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Enzyme-assisted hairpin probe remodeled label-free colorimetric sensor based on target trigger and application of sensor

A colorimetric sensor and hairpin probe technology, applied in the field of nucleic acid visualization detection, to achieve the effect of reducing complexity, improving reaction speed and realizing recycling

Active Publication Date: 2020-03-31
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention proposes a label-free colorimetric sensor based on target-triggered enzyme-assisted hairpin probe remodeling and its application, which solves the problem of highly sensitive detection of Pax-5

Method used

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  • Enzyme-assisted hairpin probe remodeled label-free colorimetric sensor based on target trigger and application of sensor
  • Enzyme-assisted hairpin probe remodeled label-free colorimetric sensor based on target trigger and application of sensor
  • Enzyme-assisted hairpin probe remodeled label-free colorimetric sensor based on target trigger and application of sensor

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Embodiment 1

[0033] 1. Preparation of a label-free colorimetric sensor based on target-triggered enzyme-assisted hairpin probe remodeling. The preparation principle is shown in figure 1 , the preparation steps are as follows:

[0034] (1) DNA pretreatment: All DNA (hairpin probes HGP, LCP, and LGP) must be pretreated before use. The sequence of the hairpin probe HGP is shown in SEQ ID No.1, and its 5' end is modified by a Phosphate group; the sequence of LCP is shown in SEQ ID No.2, and the sequence of LGP is shown in SEQ ID No.3; the processing method is: put the microcentrifuge tube containing the DNA sequence in a centrifuge at 5000rpm, centrifuge for 3 min, and use Tris-EDTA buffer with a pH of 8.0 was diluted to 10 μM; DNA with a hairpin structure was heated at 95°C for 5 min, and then slowly cooled to room temperature to allow the probe to form a hairpin structure.

[0035] (2) Mix the same volume of 10 μM LGP and 10 μM LCP in a reaction tube, incubate for 2 h, and store at 4 °C for...

Embodiment 2

[0042] Specific detection by label-free colorimetric sensors

[0043] Considering the requirements of practicability, in order to evaluate the specificity of the method of the present invention, the standard solution of different concentrations of Pax-5 is replaced by a DNA solution containing different mismatched bases with a final concentration of 250 nM. Repeat the steps of Example 1, with reference to Example 1 Experimental steps The single-base mismatch sequence MT1 sequence is shown in SEQ ID No.4, the three-base mismatch sequence MT2 sequence is shown in SEQ ID No.5, and the five-base mismatch sequence MT3 sequence is shown in SEQ ID The NT sequence shown in No.6, the complete mismatch sequence is shown in SEQ ID No.7, and the detection is carried out, and the detection result is as follows Figure 4 As shown, to examine the specificity and selectivity of the method for specific targets, the single-base mismatch sequence (MT1), three-base mismatch sequence (MT2), five-b...

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Abstract

The invention provides an enzyme-assisted hairpin probe remodeled label-free colorimetric sensor based on target trigger and an application of the sensor. The invention also provides a method for visual detection of nucleic acid. According to the method, the nucleic acid is used as a target, a reaction is performed with the help of enzymes, G-quadruplex sequences that can produce colorimetric signals are formed through multiple ways, and the G-quadruplex sequences can be used for visual detection of the nucleic acid; the method controls the reaction conditions, a hairpin probe (HGP) opened bya target gene under the action of a T4 DNA ligase is connected with another double-stranded DNA (DGP) to complete remodeling, under the synergy of a DNA polymerase and a restriction enzyme, the targetDNA is recycled, and multiple channels for generating the G-quadruplex sequences are generated. According to the method, the enzymes are used to assist the DNA reaction to form a large amount of G-quadruplexes, and the G-quadruplexes can be used for detection by using an ultraviolet spectrophotometer, and can be used for visual detection by using naked eyes; and the detection method is convenient, the materials used in the method are commercialized, and the biological safety is high.

Description

technical field [0001] The invention belongs to the field of nucleic acid visualization detection, in particular to a label-free colorimetric sensor based on target-triggered enzyme-assisted hairpin probe remodeling and an application thereof. Background technique [0002] The highly sensitive detection of nucleic acids has great potential in the field of biomedical applications. DNA and RNA contain important information in the pathological characteristics and development of diseases, and can be used as important biomarkers for studying cancer pathogenesis, identifying infection and drug resistance. Acute lymphoblastic leukemia (ALL), as one of the most common human malignancies, has the highest prevalence among children. Pax-5 is the only paired-box (PAX) family member found in the hematopoietic system, which can be divided into Pax-5a, Pax-5b, Pax-5c, Pax-5d and Pax-5e. Among them, Pax-5a is the most important, and Pax-5 usually refers to Pax-5a. Pax-5 is very important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6825
CPCC12Q1/6886C12Q2600/156C12Q1/6825C12Q2525/301C12Q2521/501C12Q2521/101C12Q2521/301
Inventor 黎泓波蒲嘉美刘明彬赵卫华王素琴
Owner JIANGXI NORMAL UNIV
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