Development of Tiling amplicon PCR for Ross River virus (RRV)

Abstract

The Ross River virus (RRV), an Australian alphavirus that causes epidemic polyarthritis, remains a priority for genomic surveillance due to its sporadic outbreaks and varying strains. Comprehensive genome amplification is necessary for monitoring its molecular evolution; however, successful multiplex PCR design requires consideration of primer quality metrics that need extensive computational analysis. We obtained publicly available RRV genomes from NCBI, aligned them using the QML1 strain (GenBank GQ433354.1) as a reference, and utilized Ubuntu-based Olivar v1.3.0 to design automated tiling amplicon primers. We looked at two tiling strategies: 50-bp and 100-bp stepwise intervals. These were amplicon sizes ranging from 300 to 600 bp. The ideal balance between coverage, efficacy, and primer quality was selected as the best 400-500 bp scheme. Primer candidates were in-silico checked using Geneious, MEGA, and SnapGene to identify which primers were specific, had a suitable GC content, and tolerated mismatches in multiple isolates of RRV. Simple adjustments to the positions and degeneracy were made to address off-target sites, three industrial mismatches, and heterodimer threats through manual curation. The predicted dimerization risk and stable thermodynamic profiles were obtained by spacing the primers evenly. The problems were corrected by redesigning and repositioning problematic primers, which demonstrated multi-site binding or primer primer complementarity. Mismatches at 3 ends of a short RNA strand were repaired with minimal IUPAC substitutions. The presence of amplicon overlaps (nominal 70 bp, insert level 24 bp) maintained the integrity of the data without adding any additional copies. In contrast, regions with extremely high GC content were automatically omitted to prevent amplification issues. Of the 22,374 annotated features of the primer, the residual off-target binding was 0.05%, indicating that the assay was overall highly specific. The 100-bp interval design is effective in recovering the complete genome of a wide variety of isolates, thereby providing a strong foundation for neutralizing genomic surveillance and lineage tracing efforts, as well as outbreak response efforts. Despite optimizing primers, residual mismatches and off-target products persist. Nevertheless, the methodological framework can be easily modified for other alphaviruses that necessitate comprehensive and strain-inclusive primer coverage.