Abstract Human noroviruses (HuNoVs) are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide. As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus, detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The widely used primers and probes for RT-qPCR were established in the early 2000s. As HuNoVs are highly variant viruses, viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time. In this study, a new duplex RT-qPCR (ND-RT-qPCR) was designed for the detection of genogroup I (GI) and genogroup II (GII) HuNoVs based on an analysis of viral sequences added in the database after 2010. Using long transcribed viral RNAs, the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GI and GII HuNoVs. The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer/probe sets for RT-qPCR (Kageyama RT-qPCR) for 23 HuNoV-positive clinical samples. All five GI samples were registered as positive by ND-RT-qPCR, whereas only two samples were registered as positive by Kageyama RT-qPCR. All 18 GII samples were registered as positive by ND-RT-qPCR, while 17 samples were registered as positive by Kageyama RT-qPCR. The sensitivity reflected by the quantification cycle (Cq) value was lower in ND-RT-qPCR than in Kageyama RT-qPCR. Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.

中文翻译：

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重新设计的双链 RT-qPCR 用于检测 GI 和 GII 人类诺如病毒

摘要 人类诺如病毒 (HuNoVs) 是主要的食源性病原体，可在全球范围内引起非细菌性急性胃肠炎。由于HuNoVs的组织培养系统还不够成熟，无法进行病毒的常规检测，检测主要依赖分子方法，如逆转录聚合酶链反应（RT-PCR）和逆转录定量实时聚合酶链反应（RT）。 -qPCR）。RT-qPCR 广泛使用的引物和探针是在 2000 年代初建立的。由于 HuNoV 是高度变异的病毒，病毒基因组突变导致先前设计的引物和/或探针完美匹配，随着时间的推移工作效率降低。在这项研究中，基于对 2010 年后数据库中添加的病毒序列的分析，设计了一种新的双链 RT-qPCR (ND-RT-qPCR) 用于检测基因组 I (GI) 和基因组 II (GII) HuNoV。 使用长转录病毒 RNA结果表明，对于 GI 和 GII HuNoV，ND-RT-qPCR 的灵敏度低至一个基因组拷贝。通过与 23 个 HuNoV 阳性临床样本的 RT-qPCR 常用 Kageyama 引物/探针组 (Kageyama RT-qPCR) 进行比较，进一步评估了 ND-RT-qPCR 的性能。所有五个 GI 样本都被 ND-RT-qPCR 登记为阳性，而只有两个样本被 Kageyama RT-qPCR 登记为阳性。所有 18 个 GII 样本都被 ND-RT-qPCR 登记为阳性，而 17 个样本被 Kageyama RT-qPCR 登记为阳性。ND-RT-qPCR 中定量循环 (Cq) 值反映的灵敏度低于 Kageyama RT-qPCR。我们的数据表明 ND-RT-qPCR 可能非常适合检测 HuNoVs 的当前菌株。