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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  • 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 nea

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

Examples

Experimental program
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Example Embodiment

[0055] Example one

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

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

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

Example Embodiment

[0060] Example two

[0061] This example is an isothermal amplification detection method based on the paper microfluidic chip described in Example 1, including minimum detection limit verification and 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 acids, see Example 1 for specific steps;

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

[0065] First, 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 220 bp, and its base sequence is shown in SEQ ID NO:1 in Table 1. The LAMP-specific primers are F3 primer, B3 primer, FIP primer, and BIP primer, and th...

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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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