Molecular Genetics Department, Cuban Neuroscience Center, 190 St and 25 St, No. 2503. Cubanacan, Playa, Havana 11600, Cuba.
Molecular Genetics Department, Cuban Neuroscience Center, 190 St and 25 St, No. 2503. Cubanacan, Playa, Havana 11600, Cuba.
J Microbiol Methods. 2022 Apr;195:106453. doi: 10.1016/j.mimet.2022.106453. Epub 2022 Mar 23.
Genomic DNA preparation is a critical step for successful fingerprinting analysis by Pulsed Field Gel Electrophoresis (PFGE). This paper presents a simple and rapid protocol to prepare DNA samples from up to 24 bacterial isolates simultaneously. It involves performing the conventional PFGE sample preparation steps (cell growth and harvest, agarose-immobilization of intact cells, and DNA release and purification) into a 24-wells culture plate, without subjecting the biological material to repeated transference of containers. The single-container protocol rendered high quality genomic DNA from E. coli clinical isolates. The DNA yields obtained from samples prepared with the single-container protocol showed no differences to those obtained using the conventional DNA sample preparation for PFGE. This procedure is a cost-effective alternative that provides a larger capacity of analysis to the PFGE technique, which could be advantageous in a context of infectious disease outbreaks for pathogens molecular subtyping.
基因组 DNA 制备是成功进行脉冲场凝胶电泳（PFGE）指纹分析的关键步骤。本文提出了一种简单快速的方法，可同时从多达 24 个细菌分离株中制备 DNA 样品。它涉及将常规 PFGE 样品制备步骤（细胞生长和收获、完整细胞的琼脂糖固定化以及 DNA 释放和纯化）进行到 24 孔培养板中，而无需将生物材料反复转移到容器中。该单容器方案从大肠杆菌临床分离株中获得了高质量的基因组 DNA。使用单容器方案制备的样品的 DNA 产量与使用常规 PFGE 用 DNA 样品制备获得的产量没有差异。该方法具有成本效益，为 PFGE 技术提供了更大的分析能力，这在传染病爆发时对病原体的分子分型可能具有优势。
