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

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

Active Publication Date: 2022-05-27
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
  • Double-head triple-helix nucleic acid probe and method for detecting chloramphenicol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of double-ended triple helix nucleic acid probe

[0050] In this embodiment, the steps of preparing a chloramphenicol-dependent double-ended triple helix nucleic acid probe solution 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 ultrapure water for 17.5 s, shake well, and place at room temperature for 15 min to obtain a chloramphenicol-dependent double-head triple-helix nucleic acid probe solution.

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

Embodiment 2

[0053] Example 2 Drawing standard curve

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

[0055] (1) Prepare standard solution of chloramphenicol

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

[0057] (2) Draw the standard curve

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

[0059] ②Use the chloramphenicol standard solution with chloramphenicol...

Embodiment 3

[0061] Embodiment 3 detects chloramphenicol in tap water

[0062] In the present embodiment, adding chloramphenicol in tap water and detecting the concentration of chloramphenicol, 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 turn.

[0064] (2) Detection of chloramphenicol sample solution

[0065] ①Take 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 the 1# sample solution to it, and place it at room temperature (25°C) for 27 minutes for recognition reaction, 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 solution in ste...

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Abstract

The invention discloses a double-head triple-helix nucleic acid probe and a method for detecting chloramphenicol, belonging to the field of antibiotic detection. The double-headed triple-helix nucleic acid probe of the present invention is formed by mixing a circular probe and a stem-shaped probe in a molar ratio of (2-4): 1 in a liquid environment with a pH value of 7.5±0.5, and then incubated at room temperature; The sequence of the probe is shown in SEQ ID NO.1, and the two ends of the circular probe are covalently linked to a fluorescent dyeing group and a fluorescent quenching group; the sequence of the stem probe is shown in SEQ ID NO.2. The double-headed triple-helical nucleic acid probe of the invention has the advantages of high sensitivity, high specificity, short detection time, wide detection range, etc., and has good application prospects.

Description

technical field [0001] The invention belongs to the field of antibiotic detection. Background technique [0002] 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 feed, it is convenient to administer, and it is the first choice for the treatment of typhoid and paratyphoid fever. [0003] The harm of chloramphenicol in my country is mainly reflected in animal-derived food, 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 reason for its residues in animal-derived foods. When chloramphenicol exceeds the standard food, it is enriched into the human body through the food chain, which in turn causes chloramphenicol blood drugs in the human body. Excessive content will not only lead to...

Claims

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Application Information

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Patent Type & Authority Patents(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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