Navigating Uncertainties in RT-qPCR and Infectivity Assessment of Norovirus

IF 4.1 2区 农林科学 Q2 ENVIRONMENTAL SCIENCES
Razieh Sadat Mirmahdi, Samantha L. Dicker, Nuradeen Garba Yusuf, Naim Montazeri
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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

RT-qPCR的不确定性导航及诺如病毒的感染性评估
人类诺如病毒(HuNoV)是全球胃肠炎的主要病因。由于缺乏可靠的培养体系,RT-qPCR是检测和定量HuNoV的金标准技术。然而,PCR无法区分感染性和非感染性颗粒仍然是一个重大限制。本研究旨在通过探索基于培养(斑块测定和TCID50)和非基于培养(RT-qPCR)的HuNoV定量方法之间的关系,以杜兰病毒作为可培养的替代物,来解决这一限制。在Tris-EDTA缓冲液(pH 7.2)中,以6.7 log10 PFU/ml或5.8 log10 TCID50/ml超离心纯化杜兰病毒,依次稀释,进行RNA提取,有或没有RNase预处理,然后用RT-qPCR定量。用动态光散射和透射电子显微镜对病毒进行了进一步的物理表征。在rnase预处理和未预处理的样品中,每次PCR反应的log10个基因组拷贝数(GC)和log10个斑块形成单位(PFU)之间存在很强的相关性(Pearson相关系数为0.99)。Beta分布表明,rnase预处理和未预处理样品的中位数GC:PFU比相似,约为3.7 log10。高GC:PFU比率可能表明RT-qPCR的敏感性或存在完整的非传染性病毒颗粒。本研究结果将有助于更准确地估计食物和环境基质中感染性诺如病毒颗粒。在BioRender中创建的图形抽象。Mirmahdi, R. (2024) https://BioRender.com/l49a583
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来源期刊
Food and Environmental Virology
Food and Environmental Virology ENVIRONMENTAL SCIENCES-MICROBIOLOGY
CiteScore
6.50
自引率
2.90%
发文量
35
审稿时长
1 months
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.
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