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

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)218-223
Number of pages6
JournalJournal of Microbiological Methods
Volume88
Issue number2
DOIs
Publication statusPublished - 2012 Feb

Keywords

  • Functional genes
  • In situ whole-cell detection
  • Polynucleotide probes
  • Two-pass TSA-FISH

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Fingerprint Dive into the research topics of 'Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes'. Together they form a unique fingerprint.

Cite this