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Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation

Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near sour...

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Autores principales: Cantillon, Daire, Roberts, Adam P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9762892/
https://www.ncbi.nlm.nih.gov/pubmed/36534927
http://dx.doi.org/10.1371/journal.pone.0278784
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author Cantillon, Daire
Roberts, Adam P.
author_facet Cantillon, Daire
Roberts, Adam P.
author_sort Cantillon, Daire
collection PubMed
description Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near source tracking (NST) allows for more targeted WBE by analysing samples from individual buildings such as schools or even individual floors such as in multi-floor office buildings. Despite the public health advantages of WBE, few strategies exist for optimising NST sampling methodologies. Therefore, we developed a protocol to evaluate virus detection in NST sampling using Pepper Mild Mottle Virus (PMMoV) as a proxy for RNA viruses. PMMoV is the most abundant enteric human associated RNA virus and is present in peppers/pepper-containing foods. Two bespoke TaqMan RT-PCR assays were developed to detect a PMMoV genomic 5’ region and a capsid associated gene. To evaluate the protocol against field samples, pepper homogenates were flushed down an in-use toilet (Liverpool School of Tropical Medicine, UK) to spike wastewater with PMMoV on multiple days, and samples collected from two sewage access points to validate NST samplers. These wastewater samples were assessed for PMMoV based on Ct values and results compared to pepper and Tabasco derived PMMoV positive controls. Positive detection of PMMoV was comparable and consistent in ten independent samples across two NST samplers regardless of pepper homogenate spiking. We have developed two novel one step TaqMan assays that amplify both PMMoV targets in viral RNA extractions from peppers, Tabasco, and wastewater samples with cDNA synthesis through to RT-PCR results taking approximately 30 minutes. Pepper homogenate flushing was not required to detect PMMoV in our wastewater samples, however this strategy of flushing PMMoV containing materials outlined here could be valuable in assessing and validating NST in buildings with no previous or current sewage flow.
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spelling pubmed-97628922022-12-20 Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation Cantillon, Daire Roberts, Adam P. PLoS One Research Article Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near source tracking (NST) allows for more targeted WBE by analysing samples from individual buildings such as schools or even individual floors such as in multi-floor office buildings. Despite the public health advantages of WBE, few strategies exist for optimising NST sampling methodologies. Therefore, we developed a protocol to evaluate virus detection in NST sampling using Pepper Mild Mottle Virus (PMMoV) as a proxy for RNA viruses. PMMoV is the most abundant enteric human associated RNA virus and is present in peppers/pepper-containing foods. Two bespoke TaqMan RT-PCR assays were developed to detect a PMMoV genomic 5’ region and a capsid associated gene. To evaluate the protocol against field samples, pepper homogenates were flushed down an in-use toilet (Liverpool School of Tropical Medicine, UK) to spike wastewater with PMMoV on multiple days, and samples collected from two sewage access points to validate NST samplers. These wastewater samples were assessed for PMMoV based on Ct values and results compared to pepper and Tabasco derived PMMoV positive controls. Positive detection of PMMoV was comparable and consistent in ten independent samples across two NST samplers regardless of pepper homogenate spiking. We have developed two novel one step TaqMan assays that amplify both PMMoV targets in viral RNA extractions from peppers, Tabasco, and wastewater samples with cDNA synthesis through to RT-PCR results taking approximately 30 minutes. Pepper homogenate flushing was not required to detect PMMoV in our wastewater samples, however this strategy of flushing PMMoV containing materials outlined here could be valuable in assessing and validating NST in buildings with no previous or current sewage flow. Public Library of Science 2022-12-19 /pmc/articles/PMC9762892/ /pubmed/36534927 http://dx.doi.org/10.1371/journal.pone.0278784 Text en © 2022 Cantillon, Roberts https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Cantillon, Daire
Roberts, Adam P.
Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
title Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
title_full Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
title_fullStr Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
title_full_unstemmed Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
title_short Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
title_sort development and evaluation of taqman-based, one-step, real-time rt-pcr assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9762892/
https://www.ncbi.nlm.nih.gov/pubmed/36534927
http://dx.doi.org/10.1371/journal.pone.0278784
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