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bioRxiv · 10.1101/2024.03.21.586178

Development and Validation of Genome-Informed and Multigene-based qPCR and LAMP Assays for Accurate Detection of Dickeya solani: A Critical Quarantine Pathogen Threatening Potato Industry

Dobhal, S.·Santillana, G.·Stulberg, M. J.·Arizala, D.·Alvarez, A. M.·Arif, M.
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Abstract

Dickeya solani, one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the worlds most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed-tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions), design loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with Quadplex, triplex and singleplex, respectively. None of the assay showed any inhibitory effect after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 8.92 minutes. The reproducibility was confirmed by performing the assay in two independent laboratories. These developed assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection and epidemiological studies. IMPORTANCEDickeya solani, one of the most aggressive soft rots causing-bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to potato industry. Accurate and timely detection of this bacteria is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research we have developed a LAMP and a multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool for management of this threatening pathogen.

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Dobhal, S., Santillana, G., Stulberg, M. J., Arizala, D., Alvarez, A. M., Arif, M.. 2024-03-21. Development and Validation of Genome-Informed and Multigene-based qPCR and LAMP Assays for Accurate Detection of Dickeya solani: A Critical Quarantine Pathogen Threatening Potato Industry. https://doi.org/10.1101/2024.03.21.586178

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