Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan.
Viruses. 2021 Nov 22;13(11):2332. doi: 10.3390/v13112332.
Norovirus-associated diseases are the most common foodborne illnesses worldwide. Polymerase chain reaction-based methods are the primary diagnostics for clinical samples; however, the high mutation rate of norovirus makes viral amplification and genotyping challenging. Technological advances in mass spectrometry (MS) make it a promising tool for identifying disease markers. Besides, the superior sensitivity of MS and proteomic approaches may enable the detection of all variants. Thus, this study aimed to establish an MS-based system for identifying and typing norovirus. We constructed three plasmids containing the major capsid protein VP1 of the norovirus GII.4 2006b, 2006a, and 2009a strains to produce virus-like particles for use as standards. Digested peptide signals were collected using a nano-flow ultra-performance liquid chromatography mass spectrometry (nano-UPLC/MS) system, and analyzed by ProteinLynx Global SERVER and TREE-PUZZLE software. Results revealed that the LC/MS system had an excellent coverage rate: the system detected more than 94% of amino acids of 3.61 femtomole norovirus VP1 structural protein. In the likelihood-mapping analysis, the proportions of unresolved quartets were 2.9% and 4.9% in the VP1 and S domains, respectively, which is superior to the 15.1% unresolved quartets in current PCR-based methodology. In summary, the use of LC/MS may efficiently monitor genotypes, and sensitively detect structural and functional mutations of noroviruses.
诺如病毒相关疾病是全球最常见的食源性疾病。聚合酶链反应（PCR）为基础的方法是临床样本的主要诊断方法；然而，诺如病毒的高突变率使得病毒扩增和基因分型具有挑战性。质谱（MS）技术的进步使其成为鉴定疾病标志物的有前途的工具。此外，MS 和蛋白质组学方法的卓越灵敏度可能能够检测到所有变体。因此，本研究旨在建立一种基于 MS 的诺如病毒鉴定和分型系统。我们构建了三个包含诺如病毒 GII.4 2006b、2006a 和 2009a 株主要衣壳蛋白 VP1 的质粒，以产生病毒样颗粒作为标准。使用纳流超高效液相色谱-质谱（nano-UPLC/MS）系统收集消化后的肽信号，并使用 ProteinLynx Global SERVER 和 TREE-PUZZLE 软件进行分析。结果表明，LC/MS 系统具有出色的覆盖率：系统检测到超过 3.61 飞摩尔诺如病毒 VP1 结构蛋白的 94%以上的氨基酸。在似然映射分析中，VP1 和 S 结构域中未解决四分体的比例分别为 2.9%和 4.9%，优于当前基于 PCR 的方法的 15.1%未解决四分体。总之，LC/MS 的使用可以有效地监测基因型，并灵敏地检测诺如病毒的结构和功能突变。
