Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/146361
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- Title
- Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus
- Related
- Journal of clinical laboratory analysis, Vol. 25, No. 5, (2011), p.359-365
- DOI
- 10.1002/jcla.20486
- Publisher
- Wiley-Blackwell
- Date
- 2011
- Author/Creator
- Lawson, Thomas S
- Author/Creator
- Connally, Russell E
- Author/Creator
- Vemulpad, Subramanyam
- Author/Creator
- Piper, James A
- Description
- Background: Aspects of the fluorescence in situ hybridization (FISH) method for the detection of clinically important bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, were investigated for optimization. Methods: Various approaches to optimizing the FISH procedure were taken and different methods were compared. To save time, hybridization and washing buffers were prepared beforehand and stored at -20°C and mixed to their final formamide and NaCl concentrations just before use. The use of 50-ml tubes for hybridizationincubation reduced drying out, reagent wastage, and reaction times. Results: A two-step permeabilization FISH assay was developed that used phosphate-buffered saline as a buffer for lysostaphin. It could detect bacteria with DNA probes conjugated to fluorophores with a higher signal intensity and the less expensive biotinylated DNA probes with minimal cell lysis in 1hr. Conclusions: The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules.
- Description
- 7 page(s)
- Subject Keyword
- FISH
- Subject Keyword
- Fluorescence in situ hybridization
- Subject Keyword
- Gram-positive bacteria
- Subject Keyword
- Molecular diagnostic techniques
- Subject Keyword
- Staphylococci
- Subject Keyword
- Staphylococcus aureus
- Subject Keyword
- Staphylococcus epidermidis
- Resource Type
- journal article
- Organisation
- Macquarie University. Dept. of Physics and Astronomy
- Organisation
- Macquarie University. Dept. of Chiropractic
- Organisation
- Macquarie University. Office of the Deputy Vice-Chancellor (Research)
- Identifier
- http://hdl.handle.net/1959.14/146361
- Identifier
- ISSN:0887-8013
- Identifier
- mq_res-ext-2-s2.0-80052855443
- Language
- eng
- Reviewed
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