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

✍ Scribed by Thomas S. Lawson; Russell E. Connally; Subramanyam Vemulpad; James A. Piper

Publisher: John Wiley and Sons
Year: 2011
Tongue: English
Weight: 169 KB
Volume: 25
Category: Article
ISSN: 0887-8013
DOI: 10.1002/jcla.20486

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✦ Synopsis

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 1 hr. 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. J. Clin. Lab. Anal. 25:359–365, 2011. © 2011 Wiley‐Liss, Inc.

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