Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Fungal Biol. 2013 Feb;117(2):156-62. doi: 10.1016/j.funbio.2013.01.004. Epub 2013 Jan 17.
Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10(®) Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.
检测致命的两栖动物真菌壶菌依赖于基于 PCR 的技术。尽管这些方法非常准确和敏感，但它们无法区分活细胞和死细胞。在这项研究中，提出了一种结合 DNA 嵌入染料吖啶橙（EMA）和实时 PCR 的新方法，该方法允许定量检测活的 B. dendrobatidis 细胞，而无需培养。开发的方法能够抑制热致死的 B. dendrobatidis 游动孢子的实时 PCR 信号达 99.9%，并且能够区分混合样品中的活孢子和热致死的 B. dendrobatidis 游动孢子。此外，该新方法还用于评估兽医消毒剂 F10(®) 抗菌溶液的抗真菌活性。这种消毒剂在低至 1:6400 的浓度下，在 1 分钟内就能有效杀死 B. dendrobatidis 游动孢子。
