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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
Author(s) -
Dobhal S.,
Boluk G.,
Babler B.,
Stulberg M.J.,
Rascoe J.,
Nakhla M.K.,
Chapman T.A.,
Crockford A.B.,
Melzer M.J.,
Alvarez A.M.,
Arif M.
Publication year - 2020
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.14579
Subject(s) - multiplex , biology , taqman , multiplex polymerase chain reaction , computational biology , microbiology and biotechnology , polymerase chain reaction , virology , genetics , gene
Aims Dickeya species are high consequence plant pathogenic bacteria; 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, Dickeya 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 a 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 of the Study 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 programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.