Genome-informed diagnostics for specific and rapid detection of Pectobacterium species using recombinase polymerase amplification coupled with a lateral flow device

Abstract

Pectobacterium species cause serious bacterial soft rot diseases worldwide on economically important fruit and vegetable crops including tomato and potato. Accurate and simple methods are essential for rapid pathogen identification and timely management of the diseases. Recombinase polymerase amplification (RPA) combined with a lateral flow device (LFD) was developed for specific detection of Pectobacterium sp. directly from infected plant materials with no need for DNA isolation. The specificity of RPA-LFD was tested with 26 Pectobacterium sp. strains and 12 non-Pectobacterium species and no false positive or false negative outcomes were observed. RPA primers and probe for host control were also developed to detect the host genome for enhanced reliability and accuracy of the developed assay. The detection limit of 10 fg was obtained with both sensitivity and spiked sensitivity assays. No inhibitory effects were observed on the RPA assay when targets (pathogen and host) were directly detected from infected potato and tomato sap. The developed RPA assay has numerous applications from routine diagnostics at point-of-care, biosecurity, surveillance and disease management to epidemiological studies. In addition, this tool can also be used to discover reservoir hosts for Pectobacterium species.

Figure 1

Ring image was generated to show the location of tyrR gene and genomic variation among the genomes. All genomes were retrieved from NCBI GenBank genome database. Gene tyrR was used to design recombinase polymerase amplification (RPA) primers and probe for specific detection of the genus Pectobacterium. The genome ring image from the inside out shows: genome coordinates (kbp), GC content (black), GC skew (purple/green). Other rings show BLASTn comparisons of 12 complete genomes as labelled. Pectobacterium carotovorum subsp. carotovorum (NCBI accession number NC_012917) was used to generate the ring image using BRIGS. Other genomes and their NCBI accession numbers: Pectobacterium carotovorum subsp. carotovorum (NC_18525), Pectobacterium carotovorum subsp. brasiliensis (NZ_CP020350), P. atrosepticum (NZ_CP009125), P. wasabiae (NC_013421); Dickeya zeae (NZ_CP006929) D. dadantii (NC_014500), D. solani (NZ_CP009460), Erwinia amylovora (NC_013961), Xanthomonas vesicatoria (NZ_CP018470), Ralstonia solanacearum (NC_003295) and Clavibacter michiganensis subsp. michiganensis (NC_009480). Plasmid sequences were not included in the analysis.

Figure 2

Overall orthologous average nucleotide identity (ANI) among bacterial genomes was calculated using Orthologous Average Nucleotide Identity Tool version 0.93 (OrthoANI). Values in color matrix boxes indicate the similarity percentage among the genomes. Clavibacter michiganensis subsp. michiganensis (NCBI accession number NC_009480), Pectobacterium carotovorum subsp. carotovorum (NCBI accession number NC_012917), P. carotovorum subsp. brasiliensis (NCBI accession number NZ_CP020350), P. wasabiae (NCBI accession number NC_013421), P. atrosepticum (NCBI accession number NZ_CP009125), Dickeya dadantii (NCBI accession number NC_014500), D. solani (NCBI accession number NZ_CP009460), D. zeae (NCBI accession number NZ_CP006929), Erwinia amylovora (NCBI accession number NC_013961) and Xanthomonas vesicatoria (NCBI accession number NZ_CP018470). Plasmid sequences were not included in the analysis.

Figure 3

Phylogenetic analysis. Phylogenetic analysis of strains included in validation of recombinase polymerase amplification (RPA) for specific detection of Pectobacterium species. Consensus sequences of dnaA gene were aligned and used to calculate the phylogenetic relationships among the strains using NJ tree building method. Consensus tree was generated using Bootstrap resampling method with 1000 replicates. Details of the strains are given in Table 1.

Figure 4

Consensus sequences of dnaA gene were used to generate pairwise color-code similarity matrix. All the strains used in inclusivity and exclusivity panels were included in these analyses; strain descriptions are shown in Table 1. The Sequence Demarcation Tool v1.2 was used to calculate percent pairwise similarity. Rs – Ralstonia solanacearum; Dz – Dickeya zeae; Dc – D. chrysanthemi; Ds – D. solani; Dd – D. dadantii; Pb – Pectobacterium betavasculorum; Pp – P. parmentieri; Pcc – P. carotovorum subsp. carotovorum; Pcb - P. carotovorum subsp. brasiliensis; Pa – P. atrosepticum; Pco - P. carotovorum subsp. odoriferum; Xv – Xanthomonas vesicatoria; Cmm – Clavibacter michiganensis subsp. michiganensis; Cmn - C. michiganensis subsp. nebraskensis.

