TY - JOUR

T1 - Development and validation of a duplex RT-qPCR assay for norovirus quantification in wastewater samples

AU - Alex-Sanders, Natasha

AU - Woodhall, Nick

AU - Farkas, Kata

AU - Scott, George

AU - Jones, Davey L

AU - Walker, David I

N1 - Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

PY - 2023/11/1

Y1 - 2023/11/1

N2 - Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.

AB - Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.

KW - Humans

KW - Norovirus/genetics

KW - Wastewater

KW - Biological Assay

KW - Disease Outbreaks

KW - RNA

U2 - 10.1016/j.jviromet.2023.114804

DO - 10.1016/j.jviromet.2023.114804

M3 - Article

C2 - 37643662

VL - 321

JO - Journal of Virological Methods

JF - Journal of Virological Methods

SN - 0166-0934

M1 - 114804

ER -