.
  • Tanaka T; Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
  • Murata S; Research Institute of Disaster Medicine, Chiba University, Chiba, Japan.
  • Miyabe A; Division of Laboratory Medicine, Clinical Genetics and Proteomics, Chiba University Hospital, Chiba, Japan.
  • Ishige T; Division of Laboratory Medicine, Clinical Genetics and Proteomics, Chiba University Hospital, Chiba, Japan.
  • Kawasaki K; Division of Laboratory Medicine, Clinical Genetics and Proteomics, Chiba University Hospital, Chiba, Japan.
  • Yokoyama M; Division of Laboratory Medicine, Clinical Genetics and Proteomics, Chiba University Hospital, Chiba, Japan.
  • Hashimoto N; Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
  • Yamagata K; Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
  • Nagano H; Research Institute of Disaster Medicine, Chiba University, Chiba, Japan.
  • Tojo-Nishimura S; Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
  • Matsushita K; Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
Sci Rep ; 13(1): 5731, 2023 04 07.
Article en En | MEDLINE | ID: mdl-37029177
Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bacterias / Metilación de ADN Tipo de estudio: Diagnostic_studies Idioma: En Revista: Sci Rep Año: 2023 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
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XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bacterias / Metilación de ADN Tipo de estudio: Diagnostic_studies Idioma: En Revista: Sci Rep Año: 2023 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Reino Unido