Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus

Thomas S. Lawson*, Russell E. Connally, Subramanyam Vemulpad, James A. Piper

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)359-365
Number of pages7
JournalJournal of Clinical Laboratory Analysis
Volume25
Issue number5
DOIs
Publication statusPublished - 2011

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