Hwang Hee Sung, Shin Gi Won, Chung Boram, Na Jeongkyeong, Jung Gyoo Yeol
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, South Korea.
Methods Mol Biol. 2013;919:155-63. doi: 10.1007/978-1-62703-029-8_15.
Among the molecular diagnostic methods for bacteria-induced diseases, capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) combined with 16S rRNA gene-specific PCR has enormous potential because it can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S rRNA gene-specific markers into separate peaks. 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 report, a protocol for the detection of 12 pathogenic bacteria is provided. 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 the easy identification of the pathogens.
在用于细菌感染性疾病的分子诊断方法中，基于毛细管电泳的单链构象多态性（CE-SSCP）结合16S rRNA基因特异性PCR具有巨大潜力，因为它可以通过简单的程序分离序列变体。然而，传统的CE-SSCP系统分辨率有限，无法将大多数16S rRNA基因特异性标记物分离成单独的峰。最近开发了一种使用聚（环氧乙烷）-聚（环氧丙烷）-聚（环氧乙烷）三嵌段共聚物基质的高分辨率CE-SSCP系统，该系统已证明能有效分离高度相似的PCR产物。在本报告中，提供了一种检测12种病原菌的方案。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都能很好地分离，并显示出特征性迁移率，便于病原体的轻松鉴定。
