title: Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes.
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.
authors.affiliation: Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Kawakami
authors.forename: Takuya
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imachi
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Hideki
authors.initials: H
authors.lastname: Harada
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Ohashi
authors.forename: Kengo
authors.initials: K
authors.lastname: Kubota
mesh_terms: D000693:Anaerobiosis; D019641:DNA, Archaeal / Q000032:analysis / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D003901:Desulfovibrio / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019605:Euryarchaeota / Q000235:genetics; D019766:Genes, Archaeal / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D010784:Photomicrography; D012680:Sensitivity and Specificity; D012722:Sewage / Q000382:microbiology; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D019641:DNA, Archaeal; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012722:Sewage