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DNA walker for SARS-CoV-2 detection and preparation method thereof

A sars-cov-2 and walker technology, applied in the field of biological detection, can solve problems such as low efficiency, achieve reliable methods, reduce the need for complex equipment, and achieve high sensitivity

Pending Publication Date: 2022-08-09
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, because amplification is induced by single-sequence binding, only one target nucleic acid fragment can be detected at a time, making confirmation of positive cases inefficient

Method used

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  • DNA walker for SARS-CoV-2 detection and preparation method thereof
  • DNA walker for SARS-CoV-2 detection and preparation method thereof
  • DNA walker for SARS-CoV-2 detection and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The present invention selects two RNA gene fragments ORF1ab and N from the open reading frame and nucleocapsid as the target chain T1 and the target chain according to the SARS-CoV-2 virus genome information (NCBI reference sequence: NC_045512.2) that has been disclosed in the NCBI database. T2, then according to the target strands T1, T2, the walking chain W, 3bp-locked strand L1, 4bp-locked strand L1, 5bp-locked strand L1, 5bp-locked strand L2, 6bp-locked strand L2, 7bp-locked strand L2 were designed , orbital chain Tr, the specific sequence is shown in Table 1. It was then synthesized and purified by Sangon Bioengineering Co., Ltd. (Shanghai, China).

[0065] The locked chain consists of a target recognition domain and a closed domain with different lengths.

Embodiment 2

[0067] 1. Preparation and storage of DNA and RNA solutions

[0068]Concentrated DNA stocks of the sequences described in Table 1 were prepared in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and diluted to working concentration with HEPES buffer (10 mM HEPES, 300 mM NaCl, pH 7.0).

[0069] Concentration method: Dissolve the nucleic acid sequence in microliters of TE buffer solution marked on the tube, and measure the concentration of the concentrated solution by ultraviolet absorption spectroscopy.

[0070] 2. Selection of locking chain L1 and locking chain L2

[0071] Mix 1.44 μL of 10 mM walking chain W and 1.50 μL of 10 mM locked chain (nbp-L1, nbp-L2) in annealing buffer (25 mM Tris-acetate, 200 mM NaCl, pH 8.0), respectively. The volume was 26.12 μL, the mixture was heated to 95°C for 2 min, and then cooled to 25°C to obtain an annealing mixture.

[0072] Combine 2.88 μL of 10 mM orbital chain Tr and 1 μL of 600 mM MgCl 2 Add to the annealing mixture, incubate at 25°C...

Embodiment 3

[0086] 1. The preparation method of gold nanoparticles is as follows:

[0087] Prepare 10 mL of sodium citrate aqueous solution with a concentration of 38.8 mM, and prepare 100 mL of HAuCl with a concentration of 1 mM 4 Aqueous solution; HAuCl 4 The aqueous solution was heated to boiling, then the aqueous sodium citrate solution was rapidly added to the HAuCl with vigorous stirring 4 The aqueous solution was heated and boiled for 10 min, stirred for 15 min after the reaction was completed, cooled to 25°C, and filtered through a 0.22 μm filter to obtain gold nanoparticles (AuNPs).

[0088] A drop of gold nanoparticle solution was added to the carbon-coated copper grid and dried at room temperature to prepare a sample for TEM characterization. Characterization was performed on a JSM-6700F transmission electron microscope with an accelerating voltage of 200 kV.

[0089] The UV-Vis absorption spectrum of AuNPs in this example is as follows: Figure 4 A, transmission electron m...

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Abstract

The invention relates to a DNA walker for SARS-CoV-2 detection and a preparation method of the DNA walker. The DNA walker for SARS-CoV-2 detection comprises a walking chain W, a locking chain L1, a locking chain L2, an orbital chain Tr, a target chain T1, a target chain T2 and gold nanoparticles. The invention also provides a preparation method of the DNA walker, and a method for detecting SARS-CoV-2 by using the DNA walker. According to the DNA walker provided by the invention, visual SARS-CoV-2 detection is realized, the DNA walker has the advantages of few interference factors, low price and good stability, and meanwhile, the sensitivity can reach 1nM. When the DNA walker or the kit provided by the invention is used for detection, a reverse transcription step is not needed, the use of enzymes, labels or modifications is avoided, the result is visualized, the requirement on complex equipment is reduced, and a convenient, economical, rapid and reliable method is provided for SARS-CoV-2 virus detection.

Description

technical field [0001] The invention relates to a DNA walker for SARS-CoV-2 detection and a preparation method thereof, belonging to the technical field of biological detection. Background technique [0002] Since its first appearance, the novel coronavirus pneumonia (COVID-19) has spread rapidly around the world and become a global pandemic, posing a serious threat to public health. It is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded RNA virus belonging to the family Coronaviridae. The disease is mainly transmitted through respiratory droplets and contact, and is highly contagious. Rapid and reliable detection of SARS-CoV-2 is critical to preventing the spread of the outbreak and treating infected cases early. At present, the routine detection method for SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR). However, this method requires specialized equipment to cyclically adjust the reaction temp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6816C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6816C12Q2563/137C12Q2563/155C12Q2563/173C12Q2521/345Y02A50/30
Inventor 徐晓文葛婧雯
Owner SHANDONG UNIV
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