TY - JOUR
T1 - Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes
AU - Kawakami, Shuji
AU - Hasegawa, Takuya
AU - Imachi, Hiroyuki
AU - Yamaguchi, Takashi
AU - Harada, Hideki
AU - Ohashi, Akiyoshi
AU - Kubota, Kengo
N1 - Funding Information:
This research was financially supported by research grants from the Japan Society for the Promotion of Science (JSPS), the Environment Research and Technology Development Fund (S2-03) of the Ministry of the Environment, Japan , and Kurita Water and Environment Foundation . SK was supported by JSPS research fellowships for young scientists.
PY - 2012/2
Y1 - 2012/2
N2 - 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.
AB - 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.
KW - Functional genes
KW - In situ whole-cell detection
KW - Polynucleotide probes
KW - Two-pass TSA-FISH
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U2 - 10.1016/j.mimet.2011.11.014
DO - 10.1016/j.mimet.2011.11.014
M3 - Article
C2 - 22172287
AN - SCOPUS:84856369368
VL - 88
SP - 218
EP - 223
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
SN - 0167-7012
IS - 2
ER -