PMID- 18552182
OWN - NLM
STAT- MEDLINE
DCOM- 20080919
LR  - 20211020
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Print)
IS  - 0099-2240 (Linking)
VI  - 74
IP  - 16
DP  - 2008 Aug
TI  - Quantification of target molecules needed to detect microorganisms by 
      fluorescence in situ hybridization (FISH) and catalyzed reporter deposition-FISH.
PG  - 5068-77
LID - 10.1128/AEM.00208-08 [doi]
AB  - Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide 
      probes is a method that is widely used to detect and quantify microorganisms in 
      environmental samples and medical specimens by fluorescence microscopy. 
      Difficulties with FISH arise if the rRNA content of the probe target organisms is 
      low, causing dim fluorescence signals that are not detectable against the 
      background fluorescence. This limitation is ameliorated by technical 
      modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal 
      numbers of rRNA copies needed to obtain a visible signal of a microbial cell 
      after FISH or CARD-FISH have not been determined previously. In this study, a 
      novel competitive FISH approach was developed and used to determine, based on a 
      thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell 
      required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in 
      mixed pure cultures and in activated sludge. The detection limits of conventional 
      FISH with Cy3-labeled probe EUB338-I were found to be 370 +/- 45 16S rRNA 
      molecules per cell for Escherichia coli hybridized on glass microscope slides and 
      1,400 +/- 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH 
      the values ranged from 8.9 +/- 1.5 to 14 +/- 2 and from 36 +/- 6 to 54 +/- 7 16S 
      rRNA molecules per cell, respectively, indicating that the sensitivity of 
      CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results 
      suggest that optimized FISH protocols using oligonucleotide probes could be 
      suitable for more recent applications of FISH (for example, to detect mRNA in 
      situ in microbial cells).
FAU - Hoshino, Tatsuhiko
AU  - Hoshino T
AD  - Department fur Mikrobielle Okologie, Universitat Wien, Althanstrasse 14, A-1090 
      Wien, Austria.
FAU - Yilmaz, L Safak
AU  - Yilmaz LS
FAU - Noguera, Daniel R
AU  - Noguera DR
FAU - Daims, Holger
AU  - Daims H
FAU - Wagner, Michael
AU  - Wagner M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20080613
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 0 (Sewage)
SB  - IM
MH  - Colony Count, Microbial
MH  - Escherichia coli/genetics/*isolation & purification
MH  - Fluorescence
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Kinetics
MH  - Oligonucleotide Probes
MH  - RNA, Bacterial/*genetics
MH  - RNA, Ribosomal, 16S/*genetics
MH  - Sensitivity and Specificity
MH  - Sewage/microbiology
PMC - PMC2519275
EDAT- 2008/06/17 09:00
MHDA- 2008/09/20 09:00
PMCR- 2009/02/01
CRDT- 2008/06/17 09:00
PHST- 2008/06/17 09:00 [pubmed]
PHST- 2008/09/20 09:00 [medline]
PHST- 2008/06/17 09:00 [entrez]
PHST- 2009/02/01 00:00 [pmc-release]
AID - AEM.00208-08 [pii]
AID - 0208-08 [pii]
AID - 10.1128/AEM.00208-08 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2008 Aug;74(16):5068-77. doi: 10.1128/AEM.00208-08. Epub 
      2008 Jun 13.