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Double-head triple-helix nucleic acid probe and method for detecting chloramphenicol by using same

A nucleic acid probe and triple helix technology, applied in the field of antibiotic detection, can solve the problems of reducing the stability and reliability of the detection method and low sensitivity, and achieve the effects of wide sample range, high sensitivity and improved affinity

Active Publication Date: 2020-10-30
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, aptamers are usually used directly in methods such as fluorescence or colorimetric analysis, and the sensitivity is low
In order to improve the sensitivity of aptamer probes, nucleic acid amplification methods and nanomaterial nucleic acid amplification methods are usually used to make the probe design more complicated. However, the introduction of nanomaterials often reduces the stability and reliability of the detection method.

Method used

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  • Double-head triple-helix nucleic acid probe and method for detecting chloramphenicol by using same
  • Double-head triple-helix nucleic acid probe and method for detecting chloramphenicol by using same
  • Double-head triple-helix nucleic acid probe and method for detecting chloramphenicol by using same

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Experimental program
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Effect test

Embodiment 1

[0049] The preparation of embodiment 1 double head triple helix nucleic acid probe

[0050] In this embodiment, the preparation of chloramphenicol-dependent double-ended triple-helical nucleic acid probe solution, the steps are as follows:

[0051] Add 7 μL of 10 μM circular probe stock solution and 3.5 μL of 10 μM circular probe stock solution to 3.5 μL of buffer (330 mM Tris-HCl, 660 mM KCl, 100 mM MgCl 2 , pH 7.5), add 17.5 μl of ultrapure water, shake well, and stand at room temperature for 15 minutes to obtain a chloramphenicol-dependent double-ended triple-helix nucleic acid probe solution.

[0052] Repeat the above operations to prepare several chloramphenicol-dependent double-ended triple-helical nucleic acid probe solutions, each 31.5 μL.

Embodiment 2

[0053] Embodiment 2 draws standard curve

[0054] In the present embodiment, the standard solution curve is drawn, and the steps are as follows:

[0055] (1) prepare chloramphenicol standard solution

[0056] Prepare chloramphenicol standard solutions with chloramphenicol concentrations of 0, 0.1, 0.5, 1, 20, 50, 100, 150, 200, 250, 300, and 500 nM, respectively.

[0057] (2) Draw a standard curve

[0058]① Take a portion (31.5 μL) of the chloramphenicol-dependent double-headed triple-helix nucleic acid probe solution prepared in Example 1, add 3.5 μL chloramphenicol standard solution with a chloramphenicol concentration of 0 nM to it, and place it at room temperature (25° C. ) placed for 27min to carry out the recognition reaction, then measure the fluorescence intensity of the reaction mixture, and record the peak value of the fluorescence intensity corresponding to the chloramphenicol standard solution;

[0059] ② Use chloramphenicol standard solutions with chloramphenic...

Embodiment 3

[0061] Embodiment 3 detects chloramphenicol in tap water

[0062] In this embodiment, chloramphenicol is added to tap water and the concentration of chloramphenicol is detected, the steps are as follows:

[0063] (1) Take tap water to prepare chloramphenicol sample solutions with chloramphenicol concentrations of 0, 5, 20, 50, 100, and 200 nM, respectively, and record them as 1# to 6# sample solutions in sequence.

[0064] (2) Chloramphenicol sample solution detection

[0065] ① Take a portion (31.5 μL) of the chloramphenicol-dependent double-headed triple-helix nucleic acid probe solution prepared in Example 1, add 3.5 μL of 1# sample solution to it, and place it at room temperature (25° C.) for 27 minutes to carry out the recognition reaction, and then Measure the fluorescence intensity of the reaction mixture, and record the peak fluorescence intensity corresponding to the chloramphenicol standard solution;

[0066] ②Use 2#~6# sample solution in turn to replace 1# sample ...

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Abstract

The invention discloses a double-head triple-helix nucleic acid probe and a method for detecting chloramphenicol by using the same, which belong to the field of antibiotic detection. The double-head triple-helix nucleic acid probe is formed by mixing an annular probe and a stem-shaped probe according to a molar ratio of (2-4): 1 in a liquid environment with a pH value of 7.5 + / -0.5 and incubatingat room temperature, the sequence of the annular probe is shown as SEQ ID NO.1, and two ends of the annular probe are respectively connected with a fluorescence staining group and a fluorescence quenching group through covalent bonds; the sequence of the stem-shaped probe is shown as SEQ ID NO. 2. The double-head triple-helix nucleic acid probe has the advantages of high sensitivity, high specificity, short detection time, wide detection range and the like, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of antibiotic detection. Background technique [0002] Chloramphenicol (chloramphenicol, CAP) is a broad-spectrum antibiotic produced by Streptomyces, which has many advantages, such as low price, high broad-spectrum, good drug stability, etc., and because chloramphenicol can be directly added to In the feed, it is convenient to administer, and it is the first choice for the treatment of typhoid and paratyphoid. It is often used in the treatment of rotten tail disease, red fin disease and vibriosis in fish farming. [0003] The harm of chloramphenicol in my country is mainly reflected in food of animal origin, and the abuse and pollution of antibiotics caused by it has gradually become a hot issue of food safety. The abuse of chloramphenicol in the breeding process is the main cause of its residue in animal-derived food. After eating food that exceeds the standard of chloramphenicol, it enters the human body through ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N21/64
CPCG01N33/9446G01N33/53G01N21/6428G01N21/6486G01N2021/6432
Inventor 林锋张勇邓锐杰曾珍
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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