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A method for rapid detection of miRNA based on asymmetric PCR and LAMP cycle amplification reaction

An amplification reaction, asymmetric technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low abundance, long period required for detection, inability to meet rapid detection, etc., and achieve detection speed. quick effect

Active Publication Date: 2021-06-15
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, miRNAs are usually short, have low abundance in blood, and the sequences of miRNAs of the same family are highly similar, so it is difficult to specifically detect miRNAs, and the detection period is relatively long. And being able to quickly detect miRNA is a difficult problem currently facing
[0004] At present, the commonly used miRNA detection method is qRT-PCR (quantitative reverse transcription polymerase chain reaction). Since the miRNA sequence is only about 20 bases, it is generally necessary to use a neck loop primer to form a complementary sequence with the end of the miRNA, and then The cDNA is generated under the action of reverse transcriptase, and then real-time PCR experiment is performed on the cDNA. PCR equipment is required in the two-step process, and the real-time PCR process in the second step often takes 3-4 hours, which cannot meet the requirements of fast testing needs

Method used

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  • A method for rapid detection of miRNA based on asymmetric PCR and LAMP cycle amplification reaction
  • A method for rapid detection of miRNA based on asymmetric PCR and LAMP cycle amplification reaction
  • A method for rapid detection of miRNA based on asymmetric PCR and LAMP cycle amplification reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, detection of miRNA sensitivity

[0072] Using miR-34a-5p, a member of the miRNA-34 family, to conduct related experiments, prepare the synthesized miRNA oligo chain to 1pM, then perform 10-fold gradient dilution, and simultaneously perform amplification detection on different concentrations of miRNA (for specific operations, refer to the implementation example 2).

[0073] Test results such as figure 2 and image 3 shown. Amplification time earlier than the negative control is a positive result, and it can be seen from the test results that the detection limit can reach 10aM.

Embodiment 2

[0074] Embodiment 2, the detection of miRNA specificity

[0075] General method, the design principle of this method is as follows figure 1 .

[0076] Select three members of the miRNA-34 family and a chain with a single base change (sequence as shown in Table 1) for related experiments, synthesize oligo for each miRNA, and detect four miRNAs at the same time to verify the specificity of the method for detecting miRNAs . For the four members of the miRNA 34 family, the detection system for the specific detection of miR-34a-5p is shown in Table 2.

[0077] Table 1 Sequences of four members of miRNA 34 family

[0078]

[0079] Table 2 Detection system for four members of miRNA 34 family

[0080]

[0081]

[0082] A. Asymmetric PCR to extend the template strand

[0083] First, template-miR and Template-miR-c were mixed at equimolar concentrations, named ds template-miR, incubated at 94°C for five minutes, then slowly cooled to room temperature, diluted to 200ng / μl, an...

Embodiment 3

[0095] Example 3, Detection of miRNA in cancer cell lines

[0096] This technique was used to detect miR-34a in a cancer cell line (cell line BEL-7404).

[0097] 1. Cultivation of cancer cell lines and extraction of total RNA

[0098] After the cancer cell line BEL-7404 was cultured, the total RNA was extracted by the trizol method as the sample to be tested.

[0099] 2. Design different AP primers (Table 5) according to the detection of different miRNAs, Template-miR, Template-miR-c, FIP and BIP are the same as in Example 2.

[0100] Table 5 AP primer designed for mi-34a

[0101]

[0102] 3. Detection of miRNA in cancer cells

[0103] A. Asymmetric PCR to extend the template strand

[0104] First, template-miR and Template-miR-c were mixed at equimolar concentrations, named ds template-miR, incubated at 94°C for five minutes, then slowly cooled to room temperature, diluted to 200ng / μl, and set aside.

[0105] Prepare the reaction system according to Table 6 to amplify...

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Abstract

The invention discloses a method for rapid detection of miRNA based on asymmetric PCR and LAMP cycle amplification reaction. The method for detecting miRNA provided by the present invention is based on asymmetric PCR and LAMP cycle amplification reaction to detect target miRNA. In the present invention, after the chain extension reaction based on asymmetric PCR generates a single strand capable of forming a dumbbell ring structure, the generated single strand is added to the LAMP amplification system, and the primer pair and miRNA trigger a rapid amplification reaction, thereby completing the specificity and specificity of the miRNA. Fast detection. The invention has the following advantages: high specificity; high sensitivity; fast detection speed; high throughput and simultaneous detection of multiple miRNAs.

Description

technical field [0001] The invention relates to the field of rapid detection of nucleic acid molecules, in particular to a method for rapid detection of miRNA based on asymmetric PCR and LAMP cycle amplification reactions. Background technique [0002] miRNAs are small non-coding RNA molecules (containing about 22 nucleotides) found in plants, animals and some viruses that play a role in RNA silencing and post-transcriptional regulation of gene expression. The first miRNAs were discovered in the early 1990s. However, miRNAs were not recognized as a unique class of biological regulators until the early 2000s. miRNA studies have revealed that different cell types and tissues express different amounts of miRNAs, and that aberrant miRNA expression is associated with disease states. The first human disease to be found to be associated with miRNA dysregulation was chronic lymphocytic leukemia. [0003] Recent studies have shown that miRNAs function as oncogenes and tumor suppre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
Inventor 程靓王瑞丽刘利
Owner INST OF CHEM CHINESE ACAD OF SCI