Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

Shefali Dobhal; Gamze Boluk; Brooke Babler; Michael J. Stulberg; John Rascoe; Mark Nakhla; Toni A. Chapman; Alex B. Crockford; Michael Melzer; Anne M. Alvarez; Mohammad Arif

During this study, comparative genomics identified unique region/gene in the target genomes of Dickeya species. We designed the primers from these target genes and their flanked region -this approach enhances the specificity and reliability of the developed assay since the gene arrangement among the strains within a species is conserved compared to strains from different species. The primers were selectively tested both in-silico, by searching for the homology in the nucleotide database (NCBI GenBank BLASTn), and in-vitro, by screening against the strains present in inclusivity and exclusivity panels (Table 1 ). Using BLASTn, the primers and probes were found to be specific for the target pathogen with 100% identity match and query coverage. No false negatives or false positives were observed during the validation. In our lab, the alcohol dehydrogenase gene was also used to develop a loop-mediated isothermal amplification assay for specific, on-site detection of D. dianthicola and proved to be highly specific (Ocenar et al. 2019) .