We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.Four methods are currently in routine use in the WHO Global Polio Laboratory Network (GPLN) (3, 14, 15) for differentiation between vaccine-related and wild poliovirus isolates (intratypic differentiation): (i) the enzyme-linked immunosorbent assay, using highly specific cross-absorbed antisera (13); (ii) nucleic acid hybridization, using Sabin vaccine strainspecific RNA probes (4); (iii) reverse transcription-PCR (RT-PCR), using vaccine strain-specific primers (16); and (iv) RT-PCR followed by restriction fragment length polymorphism analysis (1, 13). To achieve the required specificities for binding to variable target sequences, our RT-PCR primers were designed to contain mixed-base or inosine residues at positions of codon degeneracy (8, 9). In the poliovirus diagnostic RT-PCR kits currently distributed throughout the GPLN, identifications are based upon the mobilities of amplicons in polyacrylamide gels (8,9,15,16). This approach, while achieving the high levels of diagnostic accuracy and reliability required for global poliovirus surveillance, is especially laborious for GPLN laboratories with large workloads.Development of real-time RT-PCR (rRT-PCR) has opened the way for more-rapid and -accurate diagnostic assays (2). We have adapted our previously described poliovirus diagnostic RT-PCR methods (7-9) to the real-time format with an emphasis on high template specificities rather than quantitative determination of template concentrations. These new assays were tested against both Sabin vaccine-related isolates and wild poliovirus isolates representing all currently circulating genotypes.The enterovirus group-specific (panEV) primers used were essentially as described previously (10, 17). They target highly conserved sequences in the 5Ј untranslated region, and the antisense polarity primer (PCR-1; 10 pmol per assay) and TaqMan probe (panEV probe; 5 pmol) each have only one mixed-base residue, while the sense polarity primer (PCR-2; 10 pmol) is nondegenerate.To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan probes. Designing the degenerate TaqMan probes (a 15-pmol probe for each assay) was especially challenging because of the need to use longer sequences to obtain good hybrid stabilities while simultaneously compensating for the high level of degeneracy of sequences between primer binding sites. Although hybrid stabilities can be estimated by physicochemical calculations (12), development of the optimal primer and probe sets was a highly empirical process because variation within the target sequences was not predictable. The degen...

With poliovirus eradication nearing, few pockets of active wild poliovirus (WPV) transmission remain in the world. Intratypic differentiation (ITD) plays a crucial part in laboratory surveillance as the molecular detection method that can identify and distinguish wild and vaccine-like polioviruses isolated from acute flaccid paralysis cases or environmental sources. The need to detect new variants of WPV serotype 1 (WPV1) and the containment of all serotype 2 polioviruses (PV2) in 2015 required changes to the previous version of the method. The ITD version 5.0 is a set of six real-time reverse transcription-PCR (rRT-PCR) assays that serve as accurate diagnostic tools to easily detect and differentiate PV serotypes and genotypes. We describe the creation and properties of quantitation standards, including 16 control RNA transcripts and nine plaque-isolated viruses. All ITD rRT-PCR assays were validated using these standards, and the limits of detection were determined for each assay. We designed and pilot tested two new assays targeting recently circulating WPV1 genotypes and all PV2 viruses. The WPV1 assay had 99.1% specificity and 100% sensitivity, and the PV2 assay had 97.7% specificity and 92% sensitivity. Before proceeding to the next step in the global poliovirus eradication program, we needed to gain a better understanding of the performance of the ITD 5.0 suite of molecular assays and their limits of detection and specificities. The findings and conclusions in this evaluation serve as building blocks for future development work.

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Vaccine-derived polioviruses (VDPVs) are associated with polio outbreaks and prolonged infections in individuals with primary immunodeficiencies. VDPV-specific PCR assays for each of the three Sabin oral poliovirus vaccine (OPV) strains were developed, targeting sequences within the VP1 capsid region that are selected for during replication of OPV in the human intestine. Over 2400 Sabin-related isolates and identified 755 VDPVs were screened. Sensitivity of all assays was 100%, while specificity was 100% for serotypes 1 and 3, and 76% for serotype 2. The assays permit rapid, sensitive identification of OPV-related viruses and flag programmatically important isolates for further characterization by genomic sequencing.