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

Kawakami Shuji

Hasegawa Takuya

Imachi Hiroyuki

Yamaguchi Takashi

Harada Hideki

Ohashi Akiyoshi

Kubota Kengo

Journal of Microbiological Methods

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.

Functional genes

In situ whole-cell detection

Polynucleotide probes

Two-pass TSA-FISH

Biology [ 460 ]

eng

Elsevier Science BV

(c) 2012 Elsevier B.V. All rights reserved.

[ISSN] 0167-7012

[DOI] 10.1016/j.mimet.2011.11.014

[NCID] AA1063956X

[DOI] http://dx.doi.org/10.1016/j.mimet.2011.11.014