Fluorescent biosensor for detecting ochratoxin A as well as preparation method and application thereof
A biosensor, ochratoxin technology, applied in the field of biosensors, can solve the problems of long detection period, low specificity and sensitivity, and achieve the effect of fast detection speed, easy operation and short detection cycle
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Embodiment 1
[0068] Example 1 Preparation of Circular Template and Composite Probe
[0069] Prepared with 50 mM Tris-HCl, 10 mM MgCl 2 , T4 DNA Ligase Reaction Buffer with 10 mM DTT and 1 mM OTA. Formulated with 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 140 mM NaCl, 1 mM KCl, 1 mM MgCl 2 , 1mM CaCl 2 , PBS buffer solution of pH=7.4.
[0070] (1) Mix 42 μL sterilized water, 6 μL linear template (100 μM), 6 μL ligation probe (100 μM) and 6 μL 10× T4 DNA ligase buffer, denature at 95°C for 5 min, then Slowly cool down to room temperature to complete the hybridization, then add 3 μL of T4 DNA ligase (60 U / μL) to the reaction system, and react at 16°C for 20 hours; after that, the reaction system is placed in a water bath at 65°C for 15 minutes , inactivate the T4 DNA ligase in the system.
[0071] (2) Add 1 μL of exonuclease Ⅰ (20 U / μL) and 2 μL of exonuclease Ⅲ (100 U / μL) to the above reaction system and react at 37°C for 2 h; then put the reaction system at 85°C Heated in a water bath ...
Embodiment 2
[0073] Embodiment 2 Fluorescence intensity changes with endonuclease IV concentration
[0074] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:
[0075] (1) Mix 3 μL composite probe I (0.1 μM), 3 μL composite probe II (0.5 μM), 3 μL HP2 (0.5 μM), 3 μL HP3 (0.5 μM), 3 μL phi29 DNA polymerase (1 U / μL), 3 μL endonuclease IV (concentrations are 0.1 U / μL, 0.2 U / μL, 0.25 U / μL, 0.5 U / μL, 0.75 U / μL, 1 U / μL), 3 μL NMM ( 2.4 μM), 2 μL dNTP (1mM), in 3 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH7.5) and then add OTA (100 ng / μL) respectively, and then react at a constant temperature of 37°C for 120 min after mixing;
[0076] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 399 nm, the emission wavelength is 610 nm, and the detection range is 560 nm-640 nm, and the fluorescence signal changes are read....
Embodiment 3
[0080] Embodiment 3 Fluorescence intensity changes with the concentration of phi29 DNA polymerase
[0081] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:
[0082] (1) Mix 3 μL composite probe I (0.1 μM), 3 μL composite probe II (0.5 μM), 3 μL HP2 (0.5 μM), 3 μL HP3 (0.5 μM), 3 μL phi29 DNA polymerase (concentrations respectively 0.1 U / μL, 0.2 U / μL, 0.5 U / μL, 1 U / μL, 1.5 U / μL, 2 U / μL), 3 μL endonuclease IV (0.5 U / μL), 3 μL NMM ( 2.4 μM), 2 μL dNTP (1 mM), in 3 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5), add OTA (100 ng / μL) respectively after mixing, and react at a constant temperature of 37°C for 120 min after mixing;
[0083] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 399 nm, the emission wavelength is 610 nm, and the detection range is 560 nm-640 nm, and the fluorescence sign...
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