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

Abstract

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.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

“…Precision, dependability, and accuracy are important components of a robust and specific detection assay to be utilized in monitoring and surveillance programs. The foundation of a robust and specific assay depends on target selection 32 . The low cost of genome sequencing and availability of whole genomic data in public databases increases the use of comparative genomic approaches for identifying for signature genomic regions exclusively present in target species 32 .…”

“…The foundation of a robust and specific assay depends on target selection 32 . The low cost of genome sequencing and availability of whole genomic data in public databases increases the use of comparative genomic approaches for identifying for signature genomic regions exclusively present in target species 32 . In this study, we designed P. parmentieri primers to amplify a unique petF1 gene region, a genomic region highly conserved in all P. parmentieri strains tested, but not in other closely related bacterial strains, pathogenic or non-pathogenic (Fig.…”

“…Bacterial strains listed with “A”, “PL”, and culture collection IDs were stored at − 80 °C, and revived by streaking onto 2,3,5-triphenyltetrazolium chloride (TZC) medium (peptone 10 g 1 −1 , dextrose 5 g l −1 , 0.001% TZC and agar 17 g l −1 ) and TZC-sucrose medium (TZC-S: peptone 10 g l −1 , sucrose 5 g l −1 , 0.001% TZC and agar 17 g l −1 ), respectively (Norman and Alvarez 1989). The plates were incubated at 26 °C (± 2 °C) for 12–24 h. Single colonies were re-streaked onto a new TZC medium plate and later used to harvest pure bacterial growth for DNA isolation 32 .…”

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri

“…Traditional methods including culturing, microscopic observations, morphological, physiological, biochemical and serological assays have been reported for the identification of Pectobacterium spp. but used alone they are inadequate for new species identification (Czajkowski et al, 2015;Motyka et al, 2017;Nikitin et al, 2018), Nucleic acidbased molecular techniques have proven to be faster, reproducible, highly sensitive and able to differentiate species with greater accuracy (Czajkowski et al, 2015;Dobhal et al, 2020;Nikitin et al, 2018). Contrary to endpoint PCR, real-time qPCR permits the identification and quantification of the target pathogen in real time and offers a higher level of efficiency and sensitivity, making it a suitable tool for epidemiological and ecological studies (Mirmajlessi et al, 2016;Nikitin et al, 2018).…”

“…Among all PCR and isothermal formats, real-time qPCR TaqMan probe-based assays have been shown to be the most promising approaches for diagnostic surveys because of high accuracy, sensitivity and reliability of detection outcomes (Arif et al, 2015;Dobhal et al, 2020;Larrea-Sarmiento et al, 2019). So far, no diagnostic tool is available for specific detection of P. parmentieri.…”