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Kit and method for detecting COVID-19 virus by combining nested isothermal amplification with gene editing

A COVID-19 and gene editing technology, which is applied in the field of warm amplification combined with gene editing kits for detecting COVID-19 virus, can solve the problems of low detection limit, complex operation process, and poor specificity of the new coronavirus molecular nucleic acid detection technology. Achieve the effect of reducing the risk of contamination, improving amplification efficiency, and meeting the detection rate

Inactive Publication Date: 2020-12-22
安徽微分基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the deficiencies of the prior art, the present invention provides a kit and method for detecting COVID-19 virus by nested isothermal amplification combined with gene editing, which solves the problem of low detection limit, poor specificity and operational difficulties of the existing novel coronavirus molecular nucleic acid detection technology. The process is complex and time-consuming, while greatly improving the detection sensitivity, improving the specificity of detection, and simplifying the detection process, it is suitable for virus molecular diagnosis and screening in any scenario

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  • Kit and method for detecting COVID-19 virus by combining nested isothermal amplification with gene editing
  • Kit and method for detecting COVID-19 virus by combining nested isothermal amplification with gene editing
  • Kit and method for detecting COVID-19 virus by combining nested isothermal amplification with gene editing

Examples

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Embodiment 1

[0050] This embodiment discloses a nested isothermal amplification combined with gene editing COVID-19 virus kit, which can carry out COVID-19 virus gene sequence 1, COVID-19 virus gene sequence 2 and COVID-19 virus gene sequence 3 Efficient, fast, and simple specific detection can distinguish positive samples from negative samples in a short period of time without using complex equipment, and is suitable for various scenarios.

[0051] A nested isothermal amplification combined with gene editing COVID-19 virus kit, the kit includes:

[0052] (1) Nested constant temperature amplification system: RAA recombinase, reaction buffer V, magnesium acetate solution, nucleic acid-free water and nested RAA amplification junction COVID-19 virus primer set.

[0053] (2) Nucleic acid detection system based on gene editing: Cas13a nuclease, guide RNA, non-specific reporter, detection buffer C.

[0054] (3) Visual detection system: lateral flow test strips, detection chromatographic fluid.

...

Embodiment 2

[0081] Based on the nested isothermal amplification combined with gene editing COVID-19 virus kit described in Example 1, this example illustrates a nested isothermal amplification combined with gene editing COVID-19 virus detection method.

[0082] A nested isothermal amplification combined with gene editing COVID-19 virus detection method, comprising the following steps:

[0083] S1. Add the extracted viral RNA to the nested constant temperature amplification system for in vitro reverse transcription and the first round of cDNA amplification;

[0084] S2, adding the first-round amplification product to the nested constant temperature amplification system and then performing the second-round cDNA amplification;

[0085] S3, adding the second-round cDNA amplification product into the gene editing nucleic acid detection system to obtain a labeled product;

[0086] S4. Using a lateral flow test strip to detect the labeled product.

[0087] Further, the detection method is used...

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Abstract

The invention relates to the technical field of molecular biology, and discloses a kit and a method for detecting COVID-19 virus by combining nested isothermal amplification with gene editing. The kitcomprises: (1) a nested isothermal amplification system: adopting a nested amplification mode, wherein an outer primer pair is used for a first round of isothermal amplification, an inner primer pairis used for a second round of isothermal amplification, a product of the first round of isothermal amplification can be used as a template of the second round of isothermal amplification, and the inner primer pair is used for isothermal amplification; (2) a nucleic acid detection system based on gene editing: using a Cas13a protease and a specific probe to identify a nested RAA enriched templatewith a target sequence; and (3) a visual detection system: utilizing a transverse flow test strip to quickly judge whether virus molecules exist or not. The kit is simple and convenient to operate, does not need complex temperature control equipment, is rapid and efficient, can interpret a result in only one hour, has extremely high amplification efficiency, can detect five copied virus molecules,and is low in pollution risk.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to a kit and method for detecting COVID-19 virus by nested isothermal amplification combined with gene editing. Background technique [0002] The 2019-2020 novel coronavirus (COVID-19) epidemic has brought the world into a tense anti-epidemic state. The virus is transmitted by the respiratory tract. At present, the new crown pneumonia has become a global pandemic trend, and the number of global infections is increasing day by day. As of the deadline, the number of global infections has reached 4,618,821 (WHO Situation Report–119). The detection methods of the new coronavirus on the market are mainly divided into two categories, one is RNA nucleic acid detection based on the molecular level, the main technical means involved include RT-PCR method and genome sequencing method, mainly used for early screening of virus infection Detection; the second type is antigen-antibody...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12Q1/6804C12R1/93
CPCC12Q1/6804C12Q1/6848C12Q1/701C12Q2521/507C12Q2522/101C12Q2521/327C12Q2525/161C12Q2563/131C12Q2565/625
Inventor 聂志超张鸿李西清张慧
Owner 安徽微分基因科技有限公司
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