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

Multiplexing for specific detection of multiple targets in a single reaction is time efficient, provides enhanced reliability and further validation of the outcomes (Li et al. 2017) . The probes labelled with internal ZEN quencher along with the traditional 3'end quencher enhance quenching efficiency, provide less background noise and increase precision ). Multiplexing the reaction with different primer and probe sets can adversely affect the sensitivity and can increase the background noise (Li et al. 2017 ). However, the developed assay did not show any discrepancy in the detection limit when assays were performed individually as compared to the multiplex reaction; both detected 10 fg of genomic DNA. The presence of plant DNA mixed with pathogen DNA has been reported to interfere with the Taq DNA polymerase due to the presence of strong DNA-binding domains, reducing Taq-polymerase availability and activity, which results in higher Ct values (Hoy et al. 2001; Strayer et al. 2016) . Therefore, the spiked assays were performed by adding the DNA from healthy plants along with target pathogen DNA in the multiplex qPCR reaction. No adverse effect on the Ct values was observed -the detection limit remained the same (10 fg) as with the pure genomic DNA from D. dianthicola. The genome size of Dickeya species ranges from 4.62-5.05 Mb (supplementary Table 1 ). Based on the calculations, 10 ng of D. dianthicola genomic DNA contains 1.93X10 6 copies, therefore, 10 fg should be equivalent to ~ 2 copies. The sensitivity of the developed multiplex TaqMan qPCR is higher (10 fg per reaction; Ct=33.75±0.24 compared to the previously reported primers/assays by Potrykus et al. (2014) for Dickeya species (detection limit 10 pg per microlitre) and van der Wolf et al. (2014) for D. dianthicola (20 fg per reaction; Ct=36.5±0.38).