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

l galactoside ABC transporter ATP-binding protein MglA/MglC region was used to design the primers and probe specific for the genus Dickeya. Similarly, for D. dianthicola, the genomes of D. dianthicola (NZ_CM001838, NZ_CM001840, NZ_CM001841 and NZ_CM002023) were retrieved from the NCBI GenBank Genome database (Supplementary Table 1 ) and aligned using progressive Mauve; Geneious was used for analyzing the unique genes present in the D. dianthicola genome. The alcohol dehydrogenase gene was used to design the primers and probe specific for D. dianthicola. Table 2 . No flap sequences were added to the probes. The specificity of each primer was confirmed in silico using the BLASTn tool against the NCBI GenBank nucleotide and genome databases ( Table 2 ). All primers and double-quencher probes 5'-/6FAM/ZEN/3IBQ/-3' and 5'-/5HEX/ZEN/3IBQ/-3' were synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, IA). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/847590 doi: bioRxiv preprint wasabiae strain CFBP 3304 (NZ_CP015750), Erwinia amylovora strain CFBP1430 (NC_013961), Ralstonia solanacearum GMI 1000 (NC_003295), and Clavibacter sepedonicus strain ATCC33113 (NC_10407). The blue and green arrows indicate the location of the region for the galactose/methyl galactoside ABC transporter ATP-binding protein MgIA/MglC and alcohol dehydrogenase, respectively. The BLAST RING IMAGE GENERATOR (BRIG) 40 (Alikhan et al., 2011) was used to generate the image. Plasmids were not included in the analysis.