An efficient DNAzyme for the fluorescence detection of Vibrio cholerae

Abstract

Vibrio cholerae (Vc) causes cholera disease. Vc contamination is widely found in water and aquatic products, and therefore is a serious food safety concern, especially for the seafood industry. In this paper, we attempted the rapid detection of V. cholerae. Nine rounds of in vitro selection using an unmodified DNA library were successfully performed to find specific DNAzymes of Vc. Their activity was evaluated based on a fluorescence assay and gel electrophoresis. Finally, a DNAzyme (named DVc1) with good activity and specificity with a detection limit of 7.2 × 10³ CFU/mL of Vc was selected. A simple biosensor was constructed by immobilizing DVc1 and its substrate in shallow circular wells of a 96-well plate using pullulan polysaccharide and trehalose. When the crude extracellular mixture of Vc was added to the detection wells, the fluorescent signal was observed within 20 min. The sensor effectively detected Vc in aquatic products indicating its simplicity and efficiency. This sensitive DNAzyme sensor can be a rapid onsite Vc detection tool.

Keywords: DNAzyme; fluorescence detection; onsite detection; sensitive V. cholerae biosensor.

FIGURE 1

Scheme of the DNAzyme selection. There are 35 nt random nucleotide in the library, and nine rounds of selection was performed. Positive selection was performed in the 1st, 3rd, 5th, 7th, and 9th rounds of the selection process. Negative selection was performed only in the other rounds. The cleavage site is at the rAG junction and biotin was labeled on the 5′ end. The target molecule is crude extracellular mixture (CEM).

FIGURE 2

Activity assessment of the DNAzyme candidates. (a) Fluorescence intensity and (b) Gel‐based cleavage activity assessment of DVc1‐DVc5 DNAzymes [a‐d were considered to be statistically significant (p < .05), and the same letters were considered to be not significant (p > .05)]. The bar means: mean ± SD in the legend. D1‐D5:DVc1‐DVc5; Unclv: Uncleaved intact full‐length DNAzyme; Clv: cleaved product of the DNAzyme after the reaction. The marker lanes are completely cleaved DNA.

FIGURE 3

DNAzyme activated by CEM‐Vc. (a) DVc1 was tested with different bacterial CEM. Fluorescence biosensor‐based activity assessment of DVc1 against different bacterial CEM. The corresponding pictures is insert. (b) Gel‐based (15% dPAGE with 8 M urea) cleavage activity assessment of DVc1 against different bacterial CEM. Pseudomonas aeruginosa (Pa), Vibrio shilonii (Vs), Vibrio harveyi (Vh), Escherichia coli (Ec), Staphylococcus aureus (Sa), Bacillus subtilis (Bs), and Vibrio anguillarum (Va).

FIGURE 4

Optimization of experimental conditions. (a) pH optimization. Cleavage was performed with CEM‐Vc [a‐h were considered to be statistically significant (p < .05), and the same letters were considered to be not significant (p > .05)]. (b) Effects of Na⁺ and Mg²⁺ concentration and (c) various divalent metal ions on the cleavage activity of DVc1 [a‐e were considered to be statistically significant (p < .05), and the same letters were considered to be not significant (p > .05)]. The Buffer/EDTA reaction contained 300 mM EDTA in 2 × Selection buffer. The bar and the dot mean: mean ± SD in the legend.

FIGURE 5

Sensitivity detection of DVc1. (a) Fluorescence values at different concentrations of Vc (Blank: selection buffer without Vc). (b) Analytical calibration curve of fluorescence values at 5.5 × 10⁵, 5.5 × 10⁶, and 5.5 × 10⁷ CFU/mL of Vc. (c) Gel cleavage assay at different concentrations of Vc (Blank: selection buffer without Vc). The dot means: mean ± SD in the legend.

FIGURE 6

Properties and molecular weight of the target. (a) Proteinase K‐treated CEM‐Vc showed no signal with DVc1. (b) Detection of the whole cell, cell lysate and CEM. Molecular weight assessment of target protein by (c) fluorescence and (d) gel assays.

FIGURE 7

Biosensor board design. (a) Increasing fluorescence signal of DVc1‐S at different concentrations of Vc within 1 h of sample addition. The corresponding pictures of the fluorescence signal are shown at the top. (b) Significant differences in the fluorescence intensity was significant difference with each concentration at 10 min and 20 min [a‐i were considered to be statistically significant (p < .05), and the same letters were considered to be not significant (p > .05)]. The bar means: mean ± SD in the legend.

FIGURE 8

(a) Fluorescence intensities and corresponding photographs of the sensor for the tested samples: Jellyfish silk, crab claw, oyster, and tap water [a‐c were considered to be statistically significant (p < .05), and the same letters were considered to be not significant (p > .05)]. (b) Fluorescence intensities of gradually diluted oyster samples. Analytical calibration curve of fluorescence values at 1.57 × 10², 1.57 × 10³, and 1.57 × 10⁴ CFU/mL of Vc is insert. The bar and the dot mean: mean ± SD in the legend.