High throughput AS LNA qPCR method for the detection of a specific mutation in poliovirus vaccine strains.

Sabin Inactivated Poliovirus Vaccine (sIPV) has become one of the preferred vaccination options for the last step in the Poliovirus eradication program. Sequencing of poliovirus samples is needed during the manufacturing of poliovirus vaccines to assure the safety and immunogenicity of these vaccines. Next-generation sequencing analysis is the current costly and time-consuming gold standard for monitoring the manufacturing processes. We developed a low-cost and quick, highly sensitive, and allele-specific locked nucleic acid-probe-based reverse transcription quantitative PCR alternative that can accurately detect mutations in poliovirus vaccine samples during process development, scaling up, and release. Using the frequently in vitro occurring and viral replication-impacting VP1-E 295 K mutation as a showcase, we show that this technology can accurately detect E 295 K mutations in poliovirus 2 samples to similar levels as NGS. The qPCR technology was developed employing a synthetic dsDNA fragment-based standard curve containing mixes of E 295 K-WT (wildtype) and Mut (mutant) synthetic dsDNA fragments ranging from 1 × 10 7 copies/µL to 1 × 10 2 copies/µL to achieve a linear correlation with R 2  > 0.999, and PCR efficiencies of 95-105 %. Individual standard concentration levels achieved accuracies of ≥92 % (average 96 %) and precisions of ≤17 % (average 3.3 %) RSD. Specificity of locked nucleic acid (LNA)-probes was confirmed in the presence and absence of co-mutations in the probe-binding region. Application of the developed assay to Sabin Poliovirus type 2 production run samples, illustrated a linear relationship with an R 2 of 0.994, and an average accuracy of 97.2 % of the variant (allele)-specific AS LNA qPCR result, compared to NGS. The assay showed good sensitivity for poliovirus samples, containing E 295 K mutation levels between 0 % and 95 % (quantification range). In conclusion, the developed AS LNA qPCR presents a valuable low-cost, and fast tool, suitable for the process development and quality control of polio vaccines.