Method for eliminating dtt interference when hydrogen peroxide eliminates nucleic acid constant temperature amplification and detects mercury ions

A constant temperature amplification detection, hydrogen peroxide technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of low detection sensitivity of trace mercury ions, reduced effect of mercury ions and thymine, affecting nucleic acid problems such as the application of constant temperature amplification methods to achieve the effect of high experimental cost

Inactive Publication Date: 2020-01-14
XI AN JIAOTONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The existence of biothiol molecules will compete with thymine to capture trace mercury ions in the system, resulting in a decrease in the interaction between mercury ions and thymine, resulting in low detection sensitivity of trace mercury ions, which affects the nucleic acid constant temperature amplification method. detection applications

Method used

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  • Method for eliminating dtt interference when hydrogen peroxide eliminates nucleic acid constant temperature amplification and detects mercury ions
  • Method for eliminating dtt interference when hydrogen peroxide eliminates nucleic acid constant temperature amplification and detects mercury ions
  • Method for eliminating dtt interference when hydrogen peroxide eliminates nucleic acid constant temperature amplification and detects mercury ions

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Effect test

Embodiment 1

[0027] The method that hydrogen peroxide eliminates DTT interference when nucleic acid isothermal amplification detects mercury ions comprises the following steps:

[0028] (1) Mix the recognition sequences containing mismatched sites and primers and templates that trigger constant temperature amplification of nucleic acids at a concentration of 100nM in equal volumes. After the recognition sequences combine with mercury ions in the mercury-containing solution to be tested, they can form a polymerase-recognizable sequence. stable hybrid structure;

[0029] (2) Mix and react the stable hybrid structure after binding mercury ions with hydrogen peroxide solution, amplification substrate, DNA polymerase KF, SYBR Green I, and amplification reaction buffer at a reaction temperature of 37°C for 15 minutes; The concentration of each component in the mixture is: primer concentration 100nM, template concentration 100nM, hydrogen peroxide concentration 0.01%, amplification substrate 40μM...

Embodiment 2

[0040] The remaining steps are the same in this embodiment, the difference is that the concentration of hydrogen peroxide in the mixture described in step (2) is 0.5%.

[0041] The detection results showed that compared with the reaction system without adding hydrogen peroxide, the fluorescence signal was enhanced by nearly 5 times.

Embodiment 3

[0043] The remaining steps are the same in this embodiment, the difference is that the concentration of hydrogen peroxide in the mixture described in step (2) is 0.2%.

[0044] The detection results showed that compared with the reaction system without adding hydrogen peroxide, the fluorescence signal was enhanced by nearly 6 times.

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Abstract

Hydrogen peroxide eliminates the DTT interference method of nucleic acid constant temperature amplification and detection of mercury ions. The recognition sequence containing the mismatch site and the primer and template that triggers nucleic acid constant temperature amplification is mixed at a concentration of 100nM in equal volumes, and the recognition sequence is combined with the target to be detected. Mercury ions in mercury-containing solution can then form a stable hybrid structure that can be recognized by polymerase; mix the stable hybrid structure with hydrogen peroxide solution, amplification substrate, DNA polymerase KF, SYBR GreenⅠ, and amplification reaction buffer Under the action of polymerase, the amplification reaction is triggered to generate double-stranded DNA; SYBR Green I can specifically bind to double-stranded DNA molecules and emit fluorescent signals at the same time; the amplified sequence is characterized by melting curve and agarose gel electrophoresis , it is confirmed that hydrogen peroxide eliminates the interference of DTT, and the invention eliminates the interference of DTT on the detection of trace mercury ions, has good system compatibility, is easy to operate, and does not depend on complex equipment.

Description

technical field [0001] The invention belongs to the trace detection of mercury ions in the field of biotechnology, and in particular relates to a method for eliminating DTT interference by hydrogen peroxide during constant temperature amplification of nucleic acid and detection of mercury ions. [0002] technical background [0003] Heavy metal ions have high stability in the environment and are difficult to be degraded by microorganisms. Once they enter the environment, they are difficult to be repaired naturally, and can be continuously enriched and passed on in the ecosystem. Even a small amount of ingestion can cause great toxicity, which is a major threat to ecological balance and human health. As a typical representative of heavy metal ion pollution, the accumulation of mercury ions in biological tissues causes DNA damage, affects ligand-receptor interactions, destroys the immune system, and causes a series of diseases, such as brain damage, kidney failure, various cogn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/101C12Q2563/107C12Q2565/125
Inventor 赵永席赵越王亚玲袁慧董绘阳白凯王芳霞杨卫军魏帅刘华青
Owner XI AN JIAOTONG UNIV
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