Water bodies contaminated with the norovirus (NoV) are important vectors for its transmission. Therefore, enhanced monitoring of NoV in aqueous environments plays an active role in preventing diseases. Here, we reverse transcribed viral RNA into cDNA, and then used the constructed RPA-CRISPR/Cas13a-based platform for sensitive and quantitative monitoring of NoV GII.4 in aqueous environments. The use of glycerol as a phase separator and the direct release of nucleic acids from the virus by NaOH significantly enhanced the stability of the assay and reduced its economic cost. This assay is sensitive, specific, and stable. Based on the qualitative detection method, we established a relatively accurate quantitative detection method using the plasmid as a standard. Four water samples, totaling 64 samples, were analyzed using this method and compared with the qPCR method. The results of the two methods showed 100 % concordance with no significant difference in viral load. The entire process of our established method—from viral nucleic acid extraction to the output of the results—was completed in 30 min, much less than the time required for qPCR method. This suggests that the assay can be used as an alternative to qPCR for monitoring the change of NoV GII.4 concentration in water bodies, and shows high potential for application in the immediate detection of viruses in aqueous environments and resource-limited areas.

中文翻译：

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CRISPR/Cas13a 结合逆转录和 RPA 用于水环境中 NoV GII.4 监测


受诺如病毒 （NoV） 污染的水体是其传播的重要媒介。因此，加强对水环境中 NoV 的监测对预防疾病起着积极作用。在这里，我们将病毒 RNA 逆转录成 cDNA，然后使用构建的基于 RPA-CRISPR/Cas13a 的平台在水性环境中对 NoV GII.4 进行灵敏和定量的监测。使用甘油作为相分离器以及 NaOH 从病毒中直接释放核酸，显著提高了测定的稳定性并降低了其经济成本。该检测具有敏感性、特异性和稳定性。在定性检测方法的基础上，我们建立了以质粒为标准品的相对准确的定量检测方法。使用该方法分析 4 个水样，共 64 个样品，并与 qPCR 方法进行比较。两种方法的结果显示 100% 一致，病毒载量无显著差异。我们建立的方法的整个过程——从病毒核酸提取到结果输出——在 30 分钟内完成，远少于 qPCR 方法所需的时间。这表明该测定法可以作为 qPCR 的替代方法，用于监测水体中 NoV GII.4 浓度的变化，并显示出在水环境和资源有限地区即时检测病毒方面的巨大应用潜力。