Sthapit Niva, Thakali Ocean, Malla Bikash, Raya Sunayana, Sherchand Jeevan B, Haramoto Eiji
Department of Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi, 400-8511, Japan.
Interdisciplinary Center for River Basin Environment, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi, 400-8511, Japan.
J Environ Manage. 2025 Aug;389:126120. doi: 10.1016/j.jenvman.2025.126120. Epub 2025 Jun 12.
Digital polymerase chain reaction (dPCR) is a significant advancement in health-related water microbiology, enabling absolute quantification without standard curves. By partitioning samples into thousands of individual reactions, dPCR allows for precise quantification even in the presence of inhibitory substances common in environmental samples. This study evaluated the applicability of dPCR to detect gastroenteritis-causing enteropathogens (Salmonella spp., Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, JC and BK polyomaviruses, and human adenovirus), crAssphage, and four antibiotic resistance genes (ARGs) (sul1, bla, bla, and intI1) in wastewater and drinking water source samples. Wastewater samples were collected in 2018 from two municipal wastewater treatment plants (WWTPs) that used an oxidation ditch system (n = 12) and a stabilization pond system (n = 10) in the Kathmandu Valley, Nepal, whereas the drinking water source samples were collected from shallow wells between 2015 and 2016 (n = 22). The enteropathogens and ARGs were analyzed using the QIAcuity dPCR System. The highest detection ratio was observed by crAssphage in wastewater (100 %, 22/22) and by sul1 and intl1 in drinking water sources (91 %, 20/22). The log reduction values evaluated in both WWTPs were <1 using dPCR, consistent with those obtained in previous quantitative PCR studies, and may be used to cross-validate between methods. However, this study also observed low detection ratios and concentrations of enteropathogens, likely due to factors such as low sample volumes, dead volume, thermal conditions, and the dPCR platform used. Thus, optimizing these variables is imperative to enhance the applicability of dPCR in environmental assessments.
数字聚合酶链反应（dPCR）是健康相关水微生物学领域的一项重大进展，能够在无需标准曲线的情况下进行绝对定量。通过将样本分成数千个单独的反应，即使存在环境样本中常见的抑制物质，dPCR也能实现精确量化。本研究评估了dPCR在检测废水和饮用水源样本中引起肠胃炎的肠道病原体（沙门氏菌属、空肠弯曲菌、结肠弯曲菌、产气荚膜梭菌、JC和BK多瘤病毒以及人腺病毒）、crAssphage和四种抗生素抗性基因（ARGs）（sul1、bla、bla和intI1）方面的适用性。2018年，从尼泊尔加德满都谷地使用氧化沟系统的两座城市污水处理厂（n = 12）和稳定塘系统的两座城市污水处理厂（n = 10）采集了废水样本，而饮用水源样本则在2015年至2016年期间从浅井中采集（n = 22）。使用QIAcuity dPCR系统分析肠道病原体和ARGs。在废水中，crAssphage的检测率最高（100%，22/22），在饮用水源中，sul1和intl1的检测率最高（91%，20/22）。使用dPCR评估的两座污水处理厂的对数减少值均<1，与之前定量PCR研究获得的值一致，可用于方法间的交叉验证。然而，本研究还观察到肠道病原体的检测率和浓度较低，可能是由于样本量小、死体积、热条件和所使用的dPCR平台等因素所致。因此，优化这些变量对于提高dPCR在环境评估中的适用性至关重要。
