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

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.