Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, G.C., Tehran, Iran.
Lett Appl Microbiol. 2012 Apr;54(4):272-9. doi: 10.1111/j.1472-765X.2012.03203.x. Epub 2012 Jan 24.
To develop an optimized random amplified polymorphic DNA (RAPD) protocol for fingerprinting clinical isolates of Klebsiella pneumoniae.
Employing factorial design of experiments, repeatable amplification patterns were obtained for 54 nosocomial isolates using 1 μmol 1(-1) primer, 4 mmol 1(-1) MgCl(2), 0·4 mmol 1(-1) dNTPs, 2·5 U Taq DNA polymerase and 90 ng DNA template in a total volume of 25 μl. The optimum thermocycling program was: initial denaturation at 94°C for 4 min followed by 50 cycles of 1 min at 94°C, 2 min at 34°C, 2 min at 72°C and a final extension at 72°C for 10 min. The optimized RAPD protocol was highly discriminatory (Simpson's diversity index, 0·982), and all isolates were typable with repeatable patterns (Pearson's similarity coefficient ≈ 100%). Seven main clusters were obtained on a similarity level of 70% and 32 distinct clusters on a similarity level of 85%, reflecting the heterogeneity of the isolates.
Systematic optimization of RAPD generated reliable DNA fingerprints for nosocomial isolates of K. pneumoniae.
This is the first report on RAPD optimization based on factorial design of experiments for discrimination of K. pneumoniae.
开发一种优化的随机扩增多态性 DNA（RAPD）技术，用于对临床分离的肺炎克雷伯菌进行指纹图谱分析。
采用实验因子设计，使用 1μmol 1(-1)引物、4mmol 1(-1)MgCl(2)、0·4mmol 1(-1)dNTPs、2·5UTaqDNA 聚合酶和 90ngDNA 模板，在 25μl 总体积中，可获得 54 株医院分离株的可重复扩增图谱。最佳的热循环方案为：94°C 初始变性 4min，然后进行 50 个循环，每个循环包括 1min94°C、2min34°C、2min72°C 和最后的延伸在 72°C 持续 10min。优化的 RAPD 方案具有高度的分辨力（辛普森多样性指数为 0·982），所有分离株都具有可重复的图谱（皮尔逊相似系数≈100%）。在相似度为 70%的水平上获得了 7 个主要聚类，在相似度为 85%的水平上获得了 32 个独特聚类，反映了分离株的异质性。
系统优化的 RAPD 为医院分离的肺炎克雷伯菌产生了可靠的 DNA 指纹图谱。
这是首次基于实验因子设计报道 RAPD 优化用于肺炎克雷伯菌的鉴别。
