A colorimetric method for the detection of tobramycin based on double-strand displacement and three-way DNA structure
A technology for tobramycin and structure detection, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of complicated personnel training and sample preparation, long test cycle, expensive equipment, etc., to expand the detection range , Improve the detection sensitivity, the effect of simple operation
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Embodiment 1
[0036] Example 1 Drawing of tobramycin concentration standard curve
[0037] T1 and T2 were mixed at the same concentration, denatured at 95 °C for 5 min, and then renatured at 37 °C for 120 min. 2 μL (10 μmol·L -1 ) double strands were mixed with 4 μL of tobramycin solutions of different concentrations and incubated at 37°C for 30 min. To the above mixture, add 2 μL (10 μmol·L -1 ) primer 1, 2 μL (10 μmol·L -1 ) primer 2, 5 U Nt.BstNBI nickase, 8 U Bsm DNA polymerase, and 2 μL (10 mmol·L -1 ) free deoxyribonucleoside triphosphate, 1× buffer (100 mmol L -1 NaCl, 50 mmol·L -1 Tris-HCl, 10 mmol·L -1 MgCl 2 , 0.1 mg·mL -1 BSA), incubate at 55°C for 120 min after mixing to generate a large number of reporter probes, inactivate at 75°C for 10 min, and store at 4°C for later use.
[0038] Combine 10 μL of S1, 10 μL of S2, and 10 μL of S3 (all 10 -6 mol·L -1 ) were mixed, heated at 95 °C for 5 min, and then gradually cooled to room temperature. After this, 30 μL of t...
Embodiment 2
[0040] Example 2 Determination of tobramycin content in actual water samples
[0041] In order to further verify the accuracy of this method in the determination of tobramycin content in actual samples, Taihu Lake water without pretreatment was selected to dilute tobramycin to different concentrations. The reaction was carried out in exactly the same way as the standard sample of tobramycin, and the absorbance value of the solution at 420 nm was read by the UV-vis spectrophotometer in the obtained reaction solution, and the concentration of tobramycin could be calculated by substituting it into the standard curve.
[0042] The specific samples and test results are shown in Table 1.
[0043] Table 1
[0044]
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