Development of a new RT-PCR multiplex assay for poliovirus detection

Abstract

Potent vaccines to protect individuals from poliomyelitis are available and great achievements have been reached by the Global Polio Eradication Program (GPEI). Different objectives (i.e. surveillance, outbreak events) require scalable screening of samples. Although cell culture-based analysis is still needed due to the benefits regarding detailed characterization of the isolated virus, it is also time consuming and intensive lab work is required, limiting the number of samples that can be analyzed. Reliable and sensitive molecular methods would increase the capacity for high-throughput testing. In this study we designed a poliovirus type-specific multiplex RT-PCR assay allowing swift screening of various samples by real-time or digital RT-PCR. The validation data demonstrate a reliable assay in respect of sensitivity and specificity for different clinical specimens, wastewater samples as well as virus isolates. The limit of detection of the multiplex assay ranged from 7 to 50 copies/reaction, depending on the PV serotype and PCR system used. Dilution series of spiked wastewater samples showed a 10–100-fold higher detectability of PV by digital RT-PCR compared to the real-time RT-PCR technique using the designed assay. The designed assay allows rapid PV testing for different matrices to prioritize subsequent analysis. © 2026 Robert Koch Institut.