Development of a robust, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of potato pathogen Dickeya dianthicola targeting a unique genomic region

PLoS One. 2019 Jun 24;14(6):e0218868. doi: 10.1371/journal.pone.0218868. eCollection 2019.

Abstract

Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D. dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D. dianthicola, which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase, of D. dianthicola. Assay specificity was assessed using strains present in inclusivity (16 D. dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D. dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alcohol Dehydrogenase / genetics*
  • Dickeya
  • Gammaproteobacteria / genetics*
  • Gammaproteobacteria / isolation & purification
  • Limit of Detection
  • Nucleic Acid Amplification Techniques / methods*
  • Plant Diseases / microbiology
  • Plant Proteins / genetics
  • Sensitivity and Specificity
  • Solanum tuberosum / microbiology*
  • Time Factors

Substances

  • Plant Proteins
  • Alcohol Dehydrogenase

Supplementary concepts

  • Dickeya dianthicola

Grants and funding

This work was supported by the USDA National Institute of Food and Agriculture, Hatch project 9038H, managed by the College of Tropical Agriculture and Human Resources. The strains were revived and maintained by National Science Foundation grant support (NSF-CSBR grant no. DBI-1561663). The study was also partially supported by the Department of Plant and Environmental Protection Sciences teaching funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.