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

To demonstrate the robustness of the multiplex TaqMan qPCR, a single TaqMan qPCR assay was also performed to compare the detection limit using both primer/probe sets. Primer sets DICg-F1/R1-wf and Ddia-F1/R1-wf were able to detect up to 10 fg in both yellow and green reporting channels, respectively. These results indicated that the developed multiplex assay is robust and equally sensitive as single target TaqMan real-time qPCR assay ( Figure 5D1, D2) . Figure 5 . Multiplex and single TaqMan qPCR standard curves and graphs. The graphs and curves were generated using ten-fold serial diluted genomic DNA (10 ng to 1 fg) of Dickeya dianthicola (A, B, D) and ten-fold serially diluted genomic DNA mixed with host plant DNA (C). The Green and yellow channels correspond to the different reporter dye 6-FAM (495/520) and HEX (535/554) (excitation/emission spectra in nm), respectively. A1/A2 multiplex TaqMan qPCR using DICg-F1/R1wf and Ddia F1/R1 wf (wf -with flap primers) primer sets in the reaction mix. B1/B2 multiplex TaqMan qPCR using DICg-F1/R1 and Ddia F1/R1 (no flap primers) primer sets in the reaction mix. C1/C2 spiked multiplex TaqMan qPCR using DICg-F1/R1wf and Ddia F1/R1 wf (wf -with flap primers) primer sets in the reaction mix (the spiked assay was done by adding 1 μl of healthy plant-potato DNA extracted from tubers to each serial dilution to simulate natural infection and then performing the multiplex qPCR assay). D1 single TaqMan qPCR with Ddia F1/R1 wf primer set in a reaction mix. D2 single TaqMan qPCR with DICg F1/R1 wf primer set in a reaction mix. The CT values represent the average of three replicates ±SD. Slopes (Y = threshold cycles (Ct) of target DNA detected), R 2 (correlation coefficient), and E (= amplification efficiency of the real-time PCR assay) of each reaction are presented in the respective curves. X axis represents the number of cycles, and Y axis is normalized fluorescence. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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