# Development and Validation of a Quantitative RT-qPCR Panel for the Detection and Monitoring of Polioviruses in Wastewater Samples

## TL;DR

This study develops a sensitive RT-qPCR method to detect and monitor polioviruses in wastewater, improving surveillance for both vaccine-derived and wild-type strains.

## Contribution

A novel one-step RT-qPCR panel with improved sensitivity for detecting multiple poliovirus types in wastewater.

## Key findings

- One-step RT-qPCR with TaqMan MM showed improved sensitivity for detecting Sabin and wild-type polioviruses.
- The LOD for Sabin 1 in IPV was 2.49 copies/PCR, and for eDNAf ranged from 1.06 to 3.12 copies/PCR.
- Sabin 1 recovery from spiked wastewater ranged from 10.26% to 57.27%.

## Abstract

Clusters of acute flaccid paralysis (AFP) caused by oral vaccine-derived poliovirus (VDPV) in 2022 and sporadic outbreaks in New York and Gaza highlight the ongoing risk of polio, alongside the persistent global threat posed by wild-type poliovirus. This study aims to develop and validate a quantitative reverse transcription PCR (RT-qPCR) panel that employs different primer–probe sets to simultaneously detect vaccine and wild-type poliovirus (WPV) in wastewater. Using an inactivated poliovirus vaccine (IPV) and engineered DNA fragments (eDNAf), the qPCR master mix (MM) performance, assay specificity, sensitivity (limit of detection, LOD), and recovery from IPV-spiked wastewater were evaluated. Compared with two-step RT-qPCR and qScript MM, one-step RT-qPCR with TaqMan MM improved sensitivity for the following polioviruses (PV): Sabin 1 in IPV and the eDNAf of Sabin 1, 2, and 3; WPV1 and WPV3; and poliovirus type 2 (any serotype 2). The LOD for Sabin 1 in IPV was 2.49 copies/PCR, while LODs for eDNAf of polio targets ranged from 1.06 to 3.12 copies/PCR. Sabin 1 recovery from IPV-spiked wastewater ranged from 10.26% to 57.27%. The RT-qPCR panel for poliovirus exhibited good specificity and sensitivity, with moderate viral recovery, enabling rapid implementation of wastewater monitoring for PV as needed.