Table 1.
Plant samples used in this study, and numbers that were positive in the LAMP, PCR and tissue separation assays.
Table 2.
Species or populations of major plant pathogens used to evaluate the analytical specificity of the LAMP assay.
Figure 1.
Design of LAMP primers for detection of B. cinerea.
(a) Nucleotide sequence alignment of the target region Bcos5 in B. cinerea and Ssos5 in S. sclerotiorum. The sequences used for LAMP primers are indicated by bold lines. (b) Schematic representation of the LAMP primers used in this study. Construction of the inner primers FIP and BIP are shown. F1c and B1c are complementary to F1 and B1, respectively.
Table 3.
Information on the primers used in this study.
Figure 2.
LAMP detection of the Bcos5 gene in B. cinerea and digestion of positive LAMP products.
(a) LAMP for detection of B. cinerea using HNB as a visual indicator. The reaction becomes sky blue if the Bcos5 gene is present but remains violet if the gene is absent; (b) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. In (a) and (b), the positive reaction (with target DNA) is labeled “1″, and the negative reaction (without target DNA) is labeled “2″; (c), LAMP products were digested with Kpn I, and two fragments (153 bp, 49 bp) were observed by 3.0% agarose gel. M = 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Kpn I.
Figure 3.
Optimization of LAMP reaction temperature (a, b) and reaction time (c, d).
Assessment was based on HNB visualization of color change in (a) and (c) and on gel electrophoresis in (b) and (d). In (a) and (b), 1 = 61°C, 2 = 62°C, 3 = 63°C, 4 = 64°C, and 5 = 65°C. In (c) and (d), 1 = 15 min, 2 = 30 min, 3 = 45 min, 4 = 60 min, and 5 = 90 min.
Figure 4.
Specificity of LAMP detection of B. cinerea.
Assessment was based on (a) HNB visualization of color change or (b) agarose gel electrophoresis analysis of the LAMP products. M indicates a 100-bp ladder; 1, B. cinerea; 2, S. sclerotiorum; 3, F. graminearum; 4, R. cerealis; 5, R. solani; 6, V. dahliae; 7, A. alternata; 8, C. gloesporioides; 9, M. grisea.
Figure 5.
Sensitivity of LAMP vs. conventional PCR for detection of B. cinerea genomic DNA.
Detection by (a) LAMP and HNB visualization, (b) LAMP and gel electrophoresis, and (c) conventional PCR. Concentrations of template DNA (ng µL−1) per reaction in (a), (b), and (c) were: 1 = 10°, 2 = 10−1, 3 = 10−2, 4 = 10−3, 5 = 10−4, 6 = 10−5, 7 = 10−6, and 8 = 10−7. In (b) and (c), M indicates 100-bp, and DL 5000 ladder, respectively.