New Dickeya species are continually being described due to the broad natural diversity within the genus. To date, descriptions of 12 Dickeya species have been published and their genomes are available15, including the most recent additions of D. poaceiphila31, D. parazeae32, D. oryzae sp. nov.33 and D. colocasiae16. A previously published LAMP assay, designed to detect Dickeya at the genus level, successfully detected eight known Dickeya spp.34. The LAMP assay developed in the present study utilized a unique genomic region to provide a rapid, and cost-effective diagnostic assay for specific detection of D. fangzhongdai which can be applied easily at point-of-need. The assay was thoroughly validated with all Dickeya and Pectobacterium species and strains of other bacterial species.
A robust diagnostic assay requires stringent selection of a unique and specific genomic region that is well conserved within the target pathogen genomes25,30,35. In silico validation is required to confirm that the target region is present in all strains of the same species and absent in closely related species of Dickeya and Pectobacterium, and other species that occupy the same ecological niche21. The unpublished genome of a locally isolated strain of D. fangzhongdai (PL145) was used to increase the breadth of in silico analysis, as the publicly available genomes of the species do not currently include Hawaiian strains (Table 1). Four regions were specific to D. fangzhongdai based on preliminary in silico genome alignment and analyses, and in vitro endpoint PCR tests (data not shown). High band intensity maintained across different strains of D. fangzhongdai and the lack of off-target amplification, even at the relatively low annealing temperature (57ºC), indicated that the MFS transporter was an ideal region for further LAMP assay development. This held true for both sets of primers (endpoint PCR and LAMP assays) designed for this region. LAMP remains an ideal option for diagnostic assay development; it is rapid, comparatively resistant to inhibition, and field deployable20. Results can be visualized in several ways, including colorimetric indicators, turbidity, and gel electrophoresis, which enables LAMP to be used effectively in many laboratory settings25.
The validation of the designed primers with the members of inclusivity and exclusivity panels is mandatory for confirmation of assay specificity25. The positive LAMP results with members of the inclusivity panel shown in Fig. 2; the LAMP assay detected all D. fangzhongdai strains—suggest a broad range detection capability of D. fangzhongdai. In the exclusivity panel, clear negative results occurred for the other species of Dickeya and Pectobacterium (Table 3; Fig. 2). Although two recently described Dickeya species, D. poaceiphila and D. parazeae, were absent from the exclusivity panel, our genomic-informed strategy of selecting the MFS target gene for LAMP development suggests the sequence of the MFS transporter gene is not present in their genomes (Fig. 1A). Hence, the developed LAMP possesses high specificity for distinguishing the species, D. fangzhongdai, from phylogenetically closely related species, plant associated niche-sharing species of additional genera, and common habitat matrices. The detection of a target pathogen can be affected adversely by the presence of plant inhibitors, but inhibitors do not affect all assays (developed using different techniques) equally20,36. A LAMP assay developed for specific detection of a Gram-positive bacterial pathogen, Rathayibacter toxicus, using the OptiGene Master Mix, was less affected by plant inhibitors than an endpoint PCR but more affected than an RPA assay20. The currently described assay showed no effect of plant inhibitors when crude extracts of artificially infected taro, orchid plant materials, and naturally infected taro (prepared using Plant Lysis Kit)25 were tested using the developed LAMP assay (Fig. 8–10). In contrast to other developed diagnostic methods37,38, this D. fangzhongdai LAMP assay is rapid, requiring only about 15–30 min from sample preparation to detection and does not require sophisticated lab equipment (Fig. 7C).
High sensitivity is a prerequisite for a diagnostic assay that eliminates the probability of false negatives that could have serious negative consequences if a pathogen enters and becomes established in new geographical locations. Therefore, we evaluated the detection limit of the D. fangzhongdai LAMP assay in the presence of three hosts (taro, onion, orchid) matrices. The 5 µl of crude extract of either taro, onion, or orchid was added to preparations of 10-fold serially diluted genomic D. fangzhongdai DNA, but no effect of host matrices was observed, and the detection limit remained at 100 fg, which is equivalent to 18–20 genome copies (Figs. 4A-4C, 5). These results indicate that the developed assay is highly sensitive, robust, and specific. In comparison, endpoint PCR was performed with the primer set designed using the same unique MFS transporter gene region added to 10-fold serially diluted DNA of D. fangzhondai. The target pathogen was detected in all dilutions down to 10 pg (Fig. 4D). An additional spiked assay was performed to assess inhibition by adding 1 µl of plant genomic DNA extracted from healthy taro corm to each PCR reaction mixture containing 1 µl of 10-fold serially diluted DNA of D. fangzhondai (Fig. 4E). A 10 pg limit of detection was maintained, but it was 100-fold less sensitive than LAMP detection.
Evaluation of the LAMP reaction by multiple operators on the Rotor-Gene platform provided confidence in detection consistency, robustness, and sensitivity (Fig. 6). Assay performance levels were achieved on multiple operating devices, showing consistency across differing amplification platforms (Fig. 7). Finally, our assay was not inhibited by the presence of plant cellular matrices, indicating that assay functionality is not necessarily dependent on sample purity and suggesting that less-stringent extraction methods may be used. These findings suggest that the LAMP assay can be used with confidence in laboratories as well as at point-of-need.