Nucleic acid detection method of digital micro-fluidic chip based on isothermal amplification and gene editing

A technology of constant temperature amplification and digital microfluidics, which is applied in the field of molecular biology, can solve the problems of complex steps, missing nucleic acid detection, and long time-consuming nucleic acid, so as to eliminate pollution and reduce costs

Inactive Publication Date: 2020-03-17
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the invention is to provide a nucleic acid detection method based on a digital microfluidic chip based on constant temperature amplification and gene editing, which solves the current nucleic acid detection time-consuming, complicated steps, high cost, and the possibility of missing a very small amount of nucleic acid. Problems such as

Method used

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  • Nucleic acid detection method of digital micro-fluidic chip based on isothermal amplification and gene editing
  • Nucleic acid detection method of digital micro-fluidic chip based on isothermal amplification and gene editing
  • Nucleic acid detection method of digital micro-fluidic chip based on isothermal amplification and gene editing

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Detection of Mycoplasma pneumoniae in nasopharyngeal swabs

[0030] Prepare 1 copy of Mycoplasma pneumoniae virus culture medium (inactivated) as a positive control, 1 copy of nasopharyngeal swab sample of Mycoplasma pneumoniae infection confirmed by fluorescence quantitative PCR and 1 copy of nasopharyngeal swab sample of healthy person as negative control ( The swab is stored in hanks solution or normal saline). After fully shaking, press the swab along the wall of the centrifuge tube to completely press out the liquid on the swab. Then use the Tiangen Kit Hi-Swab DNA Kit High-Efficiency Oral Swab Genomic DNA Extraction Kit (DP362) to extract the nucleic acids in the above three groups of samples, and the operation strictly follows the steps described in the instructions.

[0031] Prepare a constant temperature amplification mixture in a PCR tube. The constant temperature amplification solution used contains recombinase T4 phage uvs X protein, accessory protein ...

Embodiment 2

[0037] Example 2: Detection of influenza A and B viruses in respiratory secretions

[0038] Prepare 1 influenza A virus culture medium (inactivated) as a positive control, 1 secretion sample of influenza A infection confirmed by fluorescence quantitative PCR, and 1 influenza B infection confirmed by fluorescence quantitative PCR A secretion sample, and a respiratory secretion sample of a healthy person as a negative control. Next, the Tiangen Kit Viral Genomic DNA / RNA Extraction Kit (DP315) was used to extract the nucleic acids in the above four groups of samples, and the operation strictly followed the steps described in the instructions.

[0039] Prepare a constant temperature amplification mixture in a PCR tube. The constant temperature amplification solution used contains recombinase T4 phage uvs X protein, accessory protein T4 phage uvs Y protein, single-strand binding protein gp32, DNA polymerase bsu, reverse transcriptase MMLV, Forward primer, reverse primer, and reaction b...

Embodiment 3

[0045] Example 3: Detection of hepatitis B virus in serum

[0046] Prepare 1 hepatitis B virus culture medium (inactivated) as a positive control, 1 serum sample of hepatitis B virus infection confirmed by fluorescence quantitative PCR and 1 serum sample of a healthy person as a negative control. Then use the TIANamp Blood DNA Kit TIANamp Blood DNA Kit (DP316) to extract the nucleic acids in the above three sets of samples, and the operation strictly follows the steps described in the instructions.

[0047] Prepare a constant temperature amplification mixture in a PCR tube. The constant temperature amplification solution used contains recombinase T4 phage uvs X protein, accessory protein T4 phage uvs Y protein, single-strand binding protein gp32, DNA polymerase bsu, forward primer, reverse Directional primers, and reaction buffer; reaction buffer includes MgAc, Tris-HCl, DTT, polyethylene glycol, potassium acetate, ATP, dNTPs, creatine phosphate, and creatine kinase. The mass conc...

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Abstract

The invention discloses a nucleic acid detection method of a digital micro-fluidic chip based on isothermal amplification and gene editing. The method comprises the following reagents and steps: injecting nucleic acid of a sample to be detected, an isothermal amplification mixed solution and a gene editing nucleic acid detection solution into corresponding positions in the digital micro-fluidic chip at the same time, starting a program to automatically drive the liquid drops through current so that the nucleic acid liquid drops of the sample to be detected and the liquid drops of the isothermal amplification mixed solution are mixed at first and the mixed liquid repeatedly flows and is mixed on the chip, carrying out nucleic acid isothermal amplification at 37-42 DEG C for 10-15 minutes, automatically driving the liquid drops of the gene editing nucleic acid detection liquid through current, uniformly mixing the liquid drops with the mixed liquid drops, carrying out a reaction processat 37 DEG C for 10-30 minutes, and carrying out detection by using a fluorescence detection device to deduce whether the target nucleic acid exists or not. The method shows huge potentials of high sensitivity and rapid detection at a constant temperature, the whole detection process is shortened to be within 1 hour, the automation degree is high, uncovering is not involved, and aerosol pollution is completely eradicated.

Description

Technical field [0001] The invention relates to the field of molecular biology, in particular to a nucleic acid detection method based on a digital microfluidic chip based on constant temperature amplification and gene editing. Background technique [0002] With the rapid development of science and technology, more and more highly automated instrument platforms are invented to liberate manpower and replace human work. The digital microfluidic technology is based on the formation of droplets caused by the surface tension of the liquid. The electric field is used to generate the polar hydrophilicity of the liquid surface to flatten the droplets. The polarization position is controlled by the charge and electric field gradient to generate the tension gradient. The control droplets move on the surface of the microfluidic platform. By combining a series of levels in a series of steps and repeating multiple operations, the automation of complex experimental procedures can be realized....

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2565/629C12Q2563/107C12Q2521/507C12Q2522/101C12Q2521/107
Inventor 李博安杨朝勇张睿孙珍林康凤郭剑光洪欣欣
Owner XIAMEN UNIV
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