建立污水下水道病原體採樣及檢測方法

2020年COVID-19的爆發使全球人心惶惶，更凸顯了傳染病監測的重要性，現今已知污水流行病學(wastewater-based epidemiology, WBE)可分析污水中的生物指標及病原體。儘管各國在監測方法上進行許多污水中病原體檢測的相關研究，但對於污水的採樣方法以及從污水中萃取核酸的方法卻尚未在國內進行探討，近期發現被動採樣成本低且不受採樣空間的限制，對於收集污水中的病原體是一項具有潛力的採樣工具。因此，本研究將比較被動採樣之吸附材料(紗布、棉棒、正電濾膜及負電濾膜)對包膜病毒-傳染性支氣管炎病毒(infectious bronchitis virus, IBV)與無包膜病毒噬菌體(MS2 coliphage)的吸附效率，並檢測可代表人類糞便貢獻程度的糞便指標物(辣椒輕度斑駁病毒(pepper mild mottle virus, PMMoV)、擬桿菌屬噬菌體(crAssphage)及擬桿菌屬標記基因(Bacteroides HF183))，並將被動採樣之材料樣本與隨機樣本與組合樣本進行比較，以便在家庭的污水下水道之病原體檢測方面制定更佳的採樣方法，同時比較不同核酸純化方法的萃取效率。研究結果發現，在所有測試的核酸萃取方法中，Promega的Wizard® Enviro Wastewater TNA kit萃取效率最高，而以固相萃取-矽膠膜方法(silica membrane approach)設計的試劑盒Qiagen RNeasy PowerMicrobiome kit效果較差。在模擬被動採樣時發現PMMoV、crAssphage及Bacteroides HF183在紗布的吸附濃度高於濾膜的吸附材料；其中發現無包膜病毒MS2 coliphage在正電濾膜的吸附濃度高於負電濾膜，然而，對於包膜病毒IBV，負電濾膜的吸附效率較好，這表示濾膜的電荷差異可能會影響病毒在膜上的吸附。而三種糞便指標物中PMMoV在污水中的濃度最低。最後發現本研究經流量調整後的糞便指標物與服務人口數皆呈顯著正相關，證實PMMoV、crAssphage及Bacteroides HF183有助於反映污水下水道系統的人口規模。

關鍵字

污水流行病學；被動採樣；糞便指標物； PMMoV ； crAssphage ； Bacteroides HF183

並列摘要

The outbreak of COVID-19 has become a global pandemic since 2020 and since highlighted the need for disease prevalence monitoring. Wastewater-based epidemiology (WBE) analyzes wastewaters for biological indicators and pathogens in a population. Many studies of pathogens detection in wastewater have been developed around the world; however, sewage sampling methods in a sewer collection system and nucleic acids extraction from wastewater have not been tested domestically. For pathogen harvesting, passive sampling is a potentially cost-effective tool with little or no space limitations. The objectives of this study were to compare the nucleic acid recovery efficiencies of different commercial extraction kits and to develop a better sampling strategy for pathogen detection in a domestic sewer system. Passive sampling materials (cotton gauze, electropositive membrane, and electronegative membrane) were compared for the uptake of enveloped viruses (infectious bronchitis virus, IBV) and non-enveloped virus (MS2 coliphage) as pathogen surrogates. Other fecal indicators, including pepper mild mottle virus (PMMoV), crAssphage, and Bacteroides HF183 were also measured for human population estimation. In addition, grab samples and 24-hour composite samples were also included in the study as a comparison basis. Moreover, the efficiencies of nucleic acid recovery from different commercial kits were compared with a pig-specific Bacteroidales marker, pig-2-bac. According to the result, Promega’s Wizard® Enviro Wastewater TNA kit yielded the highest recovery rate of all commercial kits tested, whereas the extraction efficiency of Qiagen’s RNeasy PowerMicrobiome kit is lower than the other three kits. Among the adsorption materials in passive sampling, the concentrations of PMMoV, crAssphage, and Bacteroides HF183 in cotton gauze were higher than in other materials. The adsorption efficiency of MS2 coliphage was better with the electropositive membrane than with the electronegative membrane. However, for IBV, the electronegative membrane performed better. For fecal indicators, the concentration for PMMoV was the lowest among the three fecal indicators tested. Strong positive Pearson correlations were found between all indicators and indicator populations. The present study confirmed that the fecal indicators could reflect the population size served by the sewage collection system successfully.

並列關鍵字

Wastewater-based epidemiology ； passive sampling ； fecal indicator ； PMMoV ； crAssphage ； Bacteroides HF183

參考文獻

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