Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan St., Albany, CA, 94710-1105, USA.
Department of Bioengineering, Shanghai Institute of Technology, Shanghai, 100 Haiquan Road, Fengxian District, Shanghai, 201418, People's Republic of China.
Food Environ Virol. 2018 Mar;10(1):29-38. doi: 10.1007/s12560-017-9317-1. Epub 2017 Aug 30.
Human noroviruses (HuNoVs) are highly infectious viruses for which water is an important medium of transmission. In this study, we explored a new in situ capture RT-qPCR (ISC-RT-qPCR) methodology to estimate the infectivity of HuNoV in environmental water samples. This assay was based on capturing encapsidated HuNoV by viral receptors, followed by in situ amplification of the captured viral genomes by RT-qPCR. We demonstrated that the ISC-RT-qPCR did not capture and enable signal amplification of heat-denatured Tulane Virus (TV) and HuNoVs. We further demonstrated that the sensitivity of ISC-RT-qPCR was equal or better than that of conventional RT-qPCR procedures for the detection of HuNoV GI and GII. We then utilized the ISC-RT-qPCR to detect HuNoV in environmental water samples for comparison against that from a conventional RT-qPCR procedure. TV was used as a process control virus. While complete inhibition of TV genomic signal was observed in 27% of samples tested by RT-qPCR, no inhibition of TV genomic signal was observed by ISC-RT-qPCR. From 72 samples tested positive for HuNoV GI signal by RT-qPCR, only 20 (27.8%) of these samples tested positive by ISC-RT-qPCR, suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. From 16 samples tested positive for HuNoV GII signal by RT-qPCR, only one of these samples tested positive by ISC-RT-qPCR. Five samples that had initially tested negative for HuNoV GII signal by RT-qPCR, was tested as positive by ISC-RT-qPCR. Overall, ISC-RT-qPCR method provided an alternative assay to estimate infectivity of HuNoV in environmental samples.
人诺如病毒（HuNoVs）是高度传染性的病毒，水是其重要的传播介质。在本研究中，我们探索了一种新的原位捕获 RT-qPCR（ISC-RT-qPCR）方法，用于估计环境水样中 HuNoV 的感染力。该方法基于病毒受体对包裹的 HuNoV 的捕获，然后通过 RT-qPCR 对捕获的病毒基因组进行原位扩增。我们证明，ISC-RT-qPCR 不能捕获和使热变性的 Tulane 病毒（TV）和 HuNoVs 信号放大。我们进一步证明，ISC-RT-qPCR 的检测灵敏度与传统 RT-qPCR 程序相当或更好，用于检测 HuNoV GI 和 GII。然后，我们利用 ISC-RT-qPCR 检测环境水样中的 HuNoV，与传统 RT-qPCR 程序进行比较。TV 被用作过程控制病毒。虽然在通过 RT-qPCR 测试的 27%的样本中观察到 TV 基因组信号完全抑制，但在 ISC-RT-qPCR 中未观察到 TV 基因组信号抑制。从通过 RT-qPCR 检测到 GI 信号阳性的 72 个 HuNoV 样本中，只有 20 个（27.8%）通过 ISC-RT-qPCR 检测到阳性，这表明 72.2%的 RT-qPCR 阳性样本不太可能具有感染力。从通过 RT-qPCR 检测到 GII 信号阳性的 16 个 HuNoV 样本中，只有一个通过 ISC-RT-qPCR 检测到阳性。最初通过 RT-qPCR 检测到 GII 信号阴性的 5 个样本，通过 ISC-RT-qPCR 检测为阳性。总体而言，ISC-RT-qPCR 方法提供了一种替代方法，用于估计环境样本中 HuNoV 的感染力。
