Fig. 1. Alignment of partial of LasAI sequences of isolates from Florida, China, Thailand, Philippines India and Brazil, and primers designed for HLB loop-mediated isothermal amplification (LAMP). Length of 269 bp from start codon was used for primer design for LAMP PCR. Black arrows are indicated as primer target sequences and extension directions. The FIP and BIP primers consist of F1c plus F2 and B1c plus B2, respectively.
Fig. 2. Loop-mediated isothermal amplification (LAMP) for specific detection of ‘Candidatus Liberibacter asiaticus’ using the primer set from the prophage gene, LasAI in HLB-infected leaves of grapefruit according to reaction temperatures (57, 60, 62 and 65℃). condition test for HLB detection. (A) Visual detection under normal light by adding SYBR Green I dye. (B) Electrophoresis analysis on 1% agarose gel. Lanes 1-4; HLB-infected grapefruit leaves, lanes 5-8; healthy grapefruit leaves, lanes 9-11; distilled water, lane M; 100 bp DNA ladder (NEB New England Biolabs, cat# N3231S).
Fig. 3. Confirmation of loop-mediated isothermal amplification (LAMP) product sequence. (A) Electrophoresis analysis of LAMP products on 4% agarose gel. Lane M, 100 bp DNA marker. The amplified product of red square was eluted for sequence analysis. (B) Result of LAMP product sequence. The arrows were indicated as FIP and BIP primers and extension direction and, the black lines; as region of F1c and B1c primers, the red square; F3 and B3 primers for target region in LasAI gene, respectively.
Fig. 4. Sensitivity of loop-mediated isothermal amplification (LAMP), conventional PCR and real-time PCR for detecting HLB. (A) Visual examination of LAMP products by adding SYBR Green I dye. (B) Electrophoresis analysis of LAMP products on 1% agarose gel. (C) Electrophoresis analysis of conventional PCR products on 1% agarose gel. (D) Sensitivity of realtime PCR for detecting HLB using primers (LJ900p, LJ900r)/probe (LJ900p) listed in Table 1. Tube and lane 1-8, and template DNA for real-time PCR; serially diluted genomic DNA (1, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6) and distilled water as negative control, respectively, M; 100 bp DNA ladder.
Table 1. Sequence of primers/probe used in this study