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

作者：Kawakami Shuji; Hasegawa Takuya; Imachi Hiroyuki; Yamaguchi Takashi; Harada Hideki; Ohashi Akiyoshi; Kubota Kengo^*

来源：Journal of Microbiological Methods, 2012, 88(2): 218-223.

DOI：10.1016/j.mimet.2011.11.014

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

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 (%26gt;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.

• 出版日期2012-2