Genome-guided, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of Dickeya dadantii

Ludena, S. M.; Tyagi, G.; Feng, Z.; Hampson, E.; Adhikari, A.; Minaai, M.; Wong, L.; Haubner, M.; Dobhal, S.; Arizala, D.; Andreason, S. A.; Mollov, D.; Ochoa-Corona, F. M.; Bingham, J.-P.; Odani, J.; Jenkins, D.; Ma, L. M.; Fletcher, J.; Stack, J. P.; Arif, M.

doi:10.1101/2024.05.04.592507

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bioRxiv · 10.1101/2024.05.04.592507

Genome-guided, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of Dickeya dadantii

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Abstract

Potatoes, among the most economically significant crops worldwide, are susceptible to various plant pathogens that significantly impact their propagation, production, storage, and distribution. Soft rot disease, caused primarily by Dickeya and Pectobacterium, results in substantial economic losses to the agricultural industry annually. In this study, we developed a rapid, reliable, and field-deployable loop-mediated isothermal amplification (LAMP) assay for detecting D. dadantii, a common soft rot causing bacteria. The D. dadantii-specific LAMP primers were designed targeting a highly conserved genomic region within D. dadantii, the TetR/AcrR family transcriptional regulator CDS and its flanking sequences. This assay was thoroughly validated with the members of inclusivity (nine strains of D. dadantii) and exclusivity panels (85 strains, including all Dickeya species, related taxa, and host DNA), detecting no false positives or negatives. The limit of detection (LOD) was established by performing assays with 10-fold serially diluted pure gDNA of D. dadantii and gDNA spiked with host crude extract; the assay detected the target pathogen down to 1 pg (188 copies) without being adversely affected by the host crude extract. The developed LAMP assay specifically detected the target pathogen in infected plant materials. Additional multi-operator blind and multi-instrument tests were conducted to assess the assays robustness and applicability, consistently yielding accurate results without false positives or negatives. These findings demonstrate the assays potential utility for biosecurity, routine diagnostics, and epidemiological studies.

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Ludena, S. M., Tyagi, G., Feng, Z., Hampson, E., Adhikari, A., Minaai, M., Wong, L., Haubner, M., Dobhal, S., Arizala, D., Andreason, S. A., Mollov, D., Ochoa-Corona, F. M., Bingham, J.-P., Odani, J., Jenkins, D., Ma, L. M., Fletcher, J., Stack, J. P., Arif, M.. 2024-05-05. Genome-guided, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of Dickeya dadantii. https://doi.org/10.1101/2024.05.04.592507

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