Figure 5

The analytical specificity of recombinase polymerase amplification (RPA) assay for target pathogen, Pectobacterium sp. Lane 1 - P. carotovorum subsp. carotovorum (A5280); lane 2 - P. carotovorum subsp. odoriferum (A1089); lane 3 - P. carotovorum subsp. brasiliensis (A2688); lane 4 - P. atrosepticum (A1850); lane 5 - P. parmentieri (A1852); lane 6 - Dickeya zeae (A6056); lane 7 – D. zeae (A5422); lane 8 - D. dadantii (A5642); lane 9 - D. chrysanthemi (A5641); lane 10 - D. solani (A5581); lane 11 - D. solani (A5582); lane 12 - Clavibacter michiganensis subsp. michiganensis (A2058); lane 13 - C. michiganensis subsp. nebraskensis (A6095); lane 14 - Xanthomonas vesicatoria (A3617); lane 15 - Ralstonia solanacearum (A5491); and lane 16 – non-template control (water).

Figure 6

Sensitivity and spiked sensitivity of the developed RPA assay using a 10-fold serially diluted genomic DNA of Pectobacterium carotovorum subsp. carotovorum (strain A5280). In the spiked sensitivity assay, 1 $\mu \mathrm{l}$ (59 ng/ul) of host genomic DNA was added to each serially diluted genomic DNA sample to confirm the lack of an inhibitory effect of host genomic DNA on the RPA assay detection limit. No difference in sensitivity between spiked and non-spiked samples was observed. Lanes 1 to 7 are serially diluted genomic DNA (1 ng to 1 fg), and line 8 is non-template control (water).

Figure 7

Detection of Pectobacterium sp. from infected plant materials using recombinase polymerase amplification (RPA). (A) Detection of the target pathogen (Pectobacterium sp.) using Pectobacterium-specific RPA assay; (B) detection of host DNA using host specific RPA assay as a host control. Lane 1 - Pectobacterium carotovorum subsp. carotovorum (A5280); lane 2 - P. carotovorum subsp. carotovorum (A5278); lane 3 - P. carotovorum subsp. carotovorum (A5368); lane 4 - P. carotovorum subsp. brasiliensis (A2688); lane 5 - P. carotovorum subsp. brasiliensis (A3048); lane 6 - P. carotovorum subsp. carotovorum (A5278); lane 7 - P. carotovorum subsp. carotovorum (A5368); lane 8 - Dickeya chrysanthemi (A5415); lane 9 - D. solani (A5581); lane 10 – D. solani (A5582): lane 11 - P. carotovorum subsp. carotovorum (A5280; positive control – DNA from pure culture); 12) non-template control (NTC; water). Only DNAs of Pectobacterium sp. reacted postively and produced a line with the target specific RPA assay. Samples 1–5 were tomato inoculated with Pectobacterium sp., samples 6–7 were potato tubers inoculated with Pectobacterium sp. and samples 8–10 were potato tubers inoculated with Dickeya sp. Color codes: red – Pectobacterium sp. detected; blue – no detection; purple – positive control for Pectobacterium specific RPA assay but it is a negative control for host-specific RPA; green – host DNA detection; yellow – NTC.

Figure 8

Schematic representation of direct detection of Pectobacterium sp. in infected host tissue using RPA-LFD and rapid sample prep with TE buffer. The results showed no inhibitory effect of plant inhibitors on RPA assay. The rapid sample prep using TE buffer takes about 12 mins. Each inoculated sample was tested with both Pectobacterium sp. specific RPA (upper; target) and host specific RPA (lower; host control). Color codes: red – detection of Pectobacterium sp. from Pectobacterium sp. infected potato tubers and tomato fruits; green – detection of host genome; yellow non-template control (NTC; water). Lanes 1–3 are potato tubers infected with Pectobacterium sp.; lanes 4–6 are tomato fruits infected with Pectobacterium sp.; lane 7 is NTC.