PMID- 27348074
OWN - NLM
STAT- MEDLINE
DCOM- 20170908
LR  - 20201209
IS  - 1462-2920 (Electronic)
IS  - 1462-2912 (Linking)
VI  - 19
IP  - 1
DP  - 2017 Jan
TI  - Direct-geneFISH: a simplified protocol for the simultaneous detection and 
      quantification of genes and rRNA in microorganisms.
PG  - 70-82
LID - 10.1111/1462-2920.13432 [doi]
AB  - Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA 
      (rRNA)-targeted oligonucleotides is a standard method to detect and identify 
      microorganisms, the specific detection of genes in bacteria and archaea, for 
      example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. 
      Here we report a much improved protocol, direct-geneFISH, which allows specific 
      gene and rRNA detection within less than 6 h. For direct-geneFISH, catalyzed 
      amplification reporter deposition (CARD) steps are removed and 
      fluorochrome-labelled polynucleotide gene probes and rRNA-targeted 
      oligonucleotide probes are hybridized simultaneously. The protocol allows 
      quantification of gene copy numbers per cell and the signal of the directly 
      labelled probes enables a subcellular localization of the rRNA and target gene. 
      The detection efficiencies of direct-geneFISH were first evaluated on Escherichia 
      coli carrying the target gene on a copy-control vector. We could show that gene 
      copy numbers correlated to the geneFISH signal within the cells. The new protocol 
      was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in 
      cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell 
      and gene detection efficiencies by direct-geneFISH were statistically identical 
      to those obtained with the original geneFISH, demonstrating the suitability of 
      the simpler and faster protocol for environmental samples.
CI  - (c) 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.
FAU - Barrero-Canosa, Jimena
AU  - Barrero-Canosa J
AD  - Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 
      Celsiusstr. 1, Bremen, D-28359, Germany.
FAU - Moraru, Cristina
AU  - Moraru C
AD  - Department of Biology of Geological Processes, Institute for Chemistry and 
      Biology of the Marine environment (ICBM), Carl-von-Ossietzky-Strasse 9-11, 
      Oldenburg, D-26111, Germany.
FAU - Zeugner, Laura
AU  - Zeugner L
AD  - Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 
      Celsiusstr. 1, Bremen, D-28359, Germany.
FAU - Fuchs, Bernhard M
AU  - Fuchs BM
AD  - Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 
      Celsiusstr. 1, Bremen, D-28359, Germany.
FAU - Amann, Rudolf
AU  - Amann R
AD  - Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 
      Celsiusstr. 1, Bremen, D-28359, Germany.
LA  - eng
PT  - Journal Article
DEP - 20160718
PL  - England
TA  - Environ Microbiol
JT  - Environmental microbiology
JID - 100883692
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Ribosomal)
SB  - IM
CIN - Environ Microbiol. 2017 Jan;19(1):3-4. PMID: 27486074
MH  - Croatia
MH  - Escherichia coli/*genetics
MH  - Gammaproteobacteria/*genetics
MH  - Gene Dosage/*genetics
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Lakes/microbiology
MH  - Oligonucleotide Probes/*genetics
MH  - RNA, Ribosomal/*genetics
EDAT- 2016/06/28 06:00
MHDA- 2017/09/09 06:00
CRDT- 2016/06/28 06:00
PHST- 2016/06/28 06:00 [pubmed]
PHST- 2017/09/09 06:00 [medline]
PHST- 2016/06/28 06:00 [entrez]
AID - 10.1111/1462-2920.13432 [doi]
PST - ppublish
SO  - Environ Microbiol. 2017 Jan;19(1):70-82. doi: 10.1111/1462-2920.13432. Epub 2016 
      Jul 18.