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

Previously, we have demonstrated that incorporation of flap sequences at the 5' position of the primer could lead to increased sensitivity, fluorescence, overall PCR yield and optimize reaction efficiency (Arif and Ochoa-Corona 2013; Larrea et al. 2019). The 5' AT-rich flap sequences were added to both primers sets, DICg F1/R1 and Ddia F1/R1, to evaluate their effects on sensitivity and/or specificity of the developed assay. Both primers with and without 5' AT-rich flap sequences, targeting the Dickeya species and D. dianthicola, were specific for respective pathogens; no cross-reactions were observed (data not shown). Both multiplex primer sets, DICg F1/R1wf and Ddia F1/R1wf, with 5' AT-rich flap showed optimum reaction efficiency compared to the primers with no flaps ( Figure 5 ) and were sufficiently sensitive to detect the genomic DNA of D. dianthicola and Dickeya species down to 10 fg. However, primers with no flap showed some discrepancy among the replicates at the lowest concentration (10 fg; DICg F1/R1). Therefore, both primers with the 5' flap sequences were used for overall validation of the developed multiplex TaqMan qPCR assay. The addition of the 5' AT-rich flap also made the primers and probes compatible to mix and match based on user's need without losing specificity and/or sensitivity; these primers work under the "One Lab -One Protocol" concept (Arif 2019).