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Method for detecting kanamycin

A kanamycin and detection method technology, applied in the field of sensors, can solve problems such as complex instrument operation, and achieve the effects of improving sensitivity, mild reaction conditions and accurate detection

Inactive Publication Date: 2016-09-07
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a colorimetric sensor for detecting kanamycin aiming at the shortcomings of complex instrument operation and the need for professional operators in the existing detection method

Method used

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  • Method for detecting kanamycin

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preparation example Construction

[0038] The preparation method of described biosensor comprises the following steps:

[0039] (1) Specific binding of target and hairpin;

[0040] (2) Amplify the signal of the detected specific sequence;

[0041] (3) Perform colorimetric detection on the sequence.

[0042] In the preparation method, it is preferable to react the target substance with the hairpin, and the operation steps are as follows: mix 2 μL of the target substance kanamycin with 2 μL of the hairpin, and incubate at 37° C. for 0.5 h.

[0043] In the preparation method, the process of specifically amplifying the detected signal is preferably as follows:

[0044] (2) Take out the mixed solution incubated in (1), add primers, dNTPs, phi29DNA polymerase and Nt.Alwi endonuclease to the mixed solution, shake for 30s, and incubate in a 37°C incubator for 1h;

[0045] In the preparation method, the addition of the enzyme is carried out at low temperature to ensure the activity of the enzyme.

[0046] (3) Mix th...

Embodiment 1

[0051] The main steps of probe hybridization are as follows:

[0052] a. Add 2 μL of PHI29 buffer to the centrifuge tube (buffer is the supporting buffer for purchasing PHI29 DNA polymerase);

[0053] b. Add 8 μL of sterilized water into the centrifuge tube and shake evenly;

[0054] At this time, we will melt the chain again:

[0055] a. Add in order according to the number of sterilized water required to dissolve one O.D corresponding to the HAP chain;

[0056] b. Put the probe chain added with sterilized water into the centrifuge, set at 3000r / min, and centrifuge for 30 seconds;

[0057] c. Take out the centrifuged HAP chain, add 2 μL of kanamycin, tetracycline, and streptomycin to each centrifuge tube, and shake evenly.

[0058] d. Add 2 μL primers, 2 μL dNTPs, 2 μL phi29 DNA polymerase and 2 μL Nt.Alwi endonuclease to each centrifuge tube in turn, and incubate in a 37-degree incubator for one hour;

[0059] e. Mix 2 μL AP and 2 μL PP and add to each centrifuge tube, a...

Embodiment 2

[0062] The main steps of restriction enzyme optimization are as follows:

[0063] a. Add 2 μL of DNTP and 2 μL of Phhi29 DNA polymerase to the centrifuge tube where the target substance was added, and then add 1 μL of Nt.Alwi endonuclease, 1.5 μL of Nt.Alwi endonuclease, 2 μL of Nt.Alwi endonuclease and 2.5 μL of Nt to each centrifuge tube .Alwi endonuclease, 3μL Nt.Alwi endonuclease;

[0064] b. After shaking evenly, incubate at 37°C for 1 hour;

[0065] The following describes the reaction that occurs in a homogeneous solution, and the main steps in a homogeneous reaction:

[0066] c. Add sterilized water, 10× buffer, HAP (2 μL), kanamycin (2 μL), phi29 DNA polymerase (2 μL), dNTPs (2 μL) into the centrifuge tube, and shake for 30 seconds.

[0067] d. Add different concentrations of Nt.Alwi endonuclease to the mixed solution in the centrifuge tube in turn;

[0068] e. Incubate the mixed solution in (d) in an incubator at 37°C for 1 hour.

[0069] f. Add the hybrid soluti...

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Abstract

The invention provides a method for detecting kanamycin. The kanamycin is detected by using double restriction enzyme cleavage sites and color change caused by G-tetrad. The preparation method specifically comprises the following steps: carrying out specific binding on a hair pin and kanamycin; adding a color developing agent into a homogeneous phase reaction mixed liquor for display detection. According to the method, high specific detection for the target object kanamycin is realized by using the oxidability of the G-tetrad; the effect of signal amplification is reached by using nucleic acid tool enzymes.

Description

technical field [0001] The invention relates to the technical field of sensors, in particular to a colorimetric sensor for detecting kanamycin. Background technique [0002] Kanamycin is a protein biosynthesis inhibitor that causes mRNA code misreading by binding to 30S ribosomes. Bacteria can become resistant if they produce an enzyme that destroys kanamycin. Kanamycin resistance plasmids are often used in molecular cloning as selection genes or marker genes. Kanamycin is an aminoglycoside antibiotic. For most Enterobacteriaceae such as Escherichia coli, Klebsiella, Enterobacter, Proteus, Shigella, Salmonella, Citrobacter, Providencia, Yersinia Bacteria, etc. have good antibacterial effect; Haemophilus influenzae, Brucella, Neisseria meningitidis, Neisseria gonorrhoeae, etc. are also mostly sensitive to this product. Kanamycin also has a certain effect on Staphylococcus (methicillin-sensitive strain) and Mycobacterium tuberculosis, but is invalid against Pseudomonas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2525/205C12Q2537/1376C12Q2521/301C12Q2525/301C12Q2565/607
Inventor 王玉崔雪君黄加栋刘素王虹智郭玉娜
Owner UNIV OF JINAN
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