两步透化荧光原位杂交（FISH）检测金黄色葡萄球菌方法的优化。
Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus.
机构信息
Macquarie University, Faculty of Science, Sydney, New South Wales, Australia.
出版信息
J Clin Lab Anal. 2011;25(5):359-65. doi: 10.1002/jcla.20486.
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.
背景
本研究旨在对荧光原位杂交（FISH）方法检测临床重要细菌（如金黄色葡萄球菌、表皮葡萄球菌和大肠杆菌）的各个方面进行优化。
方法
本研究采用了各种优化 FISH 程序的方法，并对不同方法进行了比较。为节省时间，杂交和洗涤缓冲液事先制备并储存在-20°C，在使用前按最终甲酰胺和 NaCl 浓度混合。杂交孵育使用 50ml 管，可减少干燥、试剂浪费和反应时间。
结果
本研究开发了一种两步渗透 FISH 检测方法，该方法使用磷酸盐缓冲盐水作为溶葡萄球菌素的缓冲液。与传统方法相比，该方法使用荧光标记的 DNA 探针检测到的信号强度更高，且细胞裂解更少，检测时间更短（1 小时）。
结论
当 FISH 信号较弱、细菌数量较低或需要使用其他报告分子时，可使用两步法检测。
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