Table 1.
Fungal species and isolates used to test the specificity of the RealAmp assay.
Figure 1.
Nucleic acid sequence of intergenic spacer (IGS) region of the nuclear ribosomal operon (GenBank accession number FJ985561) used for designing inner and outer primers.
The specific sequences used for primer design and their relative positions in the genome are indicated by arrows.
Figure 2.
Specificity test of the real-time fluorescence loop mediated isothermal amplification assay (RealAmp assay) for the detection of Foc TR4.
(A) Agarose gel electrophoresis analysis of RealAmp assay amplicon showing the specificity of RealAmp assay for detection of Foc TR4. Lanes 1–3, genomic DNAs of Fusarium oxysporum f. sp. cubense (Foc) race 1, subtropical race 4 (ST4) and tropical race 4 (TR4), respectively; Lanes 4–8, the DNAs of Mycosphaerella melonis, Fusarium oxysporum f. sp. cucumerium, Fusarium oxysporum f. sp. luffae, and Fusarium oxysporum f. sp. niveum, respectively; Lane M, Trans2K plus II DNA marker. Samples shown in lanes 1 to 8 in Fig. 2B, 2C and 2D is the same as in Fig. 2A. (B) The specificity of RealAmp assay was validated by specific PCR amplification using the specific FocTR4-F/FocTR4-R primer set. (C) Visual inspection of the RealAmp amplification products. The original orange colour of SYBR green turned green in the positive reaction mixture. (D) The fluorescence units vs. time graph plotted automatically by the ESE-Quant Tube Scanner. The graph reports the fluorescence in millivolts (mV) on the y axis and time in minutes on the x axis. Results can be read in the LCD panel as either positive or negative and/or in real time using a computer with the appropriate software.
Figure 3.
The sensitivity of RealAmp assay and standard curve.
(A) Sensitivity test of RealAmp assay. Lane M, Trans2K Plus II DNA marker, lanes 1–8 correspond to serial 10-fold dilutions of Foc TR4 plasmid DNA ranging from 4.3 ng/µl to 4.3×10−5 ng/µl. Samples given in lanes 1 to 8 in Fig. 3B and 3C is the same as in Fig. 3A. (B) Visual detection of the RealAmp amplification products. The original orange colour of SYBR green turned green in the positive reaction mixture. (C) The fluorescence units vs. time amplification curves plotted automatically using an ESE-Quant Tube Scanner. (D) Standard curve for RealAmp assay. The threshold time (Tt) vs. the amount of initial template plasmid DNA were plotted using an ESE-Quant Tuber Scanner. Error bars represent standard deviations from triplicate reactions.
Figure 4.
Determination of the detection limit of the real-time PCR and standard curve.
(A) Sensitivity test of the real-time PCR. The real-time fluorescence units are plotted against concentration of initial plasmid DNA ranging from 4.3 ng/µl to 4.3×10−6 ng/µl using a PRISM® 7500 Fast real-time PCR. (B) Standard curve calculated from panel A. Standard curve generated using known concentration of 10-fold serially diluted pMD18-T-TR4 plasmid DNA and the threshold cycle (Ct) value. Every DNA concentration was measured 3-fold and error bars represent standard deviations from three replicate reactions.
Figure 5.
Detection of Foc TR4 from pure spores and spores in artificially infested soil using RealAmp assay and real-time PCR.
(A) and (B) show detection of Foc TR4 from pure spores, respectively, using RealAmp assay. (C) and (D) show detection of Foc TR4 from artificially infested soil samples, respectively, using real-time PCR. (A) and (C) show the serial 10-fold dilutions of pure spores were ranging from 106 to100 spores/µl. (B) and (D) show the serial 10-fold dilutions of artificially infested soil samples were ranging from 105 to100 spores/g soil.
Figure 6.
Detection of Foc TR4 in 6 randomly selected field soil samples using the RealAmp assay and real-time PCR method, respectively.
(A) RealAmp assay detection of random field soil samples. The amplification curves were obtained using the ESE-Quant tube scanner. The graph reports the fluorescence in millivolts (mV) on the y axis and time in minutes on the x axis. Lanes 3 to 8 are random samples collected from different geographic locations, respectively. (B) Real-time PCR method detection of Foc TR4 in 6 randomly selected field soil samples and samples in lanes 3–8 correspond to those in panel (A). (C) Comparison of the quantitative results of 6 randomly selected samples between RealAmp assay and real-time PCR. A twice-autoclaved Foc-free soil sample served as negative control and a Foc TR4 artificially infested soil sample served as positive control in this study, respectively. Data are the mean (±SD) of three replicates. ns above columns indicate no significant difference between two detection methods for each sample at P<0.05 levels (ns, not significant). Paired t-test was used to test the significance of difference between two methods for all the soil samples at P<0.05 level.