Table 1.
Summary of the genomic resources and DNA from microbial isolates used in this study.
Fig 1.
Pipeline followed to find TR4-specific sequences.
Step 1 assemblies of target and not-target genomes were used for the KEC (K-mer elimination by cross-reference) method described by Beran et al. [38] to obtain an initial list of target specific candidate sequences. These sequences were queried (Step 2) in an internal BLAST to double-check the presence/absence on non-target genomes. The remaining candidate sequences were filtered in two ways. Firstly, based on the results obtained when a query was done with BLAST on the NCBI online database (Step 3), whilst the second (Step 4) aligned Illumina reads on the candidate sequences with RattleSNP software (https://github.com/sravel/RattleSNP), and computed coverage of query reads on sequence tested. Figure created with Miro™.
Fig 2.
Genomic locations of the amplicons of previously published Foc-TR4 diagnostic systems used on the genomes of UK0001 and CAV807.
The three amplicons of the three candidates described in the current publication are written in bold. The chromosome names of the UK0001 have been simplified to fit in the Figure, being originally “VMNF010000...”.
Table 2.
In silico specificity of candidate Foc-TR4 target sequence regions and LAMP amplicons of those previously published.
Table 3.
Primer sequences and concentration used for Cand-23 LAMP assay.
Fig 3.
Position and direction of the LAMP primers on the Cand-23 region.
Table 4.
Specificity results of the LAMP markers designed in this project (Cand-23, Cand-5, and Cand-27), compared with three current Foc-TR4 diagnostic reference methods.
Fig 4.
Sensitivity of the Cand-23-LAMP system as assessed in plasmid dilutions (a) and in plasmid-spiked plant tissue extractions (b).
TTR: Time To Result (mins:ss). The colour and shape of the data points indicates the DNA volume used in the reaction; blue triangles (5 µL) or yellow squares (1 µL) for TTR means, while replicates are shown as solid circles. Error bars indicate the Standard Error (SE) of the mean. The horizontal black line indicates the threshold above which a sample is considered negative, and the dashed red horizontal line indicates the value assigned to the samples that produced null signal (negative). The asterisks on top indicate whether the mean is positive with a 95% of confidence level.
Fig 5.
Cand-23 in planta result comparison.
Comparison of the results obtained with the Cand-23 LAMP assay compared to the results with: (a) the Foc-TR4 specific qPCR as reported by Matthews et al., (2020) [21] and (b) Foc-TR4 specific Ordóñez LAMP assay [37]. The colour of the data points refers the origin of the data (naturally infected samples in Indonesia, Vietnam of Comoros, or artificially inoculated material in the Glasshouse) and indicates the average of the different replicates.
Table 5.
Summary of the results obtained by the Cand-23 LAMP compared to qPCR assay as published by Matthews et al. (2020) [21].