RT Journal Article
SR Electronic
T1 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
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 847590
DO 10.1101/847590
A1 Shefali Dobhal
A1 Gamze Boluk
A1 Brooke Babler
A1 Michael J. Stulberg
A1 John Rascoe
A1 Mark Nakhla
A1 Toni A. Chapman
A1 Alex B. Crockford
A1 Michael Melzer
A1 Anne M. Alvarez
A1 Mohammad Arif
YR 2019
UL http://biorxiv.org/content/early/2019/11/20/847590.abstract
AB Aims Dickeya species are high consequence plant pathogenic bacteria listed among the quarantine pathogens of the European Union; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate, and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, D. dianthicola.Methods and Results A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed detection limit of 10 fg DNA.Conclusion The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials.Significance and Impact The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding program. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.