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

The in-silico analyses were performed via similarity search of each primer sequence against the NCBI GenBank database using the BLASTn tool; primers showed 100% specificity with query coverage and percent identity match of 100% with the target Dickeya genus and the species . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 dianthicola ( Table 2) . During in-vitro experiments, the specificity of the developed assay was tested with the DNA from 13 and 20 strains of D. dianthicola and other species of Dickeya from distinct geographical origins, respectively ( Table 1 ). The Ct values from Dickeya strains, excluding D. dianthicola, ranged from 13.89±0.04 to 30.84±0.11 in the yellow channel (n=18), while the green channel showed amplification and Ct values from 13.76±0.06 to 23.15±0.06 with the strains of D. dianthicola (n=13). The yellow channel exhibited almost similar Ct values as the green channel for the same strains of D. dianthicola (13.93±0.06 to 23.09±0. 13) . No amplification in green or yellow channels were observed from the non-target and/or closely related species (n=34). This demonstrated that the developed primers/probes are highly specific for the genus Dickeya and specifically for D. dianthicola (Table 1) . No Ct values were observed in either green or yellow channels with any member of the exclusivity panel, healthy plant or soil sample included in the assay validation. In addition, during the specificity validation, no Ct value >35 was obtained with any member of the inclusivity panel. The amplification from each representative species from genus Dickeya is shown in Figure 4 . The primers with and without 5' AT-rich flap sequences were also tested for their specificity; no false positives or false negatives were obtained (data not shown). The assays were highly specific but still the samples with Ct value after 35 cycles were considered negative.