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Micro-RNA-21 ultra-sensitive detection method based on double-enzyme signal cascade amplification

An RNA-21 and cascade amplification technology, which is applied in the ultra-sensitive detection field of microRNA-21, can solve the problems that the stability of the probe is not optimal and cannot be commercialized, and achieves great application value and high sensitivity. and selective, easy-to-use effects

Inactive Publication Date: 2018-05-15
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, due to the two-step carboxyl-amino coupling reaction used in the preparation of the probe, the coupling of styrene microspheres with horseradish peroxidase by single-stranded DNA, the stability of the probe is not optimal and needs to be further improved.
In addition, the enzyme needs to be stored at low temperature to maintain its activity, so it is currently impossible to achieve relatively stable commercial production, and needs to be further improved and optimized

Method used

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  • Micro-RNA-21 ultra-sensitive detection method based on double-enzyme signal cascade amplification
  • Micro-RNA-21 ultra-sensitive detection method based on double-enzyme signal cascade amplification
  • Micro-RNA-21 ultra-sensitive detection method based on double-enzyme signal cascade amplification

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Effect test

Embodiment 1

[0045] (1) Mix carboxylated polystyrene microspheres and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution in phosphate buffer at a ratio of 1:4, and react at room temperature After 15 to 30 minutes, add DNA single strand (one end is aminated and the other end is carboxylated), and react overnight at room temperature; react by coupling carboxylamino group and centrifuge for purification to obtain a DNA-labeled probe.

[0046] (2) The DNA probe and horseradish peroxidase are then activated and coupled at a molar ratio of 1:100, and reacted for 2 to 3 hours at room temperature; Enzyme-labeled probes.

[0047] (3) Mix equal volumes of the above-mentioned probe and TMB one-component chromogenic solution, and react in the dark for 10-15 minutes at room temperature. An equal volume of hydrochloric acid was added to terminate the reaction, and the absorbance of the solution at 450 nm was detected with a microplate reader. According to the change of absorb...

Embodiment 2

[0050] (1) Mix carboxylated polystyrene microspheres and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution in phosphate buffer at a ratio of 1:4, and react at room temperature After 15 to 30 minutes, add DNA single strand (one end is aminated and the other end is carboxylated), and react overnight at room temperature; react by coupling carboxylamino group and centrifuge for purification to obtain a DNA-labeled probe.

[0051] (2) Then the DNA probe and horseradish peroxidase are activated and coupled according to the molar ratio of 1:200, and reacted at room temperature for 2 to 3 hours; Enzyme-labeled probes.

[0052] (3) Mix equal volumes of the above-mentioned probe and TMB one-component chromogenic solution, and react in the dark for 10-15 minutes at room temperature. An equal volume of hydrochloric acid was added to terminate the reaction, and the absorbance of the solution at 450 nm was detected with a microplate reader. According to the chan...

Embodiment 3

[0055] (1) Mix carboxylated polystyrene microspheres and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution in phosphate buffer at a ratio of 1:4, and react at room temperature After 15 to 30 minutes, add DNA single strand (one end is aminated and the other end is carboxylated), and react overnight at room temperature; react by coupling carboxylamino group and centrifuge for purification to obtain a DNA-labeled probe.

[0056] (2) Then the DNA probe and horseradish peroxidase are activated and coupled according to the molar ratio of 1:300, and reacted at room temperature for 2 to 3 hours; Enzyme-labeled probes.

[0057] (3) Mix equal volumes of the above-mentioned probe and TMB one-component chromogenic solution, and react in the dark for 10-15 minutes at room temperature. An equal volume of hydrochloric acid was added to terminate the reaction, and the absorbance of the solution at 450 nm was detected with a microplate reader. According to the chan...

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Abstract

The invention discloses a micro-RNA-21 ultra-sensitive detection method based on double-enzyme signal cascade amplification. The method includes the steps: horseradish peroxidase enzyme-labeled probepreparation: coupling carboxylation polystyrene micro-spheres and horseradish peroxidase enzyme through DNA (deoxyribonucleic acid) single chains by an EDC (ethylene dichloride) carboxyl and amino coupling method; double-chain specific nuclease assisted target recycling: specifically cutting the DNA single chains complementarily paired with RNA (ribonucleic acid) to be detected in a probe by double-chain specific nuclease, triggering target circular reaction, releasing more horseradish peroxidase enzyme by cutting and amplifying signals; horseradish peroxidase enzyme signal amplification: collecting the horseradish peroxidase enzyme released in supernatant by cutting, adding a TMB (tetramethylbenzidine) chromogenic substrate, realizing naked-eye qualitative diagnosis by color change, and realizing enzyme-labeled quantification according to change of absorbance values. The difference of micro-RNA family members is effectively distinguished, and the method is suitable for community tumorscreening of high risk groups and has great application values in biomedical research and clinical diagnosis.

Description

technical field [0001] The invention relates to the technical field of gene diagnosis, and more specifically relates to a novel ultrasensitive detection method for microRNA-21 based on a dual-enzyme signal cascade amplification system. Background technique [0002] MicroRNA (miRNA) is a small endogenous noncoding molecule with short length (approximately 22 nucleotides) and binds to messenger RNA to participate in the regulation of gene expression at the post-transcriptional level. A large number of studies have shown that the abnormal expression of microRNA is closely related to various diseases, especially human tumors and cancers, such as the overexpression of miR-21 in various cancers. Therefore, microRNA as an important tumor marker can be used in the diagnosis, treatment and prognosis of tumors, which has important clinical significance. However, it is still a great challenge to realize its rapid, simple, reliable and ultrasensitive detection due to its characteristic...

Claims

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Application Information

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IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2521/301C12Q2525/207
Inventor 常津彭伟盼宫晓群赵倩朴佳芳
Owner TIANJIN UNIV
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