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

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.