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Method and device for nucleic acid testing, and application of method and device in COVID-19 detection

A nucleic acid and detection plate technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection. It can solve the problems of low integration, complicated operation, low sensitivity, etc. Powerful, easy-to-use effects

Active Publication Date: 2020-10-02
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods still have problems such as complex operation, low sensitivity, and low degree of integration.

Method used

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  • Method and device for nucleic acid testing, and application of method and device in COVID-19 detection
  • Method and device for nucleic acid testing, and application of method and device in COVID-19 detection
  • Method and device for nucleic acid testing, and application of method and device in COVID-19 detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Figure 1a It is a schematic structural diagram of an embodiment of the nucleic acid detection device of the present invention, Figure 1b It is a schematic diagram of an embodiment of nucleic acid detection using the nucleic acid detection device of the present invention. Figure 1 and figure 2 As shown, the nucleic acid detection device of the present invention includes a cover plate 100 , a detection plate 200 and a bottom plate 300 . The bottom plate 300 is placed under the detection board 200 to support the detection board 200, and the material is such as: but not limited to polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC) , glass and polyethylene etc. The cover plate 100 is made of materials such as but not limited to polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), glass and polyethylene, etc., which are covered on the detection plate 200 , including a first hole 110 , a second hole 120 and a third...

Embodiment 2

[0074] (1) Extract nucleic acid:

[0075] Extract nucleic acid according to traditional commercial kits;

[0076] (2) Sample testing:

[0077] Add 200 μL of test strip buffer system to the second chamber 220 of the microfluidic chip.

[0078] Add 13.5L molecular crowding reagent (concentration 20wt% molecular weight 35,000 polyethylene glycol), 30μL sample RNA, and 4μL primer pairs to the PCR tube in sequence, centrifuge at low speed for 10 seconds, and transfer the liquid to the reaction dry powder In the detection tube of the enzyme preparation, cover the tube cap, mix well by inverting up and down 7-8 times, and centrifuge at low speed for 10 seconds.

[0079] Add the solution in the detection tube to the first chamber 210 to implement RT-RAA amplification (pay attention to avoid air bubbles as much as possible), and add 2.5 μL of amplification starter (280nM magnesium acetate), and start timing (simultaneously tape the first hole 210 and The second hole 220 is sealed), ...

Embodiment 3

[0082] This example verifies the feasibility of a highly integrated microfluidic chip for the amplification and detection of COVID-19 virus established in Example 2. Including the following steps:

[0083] Nucleic acid was extracted by pyrolysis to obtain 0, 10 per microliter 0 、10 1 、10 2 、10 3 、10 4 、10 5 Copy number of sample RNA. The method for RT-RAA amplification and the detection of the amplified product is the same as step (2) of Example 2. The result is as image 3As shown, for the samples containing the N gene, two clear bands appeared on the test strips, indicating that the method in Example 1 of the present invention is feasible.

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Abstract

The invention provides a device for nucleic acid testing. The device comprises a first cavity, a second cavity, a third cavity, a first connecting groove, a second connecting groove and a third connecting groove, wherein the first cavity is used for RT-RAA amplification; a buffer solution is contained in the second cavity; a test strip is placed in the third cavity; the first connecting groove communicates with the first cavity; the second connecting groove communicates with the second cavity; the third connecting groove respectively communicates with the first connecting groove, the second connecting groove and the third cavity; and a base material provides support for the first cavity, the second cavity, the third cavity, the first connecting groove, the second connecting groove and thethird connecting groove. The device provided by the invention can be used for rapidly detecting a nucleic acid amplification product through the test strip for nucleic acid testing in a physically closed environment. The device provided by the invention has the characteristics of simplicity in operation, rapidness in interpretation, pollution prevention, no toxic substances, no need of large instruments and equipment and the like, can completely meet the requirements of COVID-19 epidemic on-site detection, and is beneficial to early diagnosis and early isolation of epidemic, reduction of infection rate and control of epidemic spreading.

Description

technical field [0001] The invention relates to a method for detecting substances, in particular to a nucleic acid detection method and its device, and its application in COVID-19 virus detection. Background technique [0002] The reverse transcription recombinase-mediated isothermal nucleic acid amplification technology combined with lateral flow chromatography test strips is a combination of reverse transcription recombinase-mediated isothermal nucleic acid amplification technology (RT-RAA technology) and lateral flow chromatography test strips (Lateral flow dipstick, LFD) technology, a technology that directly detects RAA amplification products with test strips. RAA-LFD technology is based on RAA technology, mainly using a specific probe (nfo probe) with a 5' end labeled with a fluorescent group (usually FAM) and a 5' end labeled with biotin or digoxin. For the primers, after 20 minutes to 40 minutes at a constant temperature of 37°C to 42°C, a double-labeled amplificati...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/107C12Q2565/629C12Q2565/625
Inventor 杨朝勇刘丹沈海聪张语倩李博安
Owner XIAMEN UNIV
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