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Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips

A micro-pillar array and chip technology, applied in the field of emulsion digital PCR, can solve the problems of limited promotion and inconvenient counting, and achieve the effects of improving detection efficiency, simple counting, and changing size parameters

Active Publication Date: 2016-08-10
江苏纳迪芯生命科技研究院有限公司
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AI Technical Summary

Problems solved by technology

Flow cytometry is a commonly used counting method in BEAMing, but expensive instruments and reagents limit the promotion of this technology
Some studies have dispersed magnetic beads in polyacrylamide gel to make magnetic bead arrays for manual counting [J.Boulanger, L.Muresan, I.Tiemann-Boege, Massively parallelhaplotyping on microscopic beads for the high-throughput phase analysis of singlemolecules[J].Summaries of Technical Papers of Annual Meeting Architectural Institute of Japan DEEnvironmental Engineering,2012,7(4):292-294.] However, the random arrangement of magnetic beads and limited microscope field of view also cause inconvenience to the counting

Method used

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  • Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips
  • Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips
  • Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips

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Embodiment 1

[0028] 1.1 BEAMing experiments

[0029]The primer sequences of this embodiment are shown in Table 1. The 5' end of primer 1 is modified with a double biotin group, which can be stably bound to the surface of the magnetic bead and will not be detached in the subsequent thermal cycle reaction. The SA modified magnetic bead Dynabeads M-270 (Invitrogen, USA) and the primer 1 Incubate in binding buffer (5mM pH 7.5 Tris-HCl, 0.5mM EDTA, 1M NaCl) at room temperature for 15min. Excess primer 1 can ensure saturation of SA sites on the surface of magnetic beads. Afterwards, the magnetic beads were enriched by a magnetic field, washed three times with a resuspension solution (20 mM pH 8.4 Tris-HCl, 50 mM KCl), and finally resuspended with a resuspension solution. The reaction oil emulsion mixture consists of 7% (wt / vol) ABIL WE09 (Germany Evonik Degussa company), 20% (vol / vol) mineral oil (U.S. Sigma company) and 73% (vol / vol) Tegosoft DEC (Germany Evonik Degussa company) company) form...

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Abstract

The invention relates to an emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips. The emulsion droplet digital PCR quantitative method comprises the steps that firstly, the microcolumn array chips are prepared; secondly, polystyrene microspheres with the surfaces modified with streptavidin are extracted, diluted and then added into turbid liquid of target magnetic beads step by step for incubation, and a microsphere and magnetic bead composite is enriched and resuspended; thirdly, a resuspended solution of the microsphere and magnetic bead composite in the second step is taken and introduced into a device to be washed, the microsphere and magnetic bead composite intercepted on the chips is counted, and the number of the target magnetic beads is obtained, so that the purpose of high-sensitivity quantification on nucleic acid is achieved. According to the emulsion droplet digital PCR quantitative method based on the microspheres and the microcolumn array chips, the chips are easy to assemble and low in cost; the target magnetic bead counting method of a BEAMing technology is more convenient and easier to implement while the advantage of high sensitivity of a BEAMing experiment is maintained, and a practical tool easy, convenient and fast to use is provided for high-sensitivity detection of nucleic acid.

Description

technical field [0001] The invention belongs to the field of emulsion digital PCR, in particular to an emulsion digital PCR quantitative method based on microsphere and microcolumn array chips. Background technique [0002] Polymerase chain reaction (PCR reaction) is the core technology of modern molecular biology experiments. Its method of specifically amplifying target nucleic acid fragments improves the sensitivity of nucleic acid molecular detection and plays an important role in many biological researches and disease diagnosis. . According to the principle of PCR reaction amplification, many nucleic acid quantification techniques have been derived, among which BEAMing (beads, emulsion, amplification, and magnetics) is an emulsion digital PCR technology based on magnetic bead solid-phase amplification [D.Dressman, H.Yan , G. Traverso, et.al., Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations [J]. Pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2563/159C12Q2563/143C12Q2563/149C12Q2565/501
Inventor 毛红菊程祖乐王琨夏文薇金庆辉赵建龙
Owner 江苏纳迪芯生命科技研究院有限公司
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