Digital miRNA analysis method for loop-mediated isothermal amplification in emulsion

A ring-mediated isothermal and analysis method technology, applied in the field of digital miRNA analysis, can solve the problems of restricting development, limited amplification effect, and low amplification efficiency, and achieve improved efficiency, stable signal, high miRNA amplification efficiency and signal amplification effect of effect

Inactive Publication Date: 2017-07-04
HUNAN UNIV
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Problems solved by technology

[0003] However, due to the low amplification efficiency of the PCR reaction in the emulsion, the current digital PCR technology has limited signal amplification effect after the amplification product is captured by magnetic beads and fluorescently labeled. Usually, steps such as secondary amplification of the signal are required. It is a sophisticated instrument and relatively expensive temperature control equipment, which restricts its development in nucleic acid digital quantification to a certain extent.

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  • Digital miRNA analysis method for loop-mediated isothermal amplification in emulsion
  • Digital miRNA analysis method for loop-mediated isothermal amplification in emulsion
  • Digital miRNA analysis method for loop-mediated isothermal amplification in emulsion

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Embodiment 1

[0034] Example 1 Detection of miRNA based on the digital analysis method of loop-mediated isothermal amplification

[0035] (1) Primer design: design loop-mediated isothermal amplification primers for miRNA. The sequence is as follows:

[0036]

[0037] (2) Preparation of primer-labeled magnetic beads:

[0038] In a 1.5 ml microcentrifuge tube, wash 100 μL (7-12×10) with 100 μL TES buffer (20mMTris-HCl, pH 7.5; 1MNaCl) 8 ) Streptavidin-coated magnetic beads with a diameter of 1 μm. After each wash, place the tube on the magnet for 1 minute to concentrate the beads and then remove the supernatant with a pipette, wash twice and resuspend in 100 μLTES buffer. Take 10 μM biotin-labeled primers and add them to magnetic beads, and incubate at room temperature for 15-30 minutes. After the reaction, the magnetic beads were washed three times with 100 μL of TES buffer for use.

[0039] (2) Design two DNA hairpin probes H1 and H2 using the target miRNA as a template;

[0040] (3) In the prese...

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Abstract

The invention provides a digital miRNA analysis method for loop-mediated isothermal amplification in an emulsion. According to the technical scheme, primer-labeled magnetic beads are used as the carrier to undergo a loop-mediated isothermal amplification reaction in stable emulsion droplets; after reaction of the magnetic beads, lots of amplification products are generated and a complementary fluorescently-labeled primer is captured; after demulsification, reacted magnetic beads are collected; and through a microscope and a flow cytometer, digital nucleic acid analysis of target DNA/Mrna is realized. The establishment of the digital miRNA analysis method provides a digital miRNA analysis means with simple process, stable signals and high sensitivity and accuracy for the reaches on genetic diagnosis, gene therapy, etc.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a digital miRNA analysis method for loop-mediated isothermal amplification in emulsion. Background technique [0002] The principle of emulsion PCR is simply that after the single-stranded DNA template is combined with the capture magnetic beads, the PCR reaction reagent and the oil phase are mixed to form an emulsion, and a series of picoliter-sized droplets-small and independent reaction chambers are obtained. The amplification reaction is carried out in this tiny reaction chamber. The amplified products are enriched on the magnetic beads. The magnetic beads are collected by centrifugation and magnetic separation, and the PCR products on the magnetic beads are sequenced after elution. This technology can dilute the sample to the single-molecule level and distribute it evenly among tens to tens of thousands of units for reaction. Finally, the original concentrat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/119C12Q2525/207C12Q2563/143C12Q2563/149
Inventor 蒋健晖唐丽娟
Owner HUNAN UNIV
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