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Method for detecting target miRNA based on G-quadruplex molecular beacon double-enzyme cascade isothermal amplification

A purpose and polymerase technology, applied in the field of biomedicine, can solve the problem that a single miRNA cannot provide clear results for early cancer diagnosis and treatment monitoring, and achieve high accuracy, good specificity, and high sensitivity

Active Publication Date: 2020-12-15
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But most cascade enzyme amplification strategies usually require several separate steps to accommodate the different reaction conditions required for multiple enzymes to participate in the complex reaction environment
Furthermore, a single miRNA is often associated with multiple diseases, thus the detection and analysis of a single miRNA cannot provide the unambiguous results required for reliable early cancer diagnosis and treatment monitoring
Given the complexity of implementing parallel analysis protocols required to detect multiple miRNAs, few amplification protocols developed to date are capable of simultaneous detection of multiple miRNAs in a single system

Method used

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  • Method for detecting target miRNA based on G-quadruplex molecular beacon double-enzyme cascade isothermal amplification
  • Method for detecting target miRNA based on G-quadruplex molecular beacon double-enzyme cascade isothermal amplification
  • Method for detecting target miRNA based on G-quadruplex molecular beacon double-enzyme cascade isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Establishment of a method for detecting miRNA using G quadruplex molecular beacon (G4MB) dual-enzyme cascade isothermal amplification

[0070] After a lot of experiments, the inventors of the present invention have established a method for detecting miRNA using G4MB double-enzyme cascade isothermal amplification. The chain body structure is released, and the primer has the opportunity to combine with the primer at the 3' end of G4MB; under the action of the primer and Bsu DNA polymerase, a double-stranded hybrid structure is synthesized using the opened G4MB as a template, and replaces the original target miRNA; The substituted miRNA can bind to other G4MBs and participate in the next cycle. In this cycle, a single miRNA can trigger the restoration of multiple G4MBs signaling, achieving a round of amplification of miRNA signaling. At the same time, once Bsu DNA polymerase catalyzes the synthesis of hybrid double strands, Lambda exonuclease can gradually dige...

Embodiment 2

[0088] The feasibility analysis of the method that embodiment 2, embodiment 1 establish

[0089] In this example, G4MB-21 was selected as the model probe, and the feasibility of detecting the target miRNA-21 by the biosensor was explored according to the method established in Example 1.

[0090] The fluorescence emission spectra of the miRNA detection system were obtained under different conditions according to the method established in Example 1 (in step 3 specifically: incubate at 37° C. for 30 min).

[0091] Condition a: The reaction system is 100uL, which is composed of reaction buffer and G4MB-21; the concentration of G4MB-21 in the reaction system is 100nM.

[0092] Condition b: the reaction system is 100uL, which is composed of reaction buffer, G4MB-21 and miRNA-21; in the reaction system, the concentration of G4MB-21 is 100nM, and the concentration of miRNA-21 is 10nM.

[0093] Condition c: The reaction system is 100uL, consisting of reaction buffer, G4MB-21, miRNA-21...

Embodiment 3

[0098] Embodiment 3, sensitivity analysis

[0099] The fluorescence emission spectrum of the miRNA detection system was obtained under the reaction system according to the method established in Example 1 (in step 3 specifically: incubate at 37° C. for 30 min). The reaction system is 100uL, consisting of reaction buffer, G4MB-21, miRNA-21, Bsu DNA polymerase (5U), Lambda exonuclease (10U) and ribonuclease inhibitor (40U); in the reaction system, The concentration of G4MB-21 was 100 nM, and the concentration of miRNA-21 was 0 zM, 100 zM, 1 aM, 10 aM, 100 aM, 1 pM, 10 pM, 100 pM, 1 nM or 10 nM.

[0100] The relationship between the concentration of miRNA-21 and the fluorescence intensity is shown in image 3 (F 0 is the fluorescence intensity of the system in the absence of miRNA-21, F is the fluorescence intensity of the system in the presence of miRNA-21). The results showed that as the concentration of miRNA-21 increased from 0zM to 100nM, the fluorescence intensity also gr...

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Abstract

The invention discloses a method for detecting target miRNA based on G-quadruplex molecular beacon double-enzyme cascade isothermal amplification. The method comprises the following steps: taking a reaction system containing miRNA, a reaction buffer solution, Bsu DNA polymerase, Lambda exonuclease and G4MB, and incubating; detecting fluorescence intensity; and judging whether the miRNA contains the target miRNA or the content of the target miRNA according to the fluorescence intensity. The G4MB sequentially comprises a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3 from the 5' end to the 3' end; the nucleotide sequence of the DNA fragment 2 is reversely complementary with the nucleotide sequence of the target miRNA; and one end of the G4MB is modified with a fluorescence marker anda phosphate group, and the other end of the G4MB is modified with a quenching group. Experiments prove that the method provided by the invention can be used for simultaneously detecting multiple target miRNAs, and is high in accuracy, high in sensitivity and good in specificity. The method disclosed by the invention has important application values.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for detecting target miRNA based on G-quadruplex molecular beacon dual-enzyme cascade isothermal amplification. Background technique [0002] microRNAs (miRNA) is a kind of short non-coding RNA (about 18-23nt), which plays an important role in the diagnosis, treatment and prognosis of tumors. miRNA is a post-transcriptional regulator of gene expression, regulating the expression of about 30% of known protein-coding genes, and plays a key role in many basic cellular processes such as cell proliferation, migration and apoptosis. Mature miRNAs exist in various body fluids such as serum and feces. Clinical evidence shows that the abnormal expression of miRNA is often related to the onset of cancer, the invasion of cancer cells, the metastasis of lesions, etc., for example, miR-21 (miR-21-5p), miR-92a (miR-92a-3p), Oncogenic miRNAs such as miR-31 (miR-31-5p) are often...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/682
CPCC12Q1/6844C12Q1/682C12Q2563/107Y02A50/30
Inventor 蒋宇扬谭英陈俊粤谭春燕
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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