Yoo Ju Eun, Lee Cheonghoon, Park SungJun, Ko GwangPyo
Department of Environmental Health Sciences, Graduate School of Public Health, Seoul National University, Seoul 08826, Republic of Korea.
Institute of Health and Environment, Seoul National University, Seoul 08826, Republic of Korea.
J Microbiol Biotechnol. 2017 Apr 28;27(4):816-824. doi: 10.4014/jmb.1612.12026.
Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.
人诺如病毒是引发非细菌性肠胃炎的广泛传播且具有传染性的病毒。实时逆转录定量聚合酶链反应（实时RT-qPCR）是目前灵敏且准确检测这些病原体的金标准，也是疫情预防与控制的关键工具。然而，不同的监测团队可能会使用不同的检测方法，且样本条件的差异可能导致结果出现分歧。此外，诺如病毒基因组高度可变且不断进化。这些问题使得有必要在准确检测的背景下重新审视实时RT-qPCR的稳健性，并研究提高检测性能的实用策略。同时进行了四种广泛引用的实时RT-qPCR检测方法（检测方法A-D），以评估诸如PCR效率、检测限以及与RT-PCR相比的灵敏度和特异性等特征，并评估检测诺如病毒基因组I和II的最准确方法。总体而言，在本研究中检测方法D被评估为最精确和准确的检测方法。在检测方法D的探针中添加了一种ZEN内部淬灭剂，该淬灭剂可降低PCR过程中的非特异性荧光，这进一步提高了检测性能。本研究比较了几种诺如病毒检测方法，基于此类比较的改进策略为诺如病毒检测的高度优化实时RT-qPCR检测方法提供了有用的特性描述。
