Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

Shuji Kawakami, Takuya Hasegawa, Hiroyuki Imachi, Takashi Yamaguchi, Hideki Harada, Akiyoshi Ohashi, Kengo Kubota

研究成果: Article査読

10 被引用数 (Scopus)

抄録

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The . mcrA gene and the . apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (>. 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the . mcrA or . apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.

本文言語English
ページ(範囲)218-223
ページ数6
ジャーナルJournal of Microbiological Methods
88
2
DOI
出版ステータスPublished - 2012 2

ASJC Scopus subject areas

  • 微生物学
  • 分子生物学
  • 微生物学(医療)

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