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miRNA detection probe, kit and detection method thereof

A detection kit and detection probe technology, applied in the fields of molecular biology, medicine and biology, can solve the problems of high detection cost, large amount of RNA, low accuracy of results, etc., and achieve accurate and reliable detection results and improved fluorescence signal value. , the effect of increased sensitivity

Active Publication Date: 2016-10-12
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of miRNA mainly include Northern Blot, in situ hybridization, gene chip, and fluorescent quantitative probe method; however, the Northern Blot method is low in sensitivity, takes a long time and consumes a large amount of RNA, which is not suitable for high-throughput analysis; in situ hybridization technology Only a limited number of miRNAs can be detected in one experiment, which is still difficult to meet the high-throughput requirements; gene chip technology can realize high-throughput analysis of miRNAs, that is, multiple miRNAs can be detected on one chip at the same time, but the disadvantage is that the accuracy of the results is low. Poor repeatability, expensive experiments
The fluorescent quantitative probe method has high detection sensitivity, but the detection cost is expensive; while the microsphere-based flow cytometry technology immobilizes the probe on the microsphere and places it in the liquid phase, which is more conducive to capturing the miRNA sequence, thus improving the accuracy. However, due to the high homology of miRNA, short length, and low content in cells or tissues, the highly sensitive and selective detection method for it still needs to be further improved

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  • miRNA detection probe, kit and detection method thereof
  • miRNA detection probe, kit and detection method thereof
  • miRNA detection probe, kit and detection method thereof

Examples

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Effect test

Embodiment 1

[0035] Embodiment 1 miRNA detection probe

[0036] A miRNA detection probe, specifically comprising a hybridization probe and a signal probe:

[0037] 1) The hybridization probe consists of three parts, the 5' end is the tag tag sequence, the middle is the reverse complementary sequence of the complete sequence of the mature target miRNA to be detected, and the 3' end is the binding sequence, which is polyA sequence or oligoCCA Sequence, polyA sequence or oligoCCA sequence is 8-20 bases in length.

[0038] 2) The signal probe consists of two parts, the 5' end is the signal sequence, and the 3' end is polyT or oligoTGG, which is the reverse complementary sequence of the binding sequence at the 3' end of the hybridization probe, wherein the signal sequence is Oligo GT, Oligo TG or Oligo GTT sequence, the length of the Oligo GT, Oligo TG or Oligo GTT sequence is 60-90 bases.

[0039] This example exemplifies the detection of reverse complementary sequences of five mature miRNA ...

Embodiment 2

[0045] Embodiment 2. Composition of kit

[0046] 1. miRNA detection kit mainly includes:

[0047] A, in embodiment 1, aim at the detection probe that 5 kinds of miRNA constructions such as target detection internal standard one, internal standard two, hsa-miR-199b, hsa-miR-122, hsa-miR-145*;

[0048]B, the microbead that is coated with anti-tag sequence, described anti-tag sequence and target miRNA detection probe 5 ' end corresponding tag sequence complementary pairing, specifically as shown in table 2; In the present embodiment, anti-tag There are also 10 T spacer sequences connected between the sequence and the microsphere;

[0049] C. Biotin-labeled dCTP, dATP, dTTP or dGTP (choose a specific one when using).

[0050] According to the designed probe fragments, select the tag sequence to minimize the secondary structure that may be formed between the anti-tag sequences of each microsphere and between the tag and the probe fragment. The corresponding anti-tag sequences ar...

Embodiment 3

[0056] Embodiment 3 A method for detecting miRNA

[0057] Using the kit in Example 2 of the present invention, 5 kinds of miRNAs including target detection internal standard 1, internal standard 2, hsa-miR-199b, hsa-miR-122, and hsa-miR-145* were detected.

[0058] 1. Detection method

[0059] 1) Formation of the signaling marker complex:

[0060] For target detection of internal standard 1, internal standard 2, hsa-miR-199b, hsa-miR-122, hsa-miR-145* and other 5 miRNAs, take PCR tubes respectively, configure pre-binding solution according to the table below, and denature at 95°C 3min, 4°C pre-binding reaction for 1min to obtain the pre-extension product;

[0061]

[0062] Wherein, according to the actual situation that polyA or OligoCCA is selected at the 3' end of the hybridization probe in Example 1, when the corresponding hybridization probe 3' end selects polyA, the miRNA signal probe is "signal sequence+polyT", when the corresponding When the 3' end of the hybridiz...

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Abstract

The invention provides an miRNA detection probe, a kit including the detection probe and an miRNA detection method. The miRNA detection probe includes a hybridization probe and a signal probe, wherein a 5' end of the hybridization probe is a tag sequence, a 3' end of the hybridization probe is a binding sequence, and a to-be-detected target miRNA reverse compliment sequence is arranged between the tag sequence and the binding sequence; a 5' end of the signal probe is a signal sequence, and a 3' end of the signal probe is polyT or oligoTGG. The detection method comprises the following steps: forming a signal labeled complex by the hybridization probe and the signal probe, then hybridizing with a sample containing miRNA, combining with micro-beads coated with an anti-tag sequence, carrying out enzyme digestion with RNase, carrying out SA-PE incubation, and detecting. The prepared miRNA detection probe and the kit can be used for high-sensitive and high-selective detection on the target miRNA.

Description

technical field [0001] The invention belongs to the fields of molecular biology, medicine and biotechnology, and in particular relates to a miRNA detection probe, a kit and a detection method thereof. Background technique [0002] microRNA (miRNA) is a type of endogenous small RNA with a length of about 20-24 nucleotides. One miRNA can have many target genes, and each gene is regulated by multiple miRNAs. Studies have found that mutations, deletions, post-transcriptional regulatory imbalances, DNA methylation modifications in promoter regions, and abnormal binding proteins cause abnormal expression of miRNAs in genes encoding and regulating miRNAs. Abnormal miRNA expression can seriously affect the efficacy of cell signal transduction pathways, leading to uncontrolled cell proliferation and differentiation. The detection of miRNA is of great significance for the monitoring of gene expression at the molecular level, and can also provide an important basis for the study of mi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/682C12Q2525/207C12Q2537/125C12Q2563/131C12Q2563/149
Inventor 陈昌华陈菲许昌有
Owner SUREXAM BIO TECH