Fig. 1: Visual detection of fungal RNAs based on the TMSD reaction and metal ion-mediated urease catalysis.
a Schematic illustration of the colorimetric assay. The DProbe is a double-stranded DNA consisting of a cis strand and a trans strand that anneal to form a C-Ag(I)-C artificial base pair. Fungal RNAs can bind with the cis strand to release the trans strand and Ag(I) ion via the TMSD reaction. The driving force of the TMSD reaction derives from the hybridization between the forward toehold (pink domain) overhang and fungal RNAs, and the resistance force comes from the break of the reverse toehold (green domain) hybridization. The released free Ag(I) ion induced by fungal RNAs inhibits urease activity, reducing the production of NH4+, which can be visualized using of pH indicator phenol red. Based on this principle, the presence of fungal RNAs can be detected by the a colour change of the pH indicator. b Absorbance at 560 nm at each reaction of the assay. Inset: visual results of each reaction. Concentrations of DProbe, urease, urea, and phenol red were 100 nM, 1 nM, 500 mM and 250 μM. c Electrophoretic analysis of the TMSD reaction. Concentrations of DProbe, and fungal RNAs were 400 nM and 600 nM, respectively. To distinguish the DProbe from the hybrid of fungal RNAs and the cis strand in the electrophoresis ladder, 30 nt poly-T was added to the 5′ end of the trans strand. d Sensitivity estimation of the assay for detecting Pst. Visual results (upper) and absorbance (below) of each sample. lg (g/μl) indicates the negative logarithm of the concentration of Pst. e Discrimination of Pst, Pt and Pgt. The ITS sequences of Pst, Pt and Pgt (upper), absorbance at 560 nm (below left) and visual result (below right) for detecting different fungi. “a. u.” in (b and d) indicates arbitrary unit. Data in (b and d) represent means ± SD (n = 3). P values from Welch’s two-sided unpaired t test in (d): *P < 0.05. Source data are provided as a Source Data file.
