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 minimize the variations among the assays, all sensitivity experiments were performed using the same serial dilutions on the same day. The sensitivity was performed using a ten-fold serially diluted purified genomic DNA (10 ng to 1 fg) of D. dianthicola (A5569). To compare the sensitivity, two sets of assays were performed, one using the primer mix with no flap and other using the primer mix with flap primers. The limit of detection of the developed assay was determined in a host DNA background: 1 µl of host (potato) genomic DNA (10 ng µl -1 ) was added in each 10-fold serially diluted genomic DNA of D. dianthicola (A5569) from 10 ng to 1 fg. The assays were performed in triplicate. These dilutions along with a no-template control (NTC, water) were run in a Rotor-Gene Q Thermocycler; the TaqMan real-time qPCR conditions were used as . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 described above. The estimated genome size of D. dianthicola is ~ 4.86 Mb. Based on the genome size, the copy number was calculated using the formula: number of copies (molecules) = * . / ( * / ) * * / (where X = amount of amplicon (ng), N = length of dsDNA amplicon, 660 g/mole = average mass of 1 bp dsDNA) or web based software (www.scienceprimer.com/copy-number-calculator-for-realtime-pcr).