{"title":"Navigating Uncertainties in RT-qPCR and Infectivity Assessment of Norovirus","authors":"Razieh Sadat Mirmahdi, Samantha L. Dicker, Nuradeen Garba Yusuf, Naim Montazeri","doi":"10.1007/s12560-024-09632-0","DOIUrl":null,"url":null,"abstract":"<div><p>Human norovirus (HuNoV) is the primary cause of gastroenteritis globally. Due to the lack of a reliable cultivation system, RT-qPCR is a gold standard technique for the detection and quantification of HuNoV. However, the inability of PCR to differentiate between infectious from non-infectious particles remains a significant limitation. This study aims to address this limitation by exploring the relationship between culture-based (plaque assay and TCID<sub>50</sub>) and non-culture-based (RT-qPCR) methods for HuNoV quantification, using Tulane virus as a cultivable surrogate. The ultracentrifuge-purified Tulane virus at 6.7 log<sub>10</sub> PFU/ml or 5.8 log<sub>10</sub> TCID<sub>50</sub>/ml in Tris–EDTA buffer (pH 7.2), was serially diluted and subjected to RNA extraction, with or without RNase pretreatment, followed by quantification with RT-qPCR. Further physical characterization of the virus stock was performed with dynamic light scattering and transmission electron microscopy. A strong correlation (Pearson’s Correlation Coefficient of 0.99) was observed between log<sub>10</sub> genome copies (GC) and log<sub>10</sub> plaque forming units (PFU) per PCR reaction for both RNase-pretreated and unpretreated samples. Beta distributions indicated a similar median GC:PFU ratio of ca. 3.7 log<sub>10</sub> for both RNase-pretreated and unpretreated samples. The high GC:PFU ratio may indicate the sensitive nature of RT-qPCR or the presence of intact, non-infectious virus particles. The outcomes of this study will contribute to the more accurate estimation of infectious norovirus particles in food and environmental matrices.</p><h3>Graphical Abstract</h3>\n<div><figure><div><div><picture><source><img></source></picture></div><div><p>Created in BioRender. Mirmahdi, R. (2024) https://BioRender.com/l49a583</p></div></div></figure></div></div>","PeriodicalId":563,"journal":{"name":"Food and Environmental Virology","volume":"17 1","pages":""},"PeriodicalIF":4.1000,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s12560-024-09632-0.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Food and Environmental Virology","FirstCategoryId":"97","ListUrlMain":"https://link.springer.com/article/10.1007/s12560-024-09632-0","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENVIRONMENTAL SCIENCES","Score":null,"Total":0}
引用次数: 0
Abstract
Human norovirus (HuNoV) is the primary cause of gastroenteritis globally. Due to the lack of a reliable cultivation system, RT-qPCR is a gold standard technique for the detection and quantification of HuNoV. However, the inability of PCR to differentiate between infectious from non-infectious particles remains a significant limitation. This study aims to address this limitation by exploring the relationship between culture-based (plaque assay and TCID50) and non-culture-based (RT-qPCR) methods for HuNoV quantification, using Tulane virus as a cultivable surrogate. The ultracentrifuge-purified Tulane virus at 6.7 log10 PFU/ml or 5.8 log10 TCID50/ml in Tris–EDTA buffer (pH 7.2), was serially diluted and subjected to RNA extraction, with or without RNase pretreatment, followed by quantification with RT-qPCR. Further physical characterization of the virus stock was performed with dynamic light scattering and transmission electron microscopy. A strong correlation (Pearson’s Correlation Coefficient of 0.99) was observed between log10 genome copies (GC) and log10 plaque forming units (PFU) per PCR reaction for both RNase-pretreated and unpretreated samples. Beta distributions indicated a similar median GC:PFU ratio of ca. 3.7 log10 for both RNase-pretreated and unpretreated samples. The high GC:PFU ratio may indicate the sensitive nature of RT-qPCR or the presence of intact, non-infectious virus particles. The outcomes of this study will contribute to the more accurate estimation of infectious norovirus particles in food and environmental matrices.
Graphical Abstract
Created in BioRender. Mirmahdi, R. (2024) https://BioRender.com/l49a583
期刊介绍:
Food and Environmental Virology publishes original articles, notes and review articles on any aspect relating to the transmission of pathogenic viruses via the environment (water, air, soil etc.) and foods. This includes epidemiological studies, identification of novel or emerging pathogens, methods of analysis or characterisation, studies on survival and elimination, and development of procedural controls for industrial processes, e.g. HACCP plans. The journal will cover all aspects of this important area, and encompass studies on any human, animal, and plant pathogenic virus which is capable of transmission via the environment or food.