Ansari Wahedul Karim, Seo Mi-Ran, Chung Yeun-Jun
Department of Medical Sciences, The Catholic University of Korea, Seoul 06591, Republic of Korea.
Department of Microbiology, The Catholic University of Korea, Seoul 06591, Republic of Korea.
Diagnostics (Basel). 2025 Jul 25;15(15):1868. doi: 10.3390/diagnostics15151868.
Norovirus is a major cause of acute gastroenteritis worldwide. Considering its highly infectious and transmissible nature, rapid and accurate diagnostic tools are of utmost importance for the effective control of outbreaks in the context of point-of-care testing (POCT). In this study, we developed a genogroup-specific multiplex reverse transcriptase loop-mediated isothermal amplification assay to detect the human norovirus genogroups I and II (GI and GII, respectively). For the comprehensive detection of clinically relevant genotypes, two sets of primers were incorporated into the assays targeting the RdRp-VP1 junction: one against GI.1 and GI.3, and the other for GII.2 and GII.4. Following optimization of the reaction variables, we standardized the reaction conditions at 65 °C with 6 mM MgSO, 1.4 mM dNTPs, 7.5 U WarmStart RTx Reverse Transcriptase, and Bst DNA polymerase at 8 U and 10 U for GI and GII, respectively. Amplification was monitored in real-time using a thermocycler platform to ensure precise quantification and detection. Finally, the assay was evaluated through portable isothermal detection device to test its feasibility in on-site settings. Both assays detected the template down to 10-10 copies per reaction and showed high target selectivity, yielding no non-specific amplification across 39 enteric pathogens. These assays enabled prompt detection of GI within 10-12 min and of GII within 12-17 min after the reaction was initiated. Onsite validation reveals all template detection below 15 min, demonstrating its potential feasibility in point-of-care applications. Including the sample preparation time, test results were obtained in less than 1 h. This method is a rapid, reliable, and scalable solution for detecting human norovirus in POCT settings for both clinical diagnosis and public health surveillance.
诺如病毒是全球急性胃肠炎的主要病因。鉴于其高度传染性和传播性，快速准确的诊断工具对于在即时检测（POCT）背景下有效控制疫情至关重要。在本研究中，我们开发了一种基因群特异性多重逆转录环介导等温扩增检测法，用于检测人诺如病毒基因群I和II（分别为GI和GII）。为了全面检测临床相关基因型，在检测中纳入了两组针对RdRp-VP1连接区的引物：一组针对GI.1和GI.3，另一组针对GII.2和GII.4。在优化反应变量后，我们将反应条件标准化为65°C，分别使用6 mM MgSO、1.4 mM dNTPs、7.5 U WarmStart RTx逆转录酶，以及针对GI和GII分别为8 U和10 U的Bst DNA聚合酶。使用热循环仪平台实时监测扩增，以确保精确的定量和检测。最后，通过便携式等温检测设备对该检测法进行评估，以测试其在现场环境中的可行性。两种检测法均能检测到每个反应低至10-10拷贝的模板，且具有高靶标选择性，对39种肠道病原体均未产生非特异性扩增。这些检测法在反应开始后10-12分钟内能够快速检测到GI，12-17分钟内能够检测到GII。现场验证表明所有模板在15分钟内均可检测到，证明了其在即时检测应用中的潜在可行性。包括样品制备时间在内，检测结果在不到1小时内即可获得。该方法是一种快速、可靠且可扩展的解决方案，用于在POCT环境中检测人诺如病毒，适用于临床诊断和公共卫生监测。
