Navigating Uncertainties in RT-qPCR and Infectivity Assessment of Norovirus

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