TY - JOUR
T1 - Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus
AU - Lawson, Thomas S.
AU - Connally, Russell E.
AU - Vemulpad, Subramanyam
AU - Piper, James A.
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=80052855443&partnerID=8YFLogxK
U2 - 10.1002/jcla.20486
DO - 10.1002/jcla.20486
M3 - Article
C2 - 21919072
AN - SCOPUS:80052855443
SN - 0887-8013
VL - 25
SP - 359
EP - 365
JO - Journal of Clinical Laboratory Analysis
JF - Journal of Clinical Laboratory Analysis
IS - 5
ER -