Advanced molecular poliovirus diagnostics across multiple real-time PCR platforms with locked nucleic acids and primer overhangs

ABSTRACT This study details the development and validation of five real-time RT-PCR assays for the intratypic differentiation (ITD) of polioviruses and the discrimination of vaccine-derived polioviruses (VDPVs) utilizing locked nucleic acid (LNA) technology and primer overhangs. Updated assays, including wild-type 1/Qβ duplex (WPV1), VDPVs 1 and 3, wild-type 3, and novel oral poliovirus type 2 (nOPV2), were designed in response to challenges posed by genetic diversity and detection gaps in existing protocols following outbreaks, and the introduction of a new vaccine replacing the Sabin 2 oral poliovirus vaccine (OPV). The CDC’s Polio and Picornavirus Branch led assay development, supporting global surveillance efforts with kits tailored for use across multiple real-time PCR platforms. Comprehensive virus collections representing all three poliovirus serotypes, nonpolio enteroviruses, and vaccine seed strains were used in the validation. Assay performance was assessed through piloting in poliovirus reference laboratories across outbreak regions, including Central Asia and Africa. Combining primers with AT-rich overhangs enhanced fluorescence, improving interpretation of weak signals for VDPV1 and WPV1 assays, and boosting analytical sensitivity for the VDPV3 assay by 10-fold to 100-fold (100%). The new assays enabled reliable detection of extinct WPV3 (100% sensitivity and 96.97% specificity), the circulating genotype WPV1 (100% sensitivity and 98.72% specificity), and discrimination of Sabin 1 and 3 from vaccine-derived strains, and the discrimination of nOPV2 from Sabin 2 (100% specificity) from surveillance samples. These advancements mark a significant milestone in molecular diagnostics for poliovirus eradication, supporting surveillance and response efforts worldwide, with remaining pockets of circulating VDPVs and wild-type 1 viruses. IMPORTANCE Recent poliovirus detections from wastewater in non-endemic regions sparked renewed interest in the agent. Limited access to extinct polioviruses for validation studies hinders diagnostic laboratories from designing and validating real-time RT-PCR assays for specific and sensitive detection in wastewater or stools. There are currently no commercial tests that can detect and type all poliovirus serotypes that have been assessed with recent endemic or seed viruses used by vaccine manufacturers, making the intratypic differentiation unique. This work describes the redesign and validation of the five RT-PCR assays with various poliovirus strains such as vaccine seed strains, outbreak strains, and eradicated viruses. Locked nucleic acids and primer overhangs were applied to the primer and probe design, resulting in advanced laboratory capability to detect contemporary poliovirus strains, strengthening surveillance in support of the global poliovirus eradication activities.