Kendra Samuel, Czucz Varga Jarmila, Gaálová-Radochová Barbora, Bujdáková Helena
Faculty of Natural Sciences, Department of Microbiology and Virology, Comenius University in Bratislava, Bratislava, 84215, Slovakia.
Biol Methods Protoc. 2024 Nov 18;9(1):bpae081. doi: 10.1093/biomethods/bpae081. eCollection 2024.
Determining the number of viable cells by calculating colony-forming units is time-consuming. The evaluation of mixed biofilms consisting of different species is particularly problematic. Therefore, the aim of this study was to optimize a molecular method-propidium monoazide quantitative polymerase chain reaction (PMA-qPCR)-for accurate and consistent differentiation between living and dead cells. In the practical experimental example, the number of genome copies representing living cells was determined in a mixed biofilm of - inhibited by photodynamic inactivation. Optimal conditions such as PMA concentration and the duration of light exposure, the optimization of DNA isolation from the mixed biofilm and standardization of PMA-qPCR parameters were tested prior to the main experiment. The genome copy number was calculated based on the known amount of genomic DNA in the qPCR and the genome size of the respective microorganism. The results showed that photodynamic inactivation in the presence of 1 mM methylene blue decreased the total genome copy number from 1.65 × 10 to 3.19 × 10, and from 4.39 × 10 to 1.91 × 10 for and (<0.01), respectively. The main disadvantage is the overestimation of the number of living cells represented by genome copy numbers. Such cells are unable to reproduce and grow (no vitality) and are continuously dying. On the other hand, PMA-qPCR determines the copy numbers of all microbial species, including a mix of eukaryotic yeasts and prokaryotic bacteria in a biofilm in one step, which is a great advantage.
通过计算菌落形成单位来确定活细胞数量很耗时。评估由不同物种组成的混合生物膜尤其成问题。因此，本研究的目的是优化一种分子方法——单叠氮碘化丙啶定量聚合酶链反应（PMA-qPCR）——以准确、一致地区分活细胞和死细胞。在实际实验示例中，在受光动力失活抑制的混合生物膜中测定了代表活细胞的基因组拷贝数。在主要实验之前，测试了诸如PMA浓度和光照持续时间等最佳条件、从混合生物膜中分离DNA的优化以及PMA-qPCR参数的标准化。根据qPCR中已知的基因组DNA量和相应微生物的基因组大小计算基因组拷贝数。结果表明，在存在1 mM亚甲蓝的情况下进行光动力失活，使总基因组拷贝数分别从1.65×10降至3.19×10，以及从4.39×10降至1.91×10（<0.01）。主要缺点是基因组拷贝数所代表的活细胞数量被高估。此类细胞无法繁殖和生长（无活力）且不断死亡。另一方面，PMA-qPCR一步就能确定生物膜中所有微生物物种的拷贝数，包括真核酵母和原核细菌的混合物，这是一个很大的优势。
