School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea.
Electrophoresis. 2010 Jul;31(14):2405-10. doi: 10.1002/elps.201000091.
Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE-based SSCP (CE-SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene-specific markers unless combined with additional modification steps. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high-resolution CE-SSCP system using a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31-1.56 pg and a dynamic range of approximately 10(2). These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.
已经开发出几种基于 16S 核糖体 RNA 基因标记物筛选的方法，用于快速、灵敏地检测致病微生物。一种这样的方法，基于毛细管电泳的单链构象多态性分析（CE-SSCP），因为该技术可以使用简单的程序分离序列变异，所以具有巨大的潜力。然而，传统的 CE-SSCP 系统分辨率有限，除非与额外的修饰步骤结合使用，否则无法分离大多数 16S 核糖体 RNA 基因特异性标记物。最近开发了一种使用聚（环氧乙烷）-聚（丙烯氧化物）-聚（环氧乙烷）三嵌段共聚物基质的高分辨率 CE-SSCP 系统，该系统已被证明能够有效地分离高度相似的 PCR 产物。在这项研究中，我们开发了一种基于高分辨率 CE-SSCP 系统的方法，该系统使用聚（环氧乙烷）-聚（丙烯氧化物）-聚（环氧乙烷）三嵌段共聚物，能够同时定量检测 12 种临床重要的病原体。使用通用引物通过 PCR 扩增病原体标记物，并通过 CE-SSCP 进行分离；每个标记峰在基线处很好地分离，并且表现出特征性的迁移率，易于识别病原体。使用不同量的基因组病原体 DNA 进行的一系列实验表明，该方法的检测限为 0.31-1.56 pg，动态范围约为 10(2)。这些结果表明，高分辨率 CE-SSCP 系统在细菌引起的疾病的临床诊断中有很大的潜力。
