Department of Environmental and Civil Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Department of Environmental and Civil Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Sci Total Environ. 2023 Dec 15;904:166338. doi: 10.1016/j.scitotenv.2023.166338. Epub 2023 Aug 15.
Polymerase chain reaction (PCR) is widely applied for the monitoring of pathogenic viruses in water environments. To date, several pretreatments to selectively detect genes from infectious viruses via PCR have been developed. This study was aimed to characterize and validate methods for quantifying active viruses and indicators and to evaluate the proportion of their active fractions in surface water (n = 42). Active E. coli and F-specific RNA phage (FRNAPH) genogroups were quantified using culture assays. In addition to these microbes, norovirus genogroups I (GI) and II, Aichi virus 1, and pepper mild mottle virus (PMMoV) were quantified by (reverse transcription)-quantitative PCR (RT-qPCR) with and without cis-dichlorodiammineplatinum (CDDP) treatment to exclude genes in inactive viruses. CDDP-RT-qPCR showed concentrations and detection frequencies comparable to or higher than culture assays. Consequently, although CDDP-RT-qPCR can suggest the presence of an inactive virus, it can also overestimate the activity of the virus in the environment. Differences between culture and CDDP-RT-qPCR and between CDDP-RT-qPCR and RT-qPCR varied among the viruses. CDDP-RT-qPCR showed a concentration comparable to the culture assay (within 1 log difference) in 93 % of positive samples for GI-FRNAPH but in <63 % of positive samples for GII- and GIII-FRNAPHs. GII-NoV was detected from 5 and 30 out of 42 samples via CDDP-RT-qPCR and RT-qPCR, respectively, and was suggested as inactivated by 2.0 log or higher in most of the samples. By contrast, concentrations of PMMoV determined by these two assays were not notably different. It is suggested that the operational conditions of wastewater treatment plants around the sites, rather than environmental stresses, affected the microbial inactivation. To better understand the infectivity of viruses in the environment, it is important to investigate them using sensitive detection methods at various sites, including the source of contamination.
聚合酶链反应（PCR）广泛应用于水环境中病原病毒的监测。迄今为止，已经开发了几种预处理方法，通过 PCR 选择性检测感染性病毒的基因。本研究旨在对定量活性病毒和指示物的方法进行特征描述和验证，并评估其在地表水中的活性部分的比例（n=42）。使用培养法定量活性大肠杆菌和 F 特异性 RNA 噬菌体（FRNAPH）基因群。除了这些微生物外，还通过（逆转录）定量 PCR（RT-qPCR）并结合顺式二氯二氨铂（CDDP）处理来排除非活性病毒中的基因，定量检测诺如病毒基因 I（GI）和 II、Aichi 病毒 1 和辣椒轻斑驳病毒（PMMoV）。CDDP-RT-qPCR 的浓度和检测频率与培养法相当或更高。因此，尽管 CDDP-RT-qPCR 可以提示存在非活性病毒，但它也可能高估环境中病毒的活性。病毒之间的培养法与 CDDP-RT-qPCR 以及 CDDP-RT-qPCR 与 RT-qPCR 之间的差异各不相同。在 GI-FRNAPH 的阳性样本中，有 93%的样本 CDDP-RT-qPCR 与培养法的浓度相当（相差 1 个对数），而在 GII-和 GIII-FRNAPHs 的阳性样本中，这一比例<63%。通过 CDDP-RT-qPCR 和 RT-qPCR 分别从 42 个样本中的 5 个和 30 个样本中检测到 GII-NoV，并且在大多数样本中，GII-NoV 被认为被 2.0 个对数或更高的水平灭活。相比之下，这两种检测方法确定的 PMMoV 浓度没有明显差异。建议污水处理厂的运行条件，而不是环境压力，影响了微生物的失活。为了更好地了解环境中病毒的感染力，重要的是要使用敏感的检测方法在包括污染来源在内的各个地点进行调查。
