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Paper micro-fluidic chip, and nucleic acid extraction method and isothermal amplification method thereof

A microfluidic chip and isothermal amplification technology, which is applied in the field of gene detection, can solve the problems of long time steps for nucleic acid extraction and limited application, and achieve good detection performance and simple and fast operation

Active Publication Date: 2017-11-21
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the extraction of nucleic acid in the first step of nucleic acid detection still takes a long time and cumbersome steps. Existing methods such as TRIZOL method, magnetic bead method, spin column method, etc. need nearly 1 hour to complete the extraction and purification of nucleic acid. The purified nucleic acid is used for downstream amplification detection, which greatly limits the application of this method in bedside rapid diagnosis

Method used

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  • Paper micro-fluidic chip, and nucleic acid extraction method and isothermal amplification method thereof
  • Paper micro-fluidic chip, and nucleic acid extraction method and isothermal amplification method thereof
  • Paper micro-fluidic chip, and nucleic acid extraction method and isothermal amplification method thereof

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Embodiment 1

[0056] This embodiment is a paper microfluidic chip designed by the present invention.

[0057] The structure of the paper microfluidic chip: Use a cutting machine to cut a circular glass fiber paper sheet with a diameter of 35mm, and draw a circular sample area with a diameter of 5mm in the center. The main material of the glass fiber sheet is SiO 2 , preferably 100wt% SiO 2 .

[0058] The above-mentioned paper microfluidic chip can be used for the extraction and purification of nucleic acid, such as figure 1 As shown, after mixing 5-10 μL of clinical samples with 50 μL of sample lysate, drop them directly on the sample loading area, and then wash the sample loading area with 250 μL of washing solution to achieve nucleic acid extraction and purification on the paper chip. The purification process can be completed within 5mins. The above-mentioned sample lysate is guanidine hydrochloride-urea lysis solution, wherein the concentration of guanidine hydrochloride is 3-4M, the ...

Embodiment 2

[0061] This embodiment is an isothermal amplification detection method based on the paper microfluidic chip described in Embodiment 1, including the minimum detection limit verification and the specificity and sensitivity verification, which is used to detect group A rotavirus.

[0062] The establishment and amplification reaction steps of group A rotavirus LAMP system include:

[0063] (1) Extraction and purification of nucleic acid, see Example 1 for specific steps;

[0064] (2) Design of target sequence and primers;

[0065] Firstly, the conserved region of group A rotavirus was obtained by sequence alignment software, and LAMP primers were designed online according to the conserved sequence (https: / / primerexplorer.jp / lampv5 / index.html). The target sequence is the NSP5 gene sequence of group A rotavirus, the sequence length is 220bp, and its base sequence is shown as SEQ ID NO: 1 in Table 1. The LAMP-specific primers are F3 primer, B3 primer, FIP primer, and BIP primer, a...

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Abstract

The invention relates to a paper micro-fluidic chip. The paper micro-fluidic chip can integrates the extraction, purification and isothermal amplification of nucleic acids and result interpretation, the whole detection process takes only 30 minutes, a pump, a centrifuge or a thermal cycler is not needed, and results are interpreted with naked eyes. The lowest detection limit of the paper micro-fluidic chip to group A rotavirus infected samples is 10<3> copies / ml, and the sensitivity and the specificity of the chip, verified with 48 clinic samples reach 100% respectively. The microfluidic chip has the advantages of convenience, high efficiency, low cost and good detection performance; the extraction and purification time of nucleic acid is only 5 minutes, so the chip can replace traditional nucleic acid extraction methods (such as nucleic acid extraction using a magnetism bead takes 1 hour) to effectively shorten the detection time; and the paper microfluidic chip can be used in a central laboratory or at bedside, so the chip can be a powerful tool for detecting related pathogens (such as group A rotavirus).

Description

technical field [0001] The present invention relates to the technical field of gene detection, in particular to a paper microfluidic chip and its nucleic acid extraction method and isothermal amplification method; more specifically, the present invention relates to the application of the paper microfluidic chip to group A rotavirus rapid detection. Background technique [0002] Infectious diarrhea in children is a clinically frequently-occurring and common disease that seriously threatens children's lives and health. It ranks among the top three causes of child mortality all year round, and the disease is more serious in developing countries. There are many types of pathogens that cause infectious diarrhea in children, among which group A rotavirus is the most common pathogen. Currently, the methods used to detect group A rotavirus mainly include immunological methods and PCR methods. These methods often require cumbersome Operating procedures, relatively expensive instrume...

Claims

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Application Information

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IPC IPC(8): C12M1/00C12N15/10C12Q1/70C12Q1/68C12R1/93
CPCC12N15/1003C12Q1/6806C12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 叶辛方雪恩李新鑫孔继烈
Owner FUDAN UNIV
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