Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters
Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters
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DOI：
10.1128/aem.03851-14
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发表时间：
2015-03-01
影响因子：
4.4
通讯作者：
Toze, S.
中科院分区：
文献类型：
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作者：
Ahmed, W.;Harwood, V. J.;Toze, S.
全球一半以上与娱乐用水相关的胃肠炎病例是由人类致病病毒引起的。由于它们的浓度通常较低且尺寸较小，因此相对难以从环境水中浓缩。尽管通过定量 PCR (qPCR) 快速计数病毒有可能大大改善水质分析和风险评估，但从环境水源中捕获和回收病毒以及从提取的核酸中去除 PCR 抑制剂的上游步骤仍然是常规使用的巨大障碍。在这里，我们比较了三种快速方法的病毒回收效率，这些方法将一升自来水和河水样本中的两种微生物源追踪 (MST) 病毒标记物人腺病毒 (HAdV) 和多瘤病毒 (HPyV) 浓缩在 HA 膜（直径 90 毫米）上。样品中加入原污水，通过酸化（方法 A）或添加 MgCl2（方法 B 和 C）促进病毒对膜的吸附。直接从膜中提取病毒核酸（方法A），或用NaOH洗脱病毒并通过离心超滤浓缩（方法B和C）。通过方法 A 处理的样品没有观察到 qPCR 抑制，但通过 B 和 C 处理的河流样品中出现了抑制。HAdV 和 HPyV 的回收效率与方法 A 相似（31 至 78%），比方法 B 和 C（2.4 至 12%）高 10 倍。对方法 B 的膜的进一步分析表明，大多数病毒没有从膜上洗脱，导致回收率较差。对最初发表的方法 A 进行修改，包括更大直径的膜和可容纳该膜的核酸提取试剂盒，从而产生了快速病毒浓缩方法，具有良好的回收率且不含抑制化合物。常用的添加阳离子 (Mg2+) 病毒吸收和酸洗脱病毒的策略效率低下，更容易受到抑制，并且会导致低估环境水中 HAdV 和 HPyV 标记物的流行率和浓度。
Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were similar to 10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg2+) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters.