Department of Civil and Environmental Engineering, Faculty of Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan.
Department of Soil, Water, and Climate, University of Minnesota, MN 55108-6028, USA; BioTechnology Institute, University of Minnesota, MN 55108-1095, USA.
J Biosci Bioeng. 2020 Jul;130(1):76-81. doi: 10.1016/j.jbiosc.2020.02.004. Epub 2020 Mar 5.
The quantification of pathogens is important for assessing water safety and preventing disease outbreaks. Culture-independent approaches, such as quantitative PCR (qPCR) and digital PCR (dPCR), are useful techniques for quantifying pathogens in water samples. However, since pathogens are usually present at low concentrations in water, it is necessary to concentrate microbial cells before extracting their DNA. Many existing microbial concentration methods are inefficient or take a long time to perform. In this study, we applied a coagulation and foam separation method to concentrate environmental water samples of between 1000 and 5000 mL to 100 μL of DNA (i.e., a 1-5 × 10-fold concentration). The concentration process took <1 h. The DNA samples were then used to quantify various target pathogens using dPCR. One gene, the Shiga toxin gene (stx) of Shiga toxin-producing Escherichia coli, was detected at 32 copies/100 mL in a river water sample. The coagulation and foam concentration method followed by dPCR reported herein is a fast, sensitive, and reliable method to quantify pathogen genes in environmental water samples.
定量检测病原体对于评估水安全和预防疾病爆发至关重要。非培养方法，如定量聚合酶链反应（qPCR）和数字聚合酶链反应（dPCR），是定量检测水样中病原体的有用技术。然而，由于病原体在水中通常浓度较低，因此在提取其 DNA 之前需要浓缩微生物细胞。许多现有的微生物浓缩方法效率低或耗时较长。在这项研究中，我们应用了一种凝聚和泡沫分离方法，将 1000 至 5000 毫升的环境水样浓缩至 100 微升的 DNA（即浓缩 1-5 倍）。浓缩过程耗时不到 1 小时。然后，使用 dPCR 对这些 DNA 样本进行各种目标病原体的定量检测。在河流水样中，我们检测到了 32 个拷贝/100 毫升的产志贺毒素大肠杆菌的志贺毒素基因（stx）。本文报道的凝聚和泡沫浓缩方法结合 dPCR 是一种快速、灵敏、可靠的方法，可用于定量检测环境水样中的病原体基因。
