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Visual virus detection method capable of integrating nucleic acid extraction with LAMP

A technology integrating nucleic acid and virus, applied in the field of visual virus detection, can solve the problems of high viral nucleic acid purity requirements, long detection time and high cost, and achieve the effect of avoiding cross infection, simple and fast operation, and low results.

Pending Publication Date: 2021-10-29
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the global gold standard for viral nucleic acid detection is real-time fluorescent quantitative polymerase chain amplification technology (RT-PCR). With its sensitive detection limit and mature application, it has become the preferred method for virus detection, but it also exists Certain limitations, such as the need for expensive instruments, professional technicians, high requirements for the purity of virus nucleic acid to be detected, long detection time, high cost, etc.

Method used

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  • Visual virus detection method capable of integrating nucleic acid extraction with LAMP
  • Visual virus detection method capable of integrating nucleic acid extraction with LAMP
  • Visual virus detection method capable of integrating nucleic acid extraction with LAMP

Examples

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Embodiment 1

[0053] Example 1. Establishment of a method for visually detecting viruses integrating nucleic acid extraction and LAMP amplification

[0054] The inventors of the present invention have established a visual virus detection method integrating nucleic acid extraction and LAMP amplification through a large number of experiments. The specific steps are as follows:

[0055] 1. The virus releases nucleic acid in a high-concentration guanidinium salt solution and is captured by silicon hydroxyl magnetic beads

[0056] 1. mix

[0057] Take the EP tube, add 200 μL of lysate and 20 μL of silanol magnetic bead solution (BioMag) with a concentration of 25 mg / mL, and mix thoroughly.

[0058] 2. Cracking

[0059] Add 200 μL of the sample to be tested, mix well, and let stand at room temperature for 3 minutes (for lysis).

[0060] 3. Capture

[0061] Magnetically separate the beads in solution.

[0062] 4. Washing

[0063] Wash the magnetic beads twice; use 500 μL of 80% (v / v) aqueou...

Embodiment 2

[0070] Embodiment 2, adopt the method that embodiment 1 establishes to detect the sensitivity of Wenzhou shrimp virus 8

[0071] One, adopt the method that embodiment 1 establishes to detect the sensitivity of Wenzhou shrimp virus 8

[0072] 1. Take 18 nuclease-free EP tubes (specification: 1.5 mL), and add 200 μL of lysate and 20 μL of silanol magnetic bead solution (BioMag) with a concentration of 25 mg / mL to each tube.

[0073] 2. Take the 18 EP tubes that have completed step 1 and divide them into 8 groups, with 2 tubes in each group from the 1st to 7th groups, and 4 tubes in the 8th group. Do the following:

[0074] Group 1: add 200 μL of virus liquid (containing Wenzhou shrimp virus 8 about 10 7 copy number);

[0075] Group 2: add 200 μL of virus liquid (containing Wenzhou shrimp virus 8 about 10 6 copy number);

[0076] Group 3: add 200 μL of virus solution (containing Wenzhou shrimp virus 8 about 10 5 copy number);

[0077] Group 4: add 200 μL of virus solution ...

Embodiment 3

[0108] Embodiment 3, adopt the method after optimization to detect the sensitivity of SARS-CoV-2 pseudovirus

[0109] 1. Take 12 nuclease-free EP tubes (specification: 1.5 mL), and add 500 μL of lysate and 20 μL of silanol magnetic bead solution (BioMag) with a concentration of 25 mg / mL to each tube.

[0110] 2. Take the 12 EP tubes that completed step 1 and divide them into 6 groups, with 2 tubes in each group. Do the following:

[0111] Group 1: Add 500 μL virus liquid (containing about 1600 copies of SARS-CoV-2 pseudovirus) to each tube;

[0112] Group 2: Add 500 μL virus liquid (containing about 800 copies of SARS-CoV-2 pseudovirus) to each tube;

[0113] Group 3: Add 500 μL virus liquid (containing about 400 copies of SARS-CoV-2 pseudovirus) to each tube;

[0114] Group 4: Add 500 μL virus liquid (containing about 200 copies of SARS-CoV-2 pseudovirus) to each tube;

[0115] Group 5: Add 500 μL virus liquid (containing about 100 copies of SARS-CoV-2 pseudovirus) to eac...

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Abstract

The invention discloses a visual virus detection method capable of integrating nucleic acid extraction with LAMP The visual virus detection method comprises the following steps of: releasing nucleic acid from a to-be-detected sample in a high-concentration guanidine salt solution, and capturing the nucleic acid by silicon hydroxyl magnetic beads; adding a reagent for LAMP into the silicon hydroxyl magnetic beads which capture the viral nucleic acid to obtain an LAMP system; and carrying out LAMP reaction, and judging whether the to-be-detected sample contains viruses or not according to the color change of the solution. The method has the following advantages that the nucleic acid adsorbed by the magnetic beads does not need to be eluted and can be directly added into an LAMP mixed solution for amplification, meanwhile, color development is carried out by virtue of phenol red, and a detection result can be visually read only by heating at a constant temperature for 30 minutes; the whole detection process is simple, convenient and rapid in operation, and nucleic acid extraction to result reading can be realized within 35 minutes; and detection sensitivity is high. The method has an important application value in detection of viruses, especially viruses with relatively strong infectivity and / or potential risks.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for visually detecting viruses integrating nucleic acid extraction and LAMP amplification. Background technique [0002] In the process of dealing with the outbreak of infectious diseases, early screening and detection of infectious disease viruses are very important. The earlier the early detection is carried out, the more favorable the later control and treatment will be. Therefore, it is of great importance to establish a fast, convenient, accurate and highly sensitive method for detecting viruses. [0003] Viral nucleic acid detection mainly involves three main processes: (1) nucleic acid extraction and purification; (2) nucleic acid amplification; (3) amplification product detection. For nucleic acid extraction and purification, currently, the lysate is mainly used to destroy virus particles to release nucleic acid, and then the nucleic acid is purified by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6806C12N15/11
CPCC12Q1/701C12Q1/6844C12Q1/6806C12Q2531/119C12Q2563/103C12Q2527/125C12Q2563/143C12Q2563/149Y02A50/30
Inventor 童贻刚贺育敢涂启航谢贴
Owner BEIJING UNIV OF CHEM TECH
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