Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
Infect Genet Evol. 2010 Oct;10(7):866-75. doi: 10.1016/j.meegid.2010.07.023. Epub 2010 Aug 6.
Over the years, approaches to the epidemiological analysis of infectious disease have undergone a remarkable evolutionary transition moving from phenotypic to molecular in nature. As discussed here, the quest for a clearer comparison of genomic relatedness between bacterial clinical isolates has involved four generations of molecular iteration. First generation plasmid analysis gave way to a second generation use of restriction enzymes and probes. This was followed by third generation pulsed field gel electrophoresis (PFGE) and PCR-based methods with movement now to fourth-generation DNA sequence-based approaches. Remarkably, despite (or perhaps because of) its more than 20-year history as a typing method, PFGE has demonstrated exceptional staying power. The reasons for this endurance as well as the pros and cons of PFGE use are examined in this review. In broad context the history and technology behind PFGE are considered. Issues commonly influencing the quality of PFGE data and its analysis are discussed. Specifics regarding the mechanics of DNA preparation, restriction-enzyme digestion, and proper conditions for electrophoresis are detailed and, most importantly for any approach to epidemiological assessment, issues regarding the analysis and interpretation of PFGE data are explored.
多年来，传染病的流行病学分析方法已经发生了显著的进化转变，从表型分析转向了分子分析。正如这里所讨论的，为了更清楚地比较细菌临床分离株的基因组相关性，已经经历了四代分子迭代。第一代质粒分析让位于第二代使用限制酶和探针。随后是第三代脉冲场凝胶电泳（PFGE）和基于 PCR 的方法，现在已经发展到第四代基于 DNA 序列的方法。值得注意的是，尽管 PFGE 作为一种分型方法已经有 20 多年的历史，但它仍然具有出色的持久力。本文回顾了 PFGE 持久存在的原因以及使用 PFGE 的优缺点。本文从广泛的角度考虑了 PFGE 的历史和技术。讨论了影响 PFGE 数据质量及其分析的常见问题。详细介绍了 DNA 制备、酶切和电泳条件的具体细节，最重要的是，对于任何流行病学评估方法，还探讨了 PFGE 数据的分析和解释问题。
