Homogeneous colorimetric bioanalysis method for detecting kanamycin and application of homogeneous colorimetric bioanalysis method
A kanamycin and biological analysis technology, applied in the field of biological analysis, can solve the problems of low analysis efficiency, cumbersome operation, and high analysis cost, and achieve the effects of high analysis sensitivity, avoiding cumbersome operation and high sensitivity
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Embodiment 1
[0030] A kind of homogeneous colorimetric bioanalysis method that detects kanamycin of the present embodiment comprises the following steps:
[0031] (1) Pretreatment of DNA strands
[0032] Take 6 PV tubes and number them #1, #2, #3, #4, #5, and #6 respectively, add 190 μL of HEPES buffer with a concentration of 20 mM pH=7.4 to #1, #2, and #5 PV tubes solution;
[0033] Next, add 10 μL of 20 μM containing Zn to #1 PV tube 2+ The Kana nucleic acid aptamer S1 connecting the nuclease base sequence, the base sequence of the nucleic acid aptamer S1 is 5'-ACC TCA AGA CCT TTC GCT AGA TGT CCAAGT GGT CTT GAG GTT CTG-3';
[0034] Then add 10 μL of a sequence S2 complementary to the aptamer S1 at a concentration of 20 μM to the #2 PV tube. The base sequence of the S2 is 5'-CAG AAC CTG ACA TCT AGT TTT-3';
[0035] Then add 10 μL DNA probe A1 with a concentration of 80 μM and 15 A bases at the 3'-end to #3 PV tube. The base sequence of the DNA probe A1 is 5'-CGT CCA AGA TTT TTA AAA AAA...
Embodiment 2
[0048] The detection of kanamycin content in the milk powder sample of embodiment 2
[0049] According to the working curve obtained in Example 1, the content of kanamycin in the milk powder sample was detected. Weigh 1 g of commercially available milk powder and dissolve it in 5 mL of 20 mM pH=7.4 HEPES buffer solution, which contains 100 mM NaCl, 10 mM MgCl 2 and 1mM ZnCl 2, take 100 μL of the dissolved milk powder solution, add 20% acetic acid to it to adjust the pH to 4.6, centrifuge after 20 minutes to remove the coagulated protein and fat in the sample, and then filter the sample solution with a 0.22 μm filter membrane, and Readjust the sample solution to pH=7.4;
[0050] Take 10 μL of the treated sample solution, add 45 μL containing 100mM NaCl, 10mM MgCl 2 and 1mM ZnCl 2 20mM HEPES buffer solution with pH=7.4. Then add 10 μL of 1 μM hairpin DNA solution S1, 10 μL of 1 μM single-stranded DNA solution S2, and 10 μL of 1 μM Zn after the annealing treatment in step (1...
Embodiment 3
[0055] The detection of kanamycin content in the honey sample of embodiment 3
[0056] According to the working curve obtained in Example 1, the content of kanamycin in the honey sample was detected. Weigh 2 g of commercially available honey and dissolve it in 4 mL of 20 mM pH=7.4 Tris-HCl buffer solution containing 100 mM NaCl, 10 mM MgCl 2 and 1mM ZnCl 2 ; Then filter the sample solution with a 0.22 μm filter membrane; take 10 μL of the filtered sample solution, add 45 μL containing 100 mM NaCl, 10 mM MgCl 2 and 1mM ZnCl 2 20mM HEPES buffer solution with pH=7.4. Then add 10 μL of 1 μM hairpin DNA solution S1, 10 μL of 1 μM single-stranded DNA solution S2, and 10 μL of 1 μM Zn after the annealing treatment in step (1). 2+ Link 1 / 2 substrate strand L1 of nuclease, 10 μL 1 μM as Zn 2+ Connect the 3'-end phosphoimidazole-functionalized L2 of the nuclease 1 / 2 substrate chain and 5 μL 4U / μL exonuclease III, and vortex and mix at 37°C for 150 minutes; then add 50 μL GNP1 to ea...
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