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A nucleic acid detection system and its detection method and application

A nucleic acid and exonuclease technology, which is used in biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of harsh reaction conditions, high identification requirements, and poor specificity. specific effect

Active Publication Date: 2020-08-04
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the detection system contains exonuclease III, it can gradually catalyze the hydrolysis of double-stranded DNA to generate single nucleotides along the 3'→5' direction, so that the fluorescent group is separated from the DNA and free in the solution. Fluorescence is restored, and the quantitative detection of exonuclease III activity can be realized through the fluorescence intensity of the detection system, but the above-mentioned prior art steps are cumbersome and complicated, the sensitivity is not high, the specificity is poor, the reaction conditions are harsh, and the requirements for equipment are high , unable to complete the detection purpose concisely and efficiently
[0006] To sum up, the existing technology still has certain limitations for the development and application of fluorescent biosensors. Therefore, providing a simple and efficient fluorescent biosensor and its preparation method has broad application prospects and huge market value.

Method used

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  • A nucleic acid detection system and its detection method and application
  • A nucleic acid detection system and its detection method and application
  • A nucleic acid detection system and its detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] (1) Mix the nucleotide sequence such as the target DNA of SEQ ID NO.2 with the concentration of 5nM and the fluorescent probe of 10nM nucleotide sequence such as SEQID NO.1 and dissolve in 1×NE buffer 1 (New England Biolabs , buffer solution of Exonuclease III), react at a temperature of 25° C. for 10 min, then add 40 U of Exonuclease III to the reacted solution, and heat to inactivate the enzyme after 30 min of enzymatic digestion reaction;

[0059] (2) Compared with step (1), except that no target DNA is added, other conditions are the same as step (1);

[0060] (3) Compared with step (1), except that no fluorescent probe is added, other conditions are the same as step (1);

[0061] (4) Compared with step (1), except that exonuclease III is not added, other conditions are the same as step (1);

[0062] The post-reaction solutions obtained in steps (1), (2), (3) and (4), as well as separate target DNA and fluorescent probes are loaded separately for polypropylene gel ...

Embodiment 2

[0070] First, optimize the concentration of monolayer molybdenum disulfide nanosheets;

[0071] First, 10 nM of a fluorescent DNA probe with a nucleotide sequence such as SEQ ID NO.1 was dissolved in a Tris-HCl buffer solution, and then a fluorescence spectrometer was used to record its fluorescence spectrum at 505-650 nm at an excitation wavelength of 490 nm;

[0072] Add aliquots (10 μg / mL) of single-layer molybdenum disulfide nanosheets to the above solution nine times, and record the fluorescence spectrum of the tested sample for each addition. The results are shown in Figure 3(A) and Figure 3(B) ;

[0073] It can be seen from Figure 3(A) and Figure 3(B) that with the continuous increase of the concentration of monolayer molybdenum disulfide nanosheets, the fluorescence of the 10nM probe is gradually quenched, when the concentration of monolayer molybdenum disulfide nanosheets is higher than At 80 μg / mL, the fluorescence signal was almost no longer weakened, and the quenc...

Embodiment 3

[0075] Secondly, optimize the amount of exonuclease III enzyme and reaction time;

[0076] First, set up nine large experimental groups, the reaction system contains 0U, 10U, 20U, 30U, 40U, 50U, 60U, 70U and 80U of enzyme, and each experimental group includes two groups, namely the experimental group and The control group, the experimental group and the control group all contain three reaction tubes. The control group does not contain a nucleotide sequence such as the target NDA of SEQ ID NO.2, while the experimental group contains a nucleotide sequence such as the target NDA of SEQ ID NO.2. Target DNA, other conditions remain unchanged, signal-to-noise ratio data = reaction group ÷ control group;

[0077] Secondly, set up six large experimental groups, and the reaction times of adding enzymes into the system are 0min, 10min, 20min, 30min, 40min and 50min, respectively. Each large experimental group contains two groups, the experimental group and the control group, and the exp...

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Abstract

The invention provides a detection system of nucleic acid and a detection method and application thereof. The detection system comprises 30-50U of exonuclease III, fluorescence probe and 60-80 micrograms / mL of monolayer molybdenum disulfide nano-sheet with diameter of 0.5-2 microns and thickness of 0.5-2nm; by applying the monolayer molybdenum disulfide nano-sheet to the fluorescence detection system of exonuclease III, the cyclic amplification function of the exonuclease III and the quenching function of the molybdenum disulfide nano-sheet are played to the greatest extent by optimizing a reaction condition and procedures for the specific nucleotide sequence; various steps are in synergistic interaction, the sensitivity and the specificity of the detection system are improved, the operation is simple and convenient, concise and efficient, and extensive in application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid detection system and a detection method and application thereof. Background technique [0002] A biosensor (Biosensor) is a device that uses a biologically active substance to generate a signal, and converts the signal to a physical and chemical detector through a converter to display a readable signal. Biosensors include three parts: sensitive biological components (biological materials such as biological tissues, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc., biologically derived materials or bio-like materials), these sensitive biological components can be bioengineered To create, connect the two converters and detection elements (working with physical and chemical methods such as optical, piezoelectric, electrochemical, thermal or electromagnetic). [0003] Exonuclease III acts on double-stranded DNA, and gradually catalyzes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2563/107C12Q2521/319C12Q2565/607
Inventor 刘刚王乐乐李兰英闻艳丽李妍梁文罗超
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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