Table 1.
Detailed description of bacterial strains used in the inclusivity panel to validate the developed LAMP assay for specific detection of Dickeya dianthicola.
Table 2.
A detailed description of bacterial strains used in the exclusivity panel to validate the developed LAMP assay for specific detection of Dickeya dianthicola.
Fig 1.
Genome alignment, unique gene alcohol dehydrogenase and primer locations.
(A) Diagrammatic circa plot showing the presence of the target gene alcohol dehydrogenase used in the primer design for the Dickeya dianthicola specific LAMP assay. From the outermost circle to the innermost the circa plot displays: length of genomes in kilobases; name of the strains; lines in the green background depicts the presence of the target gene in D. dianthicola genomes; NCBI GenBank accession numbers for each genome used in the figure. The ribbons at the center of the circle represent the connections of the unique target gene among six strains of D. dianthicola (B) Location of all six LAMP primers and their orientations.
Table 3.
Details of LAMP primers designed using unique gene alcohol dehydrogenase for specific detection of Dickeya dianthicola.
Fig 2.
Specificity determination of LAMP assay developed for detection of Dickeya dianthicola.
In this figure, four strains (1–4; A5568, PL22, A5572 and A5573) form inclusivity panel and four (5–8; A5582, A6060, A6273 and A1084) from exclusivity panel are included. (A) Melt curve diagram–only four strains of D. dianthicola were amplified, no melt curve was observed with non-D. dianthicola strains and negative controls. (B) Visualization of LAMP products after adding 3 μL of SYBR Green I stain in amplified LAMP products; green color represents positive amplification. (C) Visualization of SYBR Green I results under UV light; fluorescence indicative of positive amplification. (D) Agarose gel electrophoresis of the LAMP products on 2% agarose gel. L, 10 kb DNA molecular weight marker; 1, D. dianthicola (A5568); 2, D. dianthicola (PL22); 3, D. dianthicola (A5572); 4, D. dianthicola (A5573); 5, D. solani (A5582); 6, D. dadantii (A6060); 7, P. carotovorum subsp. carotovorum (A6273); 8, Erwinia amylovora (A1084); 9, healthy leaf potato (negative control); NTC, non-template control (water).
Fig 3.
Dickeya dianthicola LAMP assay specificity determination with naturally and artificially infected plant samples.
A-C LAMP assay results with eight naturally infected plant samples (2–9): (A) Melt curve represents the positive amplification; (B) visualization of LAMP products after addition of SYBR Green I stain, green color represents positive amplification; (C) visualization of SYBR Green I results under UV light exposure, fluorescence indicative of positive amplification. D-F LAMP assay results with artificially infected plant tissue samples. GBp1A (1)—positive control; PL22, PL24, PL25 and PL31 (2–5) are D. dianthicola infected plant samples; A5287 and A6152 (6–7) are Pectobacterium sp. infected plant samples; (D) Melt curve results; (E) visualization of LAMP products after addition of SYBR Green I stain, green color represents positive amplification; (F) visualization of SYBR Green I results under UV light exposure, fluorescence shows positive amplification. NTC is non-template control (water); No false positives or false negatives were observed.
Fig 4.
Determination of LAMP assay’s limit of detection using 10-fold serially diluted pure culture of Dickeya dianthicola.
LAMP product visualized after addition of SYBR Green I stain, positive amplification turned orange color to bright green. Tubes 1–9 showed the detection from 108 CFU/ml to 1 CFU/ml; NTC is non-template control. Results showed positive amplification down to 10 CFU/ml.
Fig 5.
Determination of limit of detection of Dickeya dianthicola LAMP assay using 10-fold serially diluted genomic DNA.
A-D: Sensitivity of serially diluted D. dianthicola genomic DNA (L = 100 bp DNA ladder, 1 = 10 ng, 2 = 1 ng, 3 = 100 pg, 4 = 10 pg, 5 = 1 pg, 6 = 100 fg, 7 = 10 fg, 8 = 1 fg, NTC = non-template control. E-H: Sensitivity assay of serially diluted D. dianthicola genomic DNA spiked with potato genomic DNA (L = ladder, 1 = 10 ng, 2 = 1 ng, 3 = 100 pg, 4 = 10 pg, 5 = 1 pg, 6 = 100 fg, 7 = 10 fg, 8 = 1 fg, NTC = non-template control). (A, E) Melt curve of sensitivity assay; (B, F) LAMP product visualized after addition of SYBR Green I stain, positive amplification turned orange color to bright green color; (C, G) LAMP product with SYBR Green I stain under UV light, fluorescence indicated positive amplification; (D, H) agarose gel electrophoresis of LAMP product on 2% agarose gel.
Fig 6.
Validation of field applicability of Dickeya dianthicola specific LAMP assay by comparing the LAMP results using real-time qPCR and heat block.
(A) Flow diagram of the DNA extraction process of naturally infected plant samples by using the plant material lysis kits: i—plant material was processed in a tube containing iron ball and 1 mL lysis buffer; ii–macerated plant tissue after shaking vigorously for 1 min; iii–loop full of macerated supernatant was transferred to new vial containing dilution buffer; iv—five μL of diluted sample (crude DNA template) was added to LAMP assay and reaction was incubated at 65°C in a heat block for 20 minutes. (B) Visualization of LAMP products amplified using real-time qPCR machine and heat block: i–visualization after addition of SYBR Green I, bright green indicated positive amplification; ii—visualization after addition of SYBR Green I under UV, fluorescence indicated positive amplification; iii–LAMP products were electrophoresed on 2% agarose gel and visualized under UV, smear-like pattern reflected positive amplification. L, DNA molecular marker; 1, Genomic DNA of D. dianthicola (PL22, positive control); 2, D. dianthicola infected; 3, D. dianthicola infected; 4, D. dianthicola infected; 5, D. dianthicola infected; NTC, non-template control (water).