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

A total of 13 naturally infected asymptomatic and symptomatic plants were tested positive using the developed multiplex TaqMan real-time qPCR; all samples gave positive results using both the Dickeya genus and D. dianthicola specific primer sets. The Ct values in both yellow and green channels ranged from 18.02±0.04 to 32.37±0.52 and 18.16±0.01 to 34.08±1.07, respectively. The bacteria were isolated from the positive samples and sequenced with forward and reverse primers described previously to confirm their identity. The multiplex qPCR showed 100% accuracy in detection of the Dickeya infected field samples. No false positives or false negatives were detected during the experiment ( Figure 6 ). Figure 6 . Multiplex TaqMan qPCR assay using infected field samples. 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. D. dianthicola, Positive control; Infected potato plant samples 1, 3, 4, 7, 8, 10, 11, 18, 19, 20, 21, 22 and 24 ; NTC, Non-template control. Each reaction was performed in three replicates.