Development of a robust, deployable loop-mediated isothermal amplification assay for rapid and specific detection of soft rot caused by Pectobacterium

Abstract

Soft rot, caused by the phytopathogenic bacteria Pectobacterium and Dickeya , is a serious and destructive disease of potato worldwide. Symptoms caused by the two genera are indistinguishable. An effective diagnostic strategy is essential for controlling the disease and reducing losses in potato production. In this study, we examined genomes from multiple Pectobacterium and Dickeya species and selected the distinctive p4h gene to design specific primers and develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and specific detection of Pectobacterium among other genera causing soft rot. The assay was designed for use with a field-portable thermal flask at 65 °C. The specificity of the developed LAMP assay was evaluated using 15 bacterial strains comprising inclusivity and exclusivity panels. The assay detected as little as 5 fg (~9000 genomic copies) of genomic DNA. It also detected Pectobacterium in infected potato tuber samples. Results were consistent between laboratory and field conditions, with no false-positive or false-negative results. The developed LAMP assay may be applied to diagnostics, agricultural quarantine, and disease management.