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 multiplex TaqMan qPCR was performed in Rotor-Gene Q (Qiagen, Germatown, MD). The concentrations of the primers and probes were adjusted to obtain the optimal amplification. The primer mix was prepared by adding 10 µl of each forward and reverse primer (from stock: 100 µmol l -1 ) for genus Dickeya and D. dianthicola and 160 µl nuclease free water. The multiplex TaqMan reactions were initially performed in two combinations: primers without 5' AT-rich flap sequences and with 5' AT-rich flap sequences. The multiplex TaqMan qPCR reaction was carried out in 25 μl reaction mixture containing 12.5 μl of Rotor-Gene Multiplex PCR Master Mix (Qiagen), 2 μl of the primer mix (5 µmol l -1 each), 0.5 μl of each DICg-P and Ddia-P probes (5 µmol l -1 stock), 1 µl of template DNA and nuclease free water was adjusted to obtain final volume. Positive and negative controls (non-template; water) were included in each TaqMan qPCR amplification run. Each qPCR reaction was performed in three replicates; standard deviation was calculated. Temperature cycling conditions were: 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, and 60 °C for 15 s, acquiring fluorescence on both green (FAM) and yellow (HEX) channels at the end of each extension step. The data analysis was done using the Rotor-Gene Q series software 2.3.1 (Built 49) with auto threshold (Ct); dynamic tube-based normalization was used.