Molecule detection method and detection kit based on DNA self-assembly and G tetramers

A technology for detection kits and detection methods, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of troublesome storage of enzymes, increased costs and operating steps, low detection sensitivity of colorimetric methods, etc., and achieve economical Inexpensive, highly sensitive, and easy-to-operate effects

Active Publication Date: 2015-06-24
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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Problems solved by technology

However, the detection sensitivity of the colorimetric method based on G tetramer is low, and the sensitivity needs to be further improved.
The traditional signal amplification technology is mainly PCR and a series of signal amplification technologies relying on tool enzymes, but the use of enzymes not only increases the cost and operation steps, but also the storage of enzymes is more troublesome, so it is necessary to develop a tool-free enzyme signal amplification technology. technology to improve detection sensitivity

Method used

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  • Molecule detection method and detection kit based on DNA self-assembly and G tetramers
  • Molecule detection method and detection kit based on DNA self-assembly and G tetramers
  • Molecule detection method and detection kit based on DNA self-assembly and G tetramers

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Embodiment 1

[0073] The reagents used are as above Bisphenol A Detection Kit As mentioned, the specific detection method is as follows:

[0074] 1) Dissolve DNA1, DNA2, DNA3, DNA4, DNA5, and DNA6 in Tris-HCl buffer (20 mM, pH 7.4, containing 200 mM NaCl and 50 mM KCl);

[0075] 2) Mix 100 nM DNA1 and 400 nM DNA2, and react at room temperature for 20 minutes to form a DNA1-DNA2 mixture; the excess of DNA2 is to ensure that all DNA1 is blocked;

[0076] 3) Add different concentrations of bisphenol A to the DNA1-DNA2 mixture and react at room temperature for 45 minutes;

[0077] 4) Add 1 mM DNA3 and DNA4 and react at room temperature for 1 hour;

[0078] 5) Mix 1.2 mM DNA5 and 1 mM DNA6 and react at room temperature for 30 minutes to form a DNA5-DNA6 mixture; the excess of DNA5 is to ensure that all DNA6 is blocked. Then add to the reaction solution in step (3), and react at room temperature for 30 minutes;

[0079] 6) Add 0.3 mM hemin (hemin) and react at room temperature for 30 minutes...

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Abstract

The invention discloses a molecule detection method and a detection kit based on DNA self-assembly and G tetramers. Molecules and an aptamer interact to start a DNA self-assembly mediated strand displacement reaction, stem-loop structured DNAs are opened continuously and a closed G tetramer sequence are activated, thus generating a large quantity of G tetramer structures with activity. After being combined with hemin, the G tetramers have HRP catalytic activity and can be used for catalytically oxidizing TMB to generate a blue substrate so that the result can be seen with naked eyes. The molecule concentration is directly related to the depth of blue color. The molecule detection method is simple to operate; the whole reaction can be completed at room temperature without use of a tool enzyme; the sensitivity is relatively high, the detection limit for bisphenol A is 1pg/mL; the reaction has very good specificity; the detection result can be seen with naked eyes; no detection instrument is needed; the molecule detection method and the detection kit can be used for quick field analysis on molecules, especially on bisphenol A.

Description

technical field [0001] The invention relates to a molecular detection method based on ribozyme, in particular to a molecular detection method based on DNA self-assembly and G tetramer and a detection kit based on the method. Background technique [0002] There are many harmful molecules in the environment, and it is very practical to detect these harmful small molecules quickly and at low cost. [0003] Bisphenol A (Bisphenol A, BPA), the chemical name is 2,2-bis(4-hydroxyphenyl)propane, which is mainly used in the synthesis of various polymer materials such as epoxy resin and polycarbonate, and is widely used in production Various plastic products. Bisphenol A penetrates into the body mainly through food packaging materials, drinking cups, water supply pipes, baby bottles and plastic films, etc., and can be enriched in organisms and is not easily degraded. Even very low concentrations of BPA can have a series of adverse effects on the endocrine system, immune system, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈俊华周顺桂温俊林
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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