PMID- 21205104
OWN - NLM
STAT- MEDLINE
DCOM- 20150908
LR  - 20191210
IS  - 1365-2672 (Electronic)
IS  - 1364-5072 (Linking)
VI  - 110
IP  - 4
DP  - 2011 Apr
TI  - Improved DNA-FISH for cytometric detection of Candida spp.
PG  - 881-92
AB  - AIMS:   We developed improved methods for DNA-based fluorescence in situ 
      hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via 
      flow cytometry. METHODS AND RESULTS:   Two previously reported C. 
      albicans-targeted DNA probes were evaluated against whole cells of C. albicans 
      and related Candida species using a rapid, high-temperature hybridization 
      protocol. One probe (CalB2208) was shown for the first time to be suitable as a 
      FISH probe. Although cell labelling for both probes was relatively bright, we 
      were able to substantially improve our results by altering fixation and 
      hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered 
      formalin and ethanol was most effective. Probe intensity was improved as much as 
      ten-fold through the use of unlabelled helper probes, and buffer containing 0.9 
      mol l-1 NaCl plus 10% formamide yielded the best hybridizations for both 
      probe/helper cocktails. Although optimal labelling occurred with longer 
      hybridizations, we found that C. albicans could be completely differentiated from 
      the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest 
      probe (Calb-1249) and that a formal washing step was not required. Specificities 
      of probe/helper cocktails under optimal conditions were determined using a panel 
      of target and nontarget cell types, including four strains of Candida 
      dubliniensis. Calb-1249 cross-reacted slightly with Candida parapsilosis and 
      strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found 
      that CalB2208 was exclusive for C. albicans. The molecular basis of this 
      specificity was confirmed by DNA sequencing. CONCLUSIONS:   We describe DNA 
      probe-based approaches for rapid and bright labelling of Candida spp. and for 
      specific labelling of C. albicans without cross-reaction with C. dubliniensis. 
      Our work improves upon previously described methods.
FAU - Bisha, B
AU  - Bisha B
AD  - Department of Food Science & Human Nutrition, Iowa State University, Ames, IA 
      50011-1061, USA.
FAU - Kim, H J
AU  - Kim HJ
FAU - Brehm-Stecher, B F
AU  - Brehm-Stecher BF
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Appl Microbiol
JT  - Journal of applied microbiology
JID - 9706280
RN  - 0 (DNA Probes)
SB  - IM
MH  - Candida/genetics/*isolation & purification
MH  - Candida albicans/genetics/isolation & purification
MH  - Candida tropicalis/genetics/isolation & purification
MH  - *DNA Probes
MH  - Flow Cytometry
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Nucleic Acid Hybridization
MH  - Sequence Analysis, DNA
EDAT- 2011/01/06 06:00
MHDA- 2015/09/09 06:00
CRDT- 2011/01/06 06:00
PHST- 2011/01/06 06:00 [entrez]
PHST- 2011/01/06 06:00 [pubmed]
PHST- 2015/09/09 06:00 [medline]
AID - 10.1111/j.1365-2672.2011.04936.x [doi]
PST - ppublish
SO  - J Appl Microbiol. 2011 Apr;110(4):881-92. doi: 10.1111/j.1365-2672.2011.04936.x.