Department of Microbiology, Universität Innsbruck, Technikerstr. 25d, 6020, Innsbruck, Austria.
Curr Microbiol. 2019 Dec;76(12):1425-1434. doi: 10.1007/s00284-019-01772-y. Epub 2019 Sep 24.
In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.
在本研究中，在提取 DNA 之前，用吖啶橙（ethidium monoazide，EMA）处理淤泥质砂参考土壤，以区分死活细胞。为此，分别向参考土壤中添加李斯特菌细胞或细胞当量。为了评估最佳处理条件，测试了不同的 EMA 浓度。然而，结果仍然没有定论。此外，不同的暗孵育期使 EMA 穿透死细胞，但不能在下游分析中选择性地去除膜受损细胞的 DNA。与未稀释的土壤相比，当使用参考土壤的 1:10 稀释液进行 DNA 提取时，可以观察到 EMA 处理的效果；然而，该效果的选择性还不足以仅对热处理细胞起作用。虽然 EMA 在土壤中的应用需要进一步评估，但该程序有可能为改善微生物生态学研究中的基于 DNA 的方法提供未来的潜力。
