Abstract
Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.
Highlights
The Clavibacter genus is a well-known, Gram-positive plant-pathogenic bacterium belonging to the Microbacteriaceae family—contains high GC—and is responsible for several devastating diseases in staple crops worldwide[1,2]
To address the demand for a point-of-need test, we proposed the development of an isothermal multiplex recombinase polymerase amplification assay, noted for a high degree of sensitivity and specificity with the potential to be implemented in a laboratory setting, and for the portable format that enables on-site detection and differentiation of Clavibacter species from C. sepedonicus (Cs). nebraskensis
We developed two unique primer and probe sets for specific detection of the genus Clavibacter and C. nebraskensis (Fig. 1)
Summary
The Clavibacter genus is a well-known, Gram-positive plant-pathogenic bacterium belonging to the Microbacteriaceae family—contains high GC—and is responsible for several devastating diseases in staple crops worldwide[1,2]. This genus consisted of only one species subdivided into nine subspecies, but recently, six subspecies were elevated to the species level-3. RPA effectiveness is highlighted by rapid detection of multiple targets within a single reaction; additional advantages of this technique include the availability of lyophilized reagents, obviation of initial denaturation, high sensitivity, affordability, reduced equipment requirements, and operation at constant low temperature employing recombinase-primer c omplexes[11,14,16,20,21,22,23]. While RPA assays are widely used in the detection of animal and human pathogens, its use in plant pathogen detection is limited, but increasing with the availability of commercial kits[14,16,20,28,29,30,31,32]; yet, no RPA multiplex has been developed for any plant pathogens
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