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-E295K mutation as a showcase, we show that this technology can accurately detect E295K 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 E295K-WT (wildtype) and Mut (mutant) synthetic dsDNA fragments ranging from 1 × 107 copies/µL to 1 × 102 copies/µL to achieve a linear correlation with R2 > 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 R2 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 E295K 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.

Phytohormone Production by the Endophyte Bacillus safensis TS3 Increases Plant Yield and Alleviates Salt Stress.

Endophytic bacteria can be used to overcome the effect of salinity stress and promote plant growth and nutrient uptake. Bacillus safensis colonizes a wide range of habitats due to survival in extreme environments and unique physiological characteristics, such as a high tolerance for salt, heavy metals, and ultraviolet and gamma radiations. The aim of our study was to examine the salt resistance of the endophytic strain TS3 B. safensis and its ability to produce phytohormones and verify its effect on plant yield in field trials and the alleviation of salt stress in pot experiments. We demonstrate that the strain TS3 is capable of producing enzymes and phytohormones such as IAA, ABA and tZ. In pot experiments with radish and oat plants in salinization, the strain TS3 contributed to the partial removal of the negative effect of salinization. The compensatory effect of the strain TS3 on radish plants during salinization was 46.7%, and for oats, it was 108%. We suppose that such a pronounced effect on the plants grown and the salt stress is connected with its ability to produce phytohormones. Genome analysis of the strain TS3 showed the presence of the necessary genes for the synthesis of compounds responsible for the alleviation of the salt stress. Strain B. safensis TS3 can be considered a promising candidate for developing biofertilizer to alleviate salt stress and increase plant yield.