Navigating uncertainties in RT-qPCR and plaque assay for infectivity assessment of norovirus 

Navigating uncertainties in RT-qPCR and plaque assay for infectivity assessment of norovirus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Navigating uncertainties in RT-qPCR and plaque assay for infectivity assessment of norovirus Razieh Sadat Mirmahdi, Samantha L. Dicker, Yusuf Nuradeen Garba, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5477864/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Mar, 2025 Read the published version in Food and Environmental Virology → Version 1 posted 12 You are reading this latest preprint version Abstract Human norovirus (hNoV) is the primary cause of gastroenteritis globally. Due to the lack of a reliable cultivation system, RT-qPCR is a gold standard technique for detection and quantification of hNoV. 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 50 ) and non-culture based (RT-qPCR) methods for hNoV quantification, using Tulane virus as a surrogate. The Ultracentrifuge-purified Tulane virus at 6.7 log 10 PFU/ml or 5.8 log 10 TCID 50 /ml in Tris-EDTA buffer (pH 7.2), was serially diluted and subjected to RNA extraction, with or without RNase pre-treatment, followed by quantification using RT-qPCR. Further physical characterization of the 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 10 genome copies (GC) and log 10 plaque forming units (PFU) per PCR reaction for both RNase-treated and untreated samples. Beta distribution analysis indicated a similar median GC:PFU ratio of ca. 3.7 log 10 for both RNase-treated and untreated samples. The high GC:PFU ratio may indicate the sensitive nature of RT-qPCR. The outcomes of this study will contribute to the more accurate estimation of infectious norovirus particles in food and environmental matrices. Food safety Infectivity assessment Norovirus Public health Surrogate Tulane virus Full Text Additional Declarations No competing interests reported. Supplementary Files DataandRcodes.zip DataandRcodes.zip Cite Share Download PDF Status: Published Journal Publication published 08 Mar, 2025 Read the published version in Food and Environmental Virology → Version 1 posted Editorial decision: Revision requested 20 Dec, 2024 Reviews received at journal 18 Dec, 2024 Reviews received at journal 16 Dec, 2024 Reviews received at journal 11 Dec, 2024 Reviewers agreed at journal 25 Nov, 2024 Reviewers agreed at journal 24 Nov, 2024 Reviewers agreed at journal 24 Nov, 2024 Reviewers agreed at journal 24 Nov, 2024 Reviewers invited by journal 24 Nov, 2024 Editor assigned by journal 19 Nov, 2024 Submission checks completed at journal 19 Nov, 2024 First submitted to journal 18 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5477864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392856859,"identity":"30bcd805-fdae-47f9-a8ef-fd396b3400a1","order_by":0,"name":"Razieh Sadat Mirmahdi","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Razieh","middleName":"Sadat","lastName":"Mirmahdi","suffix":""},{"id":392856860,"identity":"d273cf6b-124e-4a1f-8a1f-f803251a4960","order_by":1,"name":"Samantha L. 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Due to the lack of a reliable cultivation system, RT-qPCR is a gold standard technique for detection and quantification of hNoV. 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\u003csub\u003e50\u003c/sub\u003e) and non-culture based (RT-qPCR) methods for hNoV quantification, using Tulane virus as a surrogate. The Ultracentrifuge-purified Tulane virus at 6.7 log\u003csub\u003e10\u003c/sub\u003e PFU/ml or 5.8 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/ml in Tris-EDTA buffer (pH 7.2), was serially diluted and subjected to RNA extraction, with or without RNase pre-treatment, followed by quantification using RT-qPCR. Further physical characterization of the stock was performed with dynamic light scattering and transmission electron microscopy. 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