کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2090321 | 1081494 | 2012 | 6 صفحه PDF | دانلود رایگان |
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.
► We developed a FISH protocol for the detection of functional genes in prokaryotes.
► Bright fluorescence and a high detection efficiency (> 98%) were obtained.
► The discrimination threshold was about 82–89% sequence identity.
► The protocol is applicable to environmental samples.
Journal: Journal of Microbiological Methods - Volume 88, Issue 2, February 2012, Pages 218–223