改良 PFGE 方案提高易降解 DNA 的医院获得性.. 型别率
Modified PFGE protocol for improving typeability of DNA degradation susceptible nosocomial .
机构信息
Department of Medical Microbiology, Biotechnology Research Institute, Suez Canal University, Egypt.
Department of Microbiology and Immunology, Faculty of Medicine, Minia University, Egypt.
出版信息
J Med Microbiol. 2019 Dec;68(12):1787-1792. doi: 10.1099/jmm.0.001093.
PFGE is the 'gold standard' method for bacterial subtyping. However, many strains are non-typable by this approach because of DNA degradation by nucleases action. To evaluate a modified PFGE protocol for typing nosocomial isolates of . Twenty- five isolates previously exposed to DNA degradation were used to optimize an extraction method for elimination of DNases activity before applying Xba1 enzyme. Introducing of sodium dodecyl sulfate (SDS) in different concentrations to the extraction buffer was evaluated for protecting genomic DNA molecule from degradation by nucleases. Addition of 3 % SDS in combination with 3 % N-lauryl sarcosine to the extraction buffer was found to reduce the previously experienced nuclease activity. Pre-examination of plug quality prior to the digestion phase could efficiently reduce the expense of the wasted enzyme. We have successfully devised a PFGE protocol that enhanced the typeability of nosocomial .
PFGE 是细菌分型的“金标准”方法。然而，由于核酸酶的作用，许多菌株无法通过这种方法进行分型。本研究旨在评估一种改良的 PFGE 方案，用于对医院分离株进行分型。我们使用 25 株先前经历 DNA 降解的分离株来优化提取方法，以消除 Xba1 酶应用前核酸酶的活性。评估了在提取缓冲液中加入不同浓度的十二烷基硫酸钠（SDS）以保护基因组 DNA 分子免受核酸酶降解的方法。结果发现，在提取缓冲液中加入 3% SDS 与 3% N-月桂酰肌氨酸联合使用可降低先前经历的核酸酶活性。在消化阶段之前对泳道质量进行预检查可以有效地减少浪费的酶的费用。我们已经成功设计了一种 PFGE 方案，提高了医院的 型别率。
Zotero 插件