Rolling circle amplification based miRNA (micro ribonucleic acid) assay probe, assay method and kit

A rolling circle amplification reaction and detection probe technology, applied in the field of miRNA detection probes, can solve problems affecting detection specificity

Active Publication Date: 2014-02-05
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of miRNA based on rolling circle amplification usually requires high-temperature incubation (55°C-65°C) to promote the annealing between the padlock probe and miRNA, but the temperature of the treatment is close to or higher than the melting temperature of the hairpin miRNA precursor The body itself contains the miRNA sequence, so it will form a mismatch between the miRNA precursor and the padlock probe, thereby affecting the specificity of the detection

Method used

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  • Rolling circle amplification based miRNA (micro ribonucleic acid) assay probe, assay method and kit
  • Rolling circle amplification based miRNA (micro ribonucleic acid) assay probe, assay method and kit
  • Rolling circle amplification based miRNA (micro ribonucleic acid) assay probe, assay method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Provide hairpin probes, rolling circle probes, miR-4529-3p similar in sequence to miR-486-5p to be tested, and miR-486-5p precursors based on rolling circle amplification reactions for detecting miR-486-5p, including the following step:

[0121] (1) In vitro synthesis of miR-486-5p to be tested, miR-4529-3p similar in sequence to miR-486-5p to be tested, and miR-486-5p precursor; said miR-486-5p, miR-4529 The nucleotide sequences (5' to 3') of -3p and miR-486-5p precursors are shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively;

[0122] (2) Design according to the sequence of miR-486-5p to be tested, and synthesize hairpin probes and circular probes in vitro; the nucleotide sequences (5' to 3') of the hairpin probes and circular probes are respectively As shown in SEQ ID NO:1 and SEQ ID NO:2, the nucleotide sequences (5' end to 3') of the miR-486-5p, miR-4529-3p and miR-486-5p precursor are respectively As shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5...

Embodiment 2

[0132] Figure 5 The flow chart of detecting miR-486-5p using rolling circle amplification reaction provided for the embodiment of the present invention, combined with Figure 5 , the present embodiment provides a method for detecting miR-486-5p using rolling circle amplification reaction, comprising the following steps:

[0133] (1) Sample preparation

[0134] Take the miR-486-5p synthesized in Example 1 as the sample to be tested;

[0135] (2) Rolling circle amplification reaction

[0136] (2a) Provide or prepare experimental reagents:

[0137] 10U / ul Phi29 DNA polymerase, 40U / ul ribonuclease (RNase) inhibitor, 10mmol / ul dNTPs, 50×SYBR Green II dye, 10×Phi29 DNA polymerase reaction buffer (500mmol / L Tris -hydrochloric acid pH7.5, 100mmol / L magnesium chloride, 100mmol / L ammonium sulfate and 40mmol / L dithiothreitol), phosphate buffer (137mmol / L sodium chloride, 2.7mmol / L potassium chloride, 4.3mmol / L L disodium hydrogen phosphate and 1.4mmol / L potassium dihydrogen phospha...

Embodiment 3

[0163] The effect of rolling circle amplification reaction at different temperatures on the detection sensitivity of miR-486-5p, miR-4529-3p and miR-486-5p precursors includes the following steps:

[0164] (1) Sample preparation

[0165] Take miR-486-5p, miR-4529-3p and miR-486-5p precursor synthesized in Example 1 as samples to be tested;

[0166] (2) Rolling circle amplification reaction

[0167] (2a) Provide or prepare experimental reagents: refer to step (2a) of Example 2

[0168] (2b) Formation of the initial state card-issuing probe

[0169] Take the hairpin probe synthesized in vitro in Example 1, dilute it to 50nmol / L with 1× hairpin probe annealing buffer, incubate at 100°C for 8 minutes, and then slowly cool to room temperature (cooling for 4 hours) to make it fully folded and formed The card-issuing structure, the annealed card-issuing probe is the initial state of the card-issuing probe, and has a card-issuing structure;

[0170] (2c) Perform rolling circle amp...

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Abstract

The invention provides a rolling circle amplification based miRNA (micro ribonucleic acid) assay probe, an assay method and a kit. The probe comprises a hairpin probe and a ring probe, wherein the hairpin probe comprises a 5' terminal side chain (1), a loop (2) and a 3' terminal side chain (3), the 5' terminal side chain (1), the loop (2) and the 3' terminal side chain (3) are single nucleotides; the 5' terminal side chain (1) and the loop (2) are provided with a nucleotide sequence supplementary with miRNA to be assayed; partial nucleotide sequences of the 5' terminal side chain (1) and the 3' terminal side chain (3) are supplementary; the 3' terminal side chain (3) is provided with a nucleotide sequence supplementary with partial nucleotide sequence of the ring probe. The miRNA assay method can distinguish miRNA to be assayed, miRNA similar to a target sequence and target miRNA precursor; extraction and purification of miRNA is not needed when a blood sample is assayed.

Description

technical field [0001] The invention relates to the field of nucleic acid molecule detection, in particular to a miRNA detection probe, detection method and kit based on a rolling circle amplification reaction. Background technique [0002] miRNA is a class of non-coding small molecule RNA in eukaryotic cells, which mainly regulates target genes at the post-transcriptional level. miRNA participates in the regulation of many physiological and pathological processes, especially in the occurrence and development of tumors, playing an important regulatory role. Along with the occurrence and development of cancer, the abnormal fluctuation of miRNA levels in peripheral body fluids (especially serum) has become an important physiological indicator for early diagnosis of cancer. However, miRNAs are usually small in size, low in abundance in body fluids, and the nucleic acid sequences of different miRNAs in the same family are highly similar, so it is very challenging to achieve hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/125
Inventor 张春阳李莹
Owner SHENZHEN INST OF ADVANCED TECH
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