Knüpfer Mandy, Braun Peter, Baumann Kathrin, Rehn Alexandra, Antwerpen Markus, Grass Gregor, Wölfel And Roman
Bacteriology and Toxinology, Bundeswehr Institute of Microbiology, 80937 Munich, Germany.
Microbial Genomics and Bioinformatics, Bundeswehr Institute of Microbiology, 80937 Munich, Germany.
Microorganisms. 2020 May 20;8(5):763. doi: 10.3390/microorganisms8050763.
A variety of methods have been established in order to optimize the accessibility of DNA originating from cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. Here, we started by systematically assessing the efficiencies of 20 DNA extraction kits for vegetative cells. Of these, the Epicentre MasterPure kit gave the best DNA yields and quality suitable for further genomic analysis. Yet, none of the kits tested were able to extract reasonable quantities of DNA from cores of the endospores. Thus, we developed a mechanical endospore lysis protocol, facilitating the extraction of high-quality DNA. Transmission electron microscopy or the labelling of spores with the indicator dye propidium monoazide was utilized to assess lysis efficiency. Finally, the yield and quality of genomic spore DNA were quantified by PCR and they were found to be dependent on lysis matrix composition, instrumental parameters, and the method used for subsequent DNA purification. Our final standardized lysis and DNA extraction protocol allows for the quantitative detection of low levels (<50 CFU/mL) of endospores and it is suitable for direct quantification, even under resource-limited field conditions, where culturing is not an option.
为了优化源自细胞和芽孢的DNA的可及性，以便于进行高灵敏度分子诊断，已经建立了多种方法。然而，大多数芽孢裂解技术尚未就其定量能力进行评估。在这里，我们首先系统地评估了20种用于营养细胞的DNA提取试剂盒的效率。其中，Epicentre MasterPure试剂盒的DNA产量和质量最佳，适合进一步的基因组分析。然而，所测试的试剂盒中没有一种能够从芽孢核心中提取出适量的DNA。因此，我们开发了一种机械芽孢裂解方案，便于提取高质量的DNA。利用透射电子显微镜或用指示染料单叠氮碘化丙啶标记芽孢来评估裂解效率。最后，通过PCR对基因组芽孢DNA的产量和质量进行了定量，发现它们取决于裂解基质组成、仪器参数以及用于后续DNA纯化的方法。我们最终的标准化裂解和DNA提取方案能够对低水平（<50 CFU/mL）的芽孢进行定量检测，即使在资源有限的野外条件下（无法进行培养），它也适用于直接定量。
