Qualitative Single Base Extension assay for Identification of Single Nucleotide Polymorphism in Mumps Virus Genome

Abstract

Objective: Objective of the study was to standardize and evaluate suitability of Single Base Extension (SBE) singleplex assay for rapid routine monitoring of genetic homogeneity of virus samples, from productivity improvement experiments in Mumps vaccine. A Single Base Extension assay is a simple, cost effective tool for quick identification of single nucleotide polymorphism (SNP) at the three known sites in Mumps vaccine virus genome. The assay can be developed as a singleplex or multiplex version with potential for detection of up to ten SNP bases in a single reaction.Methods: SNaPshot kit from Applied Biosystems (ThermoFisher) was used to identify the nucleotide bases at the three SNP sites in Mumps virus genome. Approximate sizing locations of individual primers were determined using the SNaPshot Primer Focus Kit, also from Applied Biosystems. Genetic analyzer 3500 was used for the capillary electrophoresis of the processed samples. Results obtained from the data collection software were analyzed using GeneMapper software. Findings: Nucleotide G was identified at locations 1073 and 11345 and nucleotide C was identified at location 5261 for all tested mumps vaccine samples, thus demonstrating consistency in genetic homogeneity at the three known SNP hotspots in experimental mumps vaccine virus samples. Conclusion: The singleplex SBE assay was found to be a reliable and relatively inexpensive tool for routine monitoring of genetic homogeneity of vaccine virus samples, with potential for multiplexing that would reduce the cost and assay time even further.