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Mercury ion detection probe group based on exonuclease III, kit and mercury ion detection method

A technology of exonuclease and detection method, which is applied in the field of mercury ion detection and mercury ion detection probe group based on exonuclease III, which can solve the problems that complex samples cannot be applied, the detection system is complicated, and the cost of equipment and instruments is expensive.

Active Publication Date: 2018-01-16
HUNAN INSTITUTE OF ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most of the traditional detection methods are instrumental analysis methods, such as atomic absorption spectrometry, atomic emission spectrometry, etc. These methods are expensive in equipment and instruments, and the experimental process is cumbersome, which is not conducive to the development of on-site and rapid detection technologies.
In recent years, many exonucleases have been developed as an auxiliary means to amplify the signal of the enzyme cleavage cycle and apply it to the highly sensitive detection of metal ions (Biosensors and Bioelectronics, 2015, 68, 266-271; Analytical Chemistry, 2014, 86, 3108-3114; Sensors&Actuators: B.Chemical, 2017, 246:896-903), but generally the sensitivity is not high or the detection system is relatively complicated, and it can only be applied to water samples, and cannot be applied to complex samples in the biomedical field, such as urine. The field of application has been greatly limited

Method used

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  • Mercury ion detection probe group based on exonuclease III, kit and mercury ion detection method
  • Mercury ion detection probe group based on exonuclease III, kit and mercury ion detection method
  • Mercury ion detection probe group based on exonuclease III, kit and mercury ion detection method

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preparation example Construction

[0028] The preparation method of the probe is not particularly limited in the present invention, and it is enough to entrust a biological company well-known to those skilled in the art to carry out the synthesis.

[0029] The present invention also provides a mercury ion detection kit based on the probe set described in the above technical solution, and the kit includes the probe set described in the above technical solution and exonuclease III. In the present invention, the kit preferably also includes a Tris-HCl buffer, which is used as a reaction solvent; the kit preferably also includes heme, ABTS and H 2 o 2 , the heme, ABTS and H 2 o 2 The G-quadruplex formation sequence dissociated due to the presence of mercury ions in the detection process can be realized, thereby realizing the detection of mercury ions.

[0030] The present invention also provides a mercury ion detection method based on the probe set described in the above technical solution or the kit described i...

Embodiment 1

[0046] Design of Hairpin DNA Probe and Mercury Ion Recognition Probe

[0047] The sequence structure of the hairpin DNA probe is as follows (5'-3'):

[0048] 5'-CGAAAAGTGT GGGTAGGGCGGGTTGGG ACCCTACCCACACTTTTCGACTTTTCG-3'

[0049] Among them, the bold underlined partial sequence (GGGTAGGGCGGGTTGGG) is the G-quadruplex forming sequence, the 5' end partial sequence of the G-quadruplex forming sequence and its 5' upstream sequence (CGAAAAGTGTGGGTAGGG) and the G-quadruplex forming sequence The downstream sequence at the 3' end (CCCTACCCACACTTTTCG) is a complementary sequence, forming a stem-like region. The 3' end of the hairpin DNA probe contains two sets of repeating sequences (ACTTTTCG), one group is in a single-stranded free state, and the other group is in the stem-like region. The state of hybridization is associated with the signal amplification cycle 1 and cycle 2 processes, respectively.

[0050] The mercury ion recognition probe sequence structure is as follows (5'-3'...

Embodiment 2

[0054] Detection of different concentrations of mercury ions in water samples

[0055] Take a sterilized 0.5mL centrifuge tube, respectively take 25 μL of the hairpin DNA probe (0.5 μM) in Example 1, and 25 μL of the mercury ion recognition probe (0.2 μM) in Example 1 in 20 mM Tris-HCl buffer Solution (pH=7.4, 70mMNaCl, 10mMMgCl 2 , 20mM KCl) to 90°C for 10-20min, then gradually cooled to room temperature (temperature setting: 70°C for 10-15min, 50°C for 25-30min, 30°C for 10-15min, take it out at 20-25°C Leave it for 20-30 minutes). Add 25 μL of detection sample (mercury ion concentration between 0.001pmol / L to 1pmol / L) and 10U Exo III to the above solution, stir evenly, and then react at 25°C for 60min to make it enter a spontaneous circulation process and generate a large amount of G - a quadruplex forming sequence. Treat at 80°C for 10 minutes to inactivate the Exo III enzyme. After the reaction was completed, 0.5 μL of 0.4 μmol / L hemin was added and reacted in the dar...

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Abstract

The invention relates to a mercury ion detection probe group based on exonuclease III, a kit and a mercury ion detection method and belongs to the technical field of heavy metal detection. The mercuryion detection probe group based on the exonuclease III, provided by the invention, comprises a hairpin DNA (Deoxyribonucleic Acid) probe and a mercury ion recognition probe. The probe group and the kit, provided by the invention, can realize ultrahigh-sensitivity detection of mercury ions in a water body sample and a urine sample, have strong specificity and high stability and are not influencedby other metal ions.

Description

technical field [0001] The invention relates to the technical field of heavy metal detection, in particular to a mercury ion detection probe group, a kit and a mercury ion detection method based on exonuclease III. Background technique [0002] Hg 2+ Belonging to heavy metals, they and their compounds are harmful to organisms and are very difficult to degrade in the natural environment. At the same time, animals cannot excrete them smoothly through the excretory system. Through the enrichment of the food chain, they can be transferred from the organism to the human body, and eventually Lead to a series of diseases in the human body, and may even lead to the generation of malignant tumors. Most of the traditional detection methods are instrumental analysis methods, such as atomic absorption spectrometry, atomic emission spectrometry, etc. These methods are expensive in equipment and equipment, and the experimental process is cumbersome, which is not conducive to the developm...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张何王青傅昕张洁
Owner HUNAN INSTITUTE OF ENGINEERING
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