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

Abstract

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.

One-Step Reverse-Transcription Recombinase-Aided Amplification CRISPR/Cas12a-Based Lateral Flow Assay for Fast Field Screening and Accurate Differentiation of Four Major Tobamoviruses Infecting Tomato and Pepper

“…The positive-sense RNA genome of tobamoviruses (6.3–6.6 kb in size) encodes four proteins: a 124–132 kDa protein (ORF1), RNA-dependent RNA polymerase (181–189 kDa protein, ORF2), MP (28–31 kDa protein, ORF3), and a coat protein (17–18 kDa, ORF4) . The coat protein gene is the most frequently used target for PMMoV, ToBRFV, ToMV, and ToMMV detection. − The ORF1 genes have also been used to detect PMMoV, ToBRFV, and ToMV . The coat protein was our first choice; however, the CP genes from virus isolates are conserved, and the sequences from the different viruses vary greatly.…”

“…The previously reported primers and TaqMan probes (Table S1) of the TaqMan real-time RT-PCR method − were employed as a control. The TaqMan real-time RT-PCR amplification was performed in a 20.0-μL reaction volume containing 10 μL of 2× PerfectStart Probe One-Step qPCR SuperMix (Cat.…”