Vinjé Jan, Hamidjaja Raditijo A, Sobsey Mark D
Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7431, USA.
J Virol Methods. 2004 Mar 15;116(2):109-17. doi: 10.1016/j.jviromet.2003.11.001.
Noroviruses (NoV), previously called "Norwalk-like viruses", have emerged as the single most important cause of acute gastroenteritis worldwide. Most diagnostic reverse transcription-polymerase chain reaction (RT-PCR) assays target the viral RNA-dependent RNA polymerase; however, the major capsid protein (VP1) is the reference genomic region for establishing genotypes. In this study, we analyzed complete NoV VP1 sequences (n=100) and determined a region (region D) that was most suitable to differentiate between genotypes. Within region D, we designed two genogroup specific, broadly reactive, degenerate primer sets (GI and GII). The region D primers were evaluated in a single-tube one-step RT-PCR assay using a panel of 81 (31 GI, 50 GII) NoV strains from both outbreaks and sporadic cases. In total, 95% of the samples tested positive using the new region D primer sets. Phylogenetic analysis of region D sequences (36 deduced amino acids for GI, 56 deduced amino acids for GII), revealed 19 clusters (7 within GI and 12 within GII) including three new genetically distinct clusters, two of which were unresolved using region A sequences. Phylogenetic analysis of the complete VP1 sequences revealed identical grouping of strains and confirmed the newly identified clusters using region D. In summary, we successfully developed and evaluated a broadly reactive RT-PCR assay for reliable genotyping of GI and GII noroviruses.
诺如病毒（NoV），以前被称为“诺沃克样病毒”，已成为全球急性胃肠炎的单一最重要病因。大多数诊断性逆转录聚合酶链反应（RT-PCR）检测针对病毒的RNA依赖性RNA聚合酶；然而，主要衣壳蛋白（VP1）是用于确定基因型的参考基因组区域。在本研究中，我们分析了完整的NoV VP1序列（n = 100），并确定了一个最适合区分基因型的区域（区域D）。在区域D内，我们设计了两组基因组特异性、广泛反应性的简并引物（GI和GII）。使用来自暴发和散发病例的81株（31株GI，50株GII）NoV毒株，在单管一步RT-PCR检测中对区域D引物进行了评估。总体而言，使用新的区域D引物组，95%的样本检测呈阳性。对区域D序列（GI为36个推导氨基酸，GII为56个推导氨基酸）的系统发育分析揭示了19个簇（GI内7个，GII内12个），包括三个新的遗传上不同的簇，其中两个使用区域A序列无法解析。对完整VP1序列的系统发育分析揭示了毒株的相同分组，并使用区域D证实了新鉴定的簇。总之，我们成功开发并评估了一种广泛反应性的RT-PCR检测方法，用于可靠地对GI和GII诺如病毒进行基因分型。
