Optimization of a Two-step Permeabilization Fluorescence in Situ Hybridization (FISH) Assay for the Detection of Staphylococcus Aureus

Overview

Journal J Clin Lab Anal

Publisher Wiley

Specialties Biomedical Engineering
Biotechnology
Pathology

Date 2011 Sep 16

PMID 21919072

Citations 2

Authors

Thomas S Lawson

Russell E Connally

Subramanyam Vemulpad

James A Piper

Affiliations

Soon will be listed here.

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 hybridization incubation 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.

Citing Articles

An Introduction to Fluorescence in situ Hybridization in Microorganisms.

Almeida C, Azevedo N Methods Mol Biol. 2021; 2246:1-15.

PMID: 33576979 DOI: 10.1007/978-1-0716-1115-9_1.

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic .

Sayyadi N, Connally R, Lawson T, Yuan J, Packer N, Piper J Molecules. 2019; 24(11).

PMID: 31159269 PMC: 6600140. DOI: 10.3390/molecules24112083.

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