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Method of detecting microRNAs used for diagnosing cerebral stroke from blood

A stroke and blood technology, applied in the field of nucleic acid detection, can solve time-consuming and labor-intensive problems

Inactive Publication Date: 2013-12-04
张飚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the conventional method for detecting microRNA in blood is the real-time quantitative polymerase chain reaction (qRT-PCR) method. This method detects a microRNA molecule and needs to synthesize a pair of primers. If several or dozens of microRNA molecules are detected, it is necessary to Synthesizing a correspondingly large number of primers and qRT-PCR reactions is time-consuming and laborious

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] This embodiment provides a fluorescently encoded microsphere, the fluorescently encoded microsphere is immobilized with a first nucleic acid fragment, and the fluorescent code of the fluorescently encoded microsphere is in one-to-one correspondence with the sequence of the first nucleic acid fragment on the fluorescently encoded microsphere Relationship.

[0021] Since the fluorescent-encoded microspheres of this embodiment are immobilized with the first nucleic acid fragment, the fluorescent-encoded microspheres can be used to bind microRNA directly or indirectly through nucleic acid hybridization, and then be used to detect microRNA from blood for diagnosing stroke. Moreover, the fluorescent code of the fluorescent coded microsphere has a one-to-one correspondence with the sequence of the first nucleic acid fragment. Therefore, one kind of fluorescent coded microsphere can detect one kind of microRNA correspondingly, that is, the type of microRNA can be distinguished b...

Embodiment 2

[0024] This embodiment provides a nucleic acid probe, the nucleic acid probe is a conjugate of biotin and a second nucleic acid fragment, and the second nucleic acid fragment includes: A first subfragment complementary to a nucleic acid fragment, and a second subfragment complementary to a microRNA; the second subfragment differs in sequence from the first nucleic acid fragment.

[0025] And the second sub-fragment on the above-mentioned nucleic acid probe is respectively complementary to a kind of microRNA in the following:

[0026]hsa-let-7a-5p, hsa-let-7c, hsa-let-7d-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR- 1182, hsa-miR-1225-3p, hsa-miR-1225-5p, hsa-miR-1228-3p, hsa-miR-1234, hsa-miR-1238, hsa-miR-126-3p, hsa-miR- 126-5p, hsa-miR-1260b, hsa-miR-1275, hsa-miR-128, hsa-miR-1280, hsa-miR-1304-3p, hsa-miR-142-3p, hsa-miR-146a- 5p, hsa-miR-148a-3p, hsa-miR-148b-3p, hsa-miR-151a-3p, hsa-miR-151a-5p, hsa-miR-151b, hsa-miR-152, hsa-miR- 15a-5p, hsa-miR-181a-5p,...

Embodiment 3

[0031] This embodiment provides a method for detecting microRNA from blood for diagnosing cerebral apoplexy, which method utilizes the above-mentioned fluorescently encoded microspheres and nucleic acid probes, which includes the following steps:

[0032] Step 1: Add the sample to be tested and various nucleic acid probes provided in Example 2 to the mixture composed of various fluorescently encoded microspheres provided in Example 1 to carry out a hybridization reaction.

[0033] The second step: adding multiple single-strand degrading enzymes to the mixture after the hybridization reaction, and performing a degradation reaction, so that one single-strand degrading enzyme degrades one microRNA.

[0034] Step 3: add fluorescently labeled streptavidin to the mixture after the degradation reaction to carry out binding reaction; wherein, the fluorescent wavelength emitted by the streptavidin is different from the fluorescent wavelength emitted by the fluorescently encoded microsph...

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Abstract

The invention relates to a method of detecting microRNAs used for diagnosing cerebral stroke form blood. The method includes: adding a sample to be tested and various nucleic acid probes into mixture of various fluorescence-encoded micro-beads to allow for hybridization; adding various single-chain degrading enzymes into the mixture which is hybridized, and allowing for degradation; adding fluorescence-marked streptavidin into the mixture which is degraded, and allowing for conjugation; cleaning the micro-beads which is conjugated; recognizing fluorescence codes of the micro-beads which are cleaned, and detecting fluorescence intensity of the streptavidin on the micro-beads; determining the type of the detected microRNAs according to the fluorescence codes of the micro-beads; determining whether the corresponding types of microRNAs are conjugated to the micro-beads or not according to the fluorescence intensity, and accordingly determining whether the corresponding types of microRNAs exist in the sample to be tested. The method has the advantage that the step of primer synthesis is omitted so that detection is time saving and labor saving.

Description

technical field [0001] The invention relates to the field of nucleic acid detection, in particular to a method for detecting microRNA from blood for diagnosing cerebral apoplexy. Background technique [0002] At present, the conventional method for detecting microRNA in blood is the real-time quantitative polymerase chain reaction (qRT-PCR) method. This method detects a microRNA molecule and needs to synthesize a pair of primers. If several or dozens of microRNA molecules are detected, it is necessary to Synthesizing a correspondingly large number of primers and the number of qRT-PCR reactions is time-consuming and labor-intensive. Contents of the invention [0003] The object of the present invention is to provide a method for detecting microRNA from blood for diagnosing stroke, so as to solve the above-mentioned problems. [0004] In an embodiment of the present invention, a method for detecting microRNA from blood for diagnosing cerebral apoplexy is provided, comprisin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张飚
Owner 张飚
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