Fig 1.
Diagrammatic representations of the insulated isothermal PCR (iiPCR) assay, the POCKIT™ Nucleic Acid Analyzer, and the R-tube™.
(A) The iiPCR, established on the basis of the Rayleigh-Bénard convective PCR method, is a rapid platform for nucleic acid amplification. (B) The iiPCR system is carried out in the R-tube™ within the user-friendly POCKIT™ Nucleic Acid Analyzer designed by GeneReach Biotechnology Corporation (Taichung City, Taiwan). The POCKIT™ analyzer (28 × 25 × 8.5 cm, W × D × H) provides isothermal heating at the bottom of the R-tube™ to generate a cycling thermal convection to drive the PCR reaction. DNA amplification and product detection can be completed automatically by the POCKIT™ analyzer with a single default program.
Table 1.
Isolates of plant pathogens used in this study and their PCR amplification results with PCR-based identification methods.
Table 2.
Molecular markers and the corresponding primers used in this study.
Fig 2.
Optimization of TaqMan probe concentration for insulated isothermal PCR (iiPCR) assay of Fusarium oxysporum f. sp. cubense (Foc) race 4.
Different concentrations (0, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 nM) of probe were tested in the Foc iiPCR to evaluate the effects of TaqMan probe concentration on fluorescent signal production. The mean S/N ratio (fluorescent intensityafter/fluorescent intensitybefore) of each reaction was plotted against the TaqMan probe concentration. Error bars represent the standard deviations from seven replicate reactions.
Fig 3.
Sensitivity evaluation of Foc race 4 TaqMan probe-based insulated isothermal PCR (iiPCR) assay.
Serial dilutions of (A) standard template pFoc242 (ranging from 106 to 1 copies) and (B) Fusarium oxysporum f. sp. cubense (Foc) race 4 genomic DNA (ranging from 105 to 1 fg) were subjected iiPCR assay. The S/N ratios (fluorescent intensityafter/fluorescent intensitybefore) of Foc race 4 iiPCR assay were calculated. Mean S/N ratio of each reaction was plotted against standard template or Foc race 4 genomic DNA. Error bars represent the standard deviations from seven replicate reactions.
Table 3.
In planta detection of Fusarium oxypsorum f. sp. cubense race 4 in field-infected banana by insulated isothermal polymerase chain reaction (iiPCR) method.
Table 4.
Comparison of Foc race 4 iiPCR and PCR-based identification for field detection.