Abstract：
Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reaction（qPCR）. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNA（tDNA）extracted from spore cells and intracellular（iDNA）and extracellular DNA（eDNA）extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xx（PMAxx）before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following: 1）amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2）lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.
摘要：
机械珠破碎是从孢子细胞中提取DNA的有效方法，用于随后通过定量聚合酶链反应（qPCR）定量孢子种群。在这项研究中,为了验证孢子DNA定位和提取效率，分级分离的DNA包括从孢子细胞中提取的总DNA（tDNA）和通过化学去污和碱性裂解缓冲液从分级分离的孢子中提取的细胞内DNA（iDNA）和细胞外DNA（eDNA）。每个随后是珠子破坏。此外，每次离心后，将碱裂解缓冲液处理的孢子细胞强烈洗涤3次和5次，以确定重复离心对DNA量的影响。该过程通过分级分离的孢子颗粒和在机械珠破碎之前用单叠氮化物丙锭xx（PMAxx）进行悬浮处理来实现。用qPCR评估三个分级和提取的DNA。eDNA的含量高于iDNA，在qPCR分析中更接近tDNA水平。这些结果表明：1）通过包括碱性裂解缓冲液和珠子破坏的组合方法，eDNA的量大于iDNA，并且占tDNA的大部分量。2）裂解缓冲液通过多个洗涤步骤部分消除eDNA片段，但这在很大程度上并不独立于离心的次数。
