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Method for identifying D614G mutation in SARS-CoV-2 based on CRISPR-Cas12a

A D614G, sars-cov-2 technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of complex operation, high difficulty and high cost, and achieve high detection sensitivity, The effect is strong, the method is simple and easy to implement and the effect is fast

Pending Publication Date: 2021-11-02
中国人民解放军疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main methods for identifying the mutation site of the new coronavirus D614G include DNA sequencing, Amplification Refractory Mutation System (ARMS)-PCR, etc. The operation is complex, difficult, time-consuming, and costly. To develop a rapid identification method The detection method of D614G mutation site is very necessary

Method used

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  • Method for identifying D614G mutation in SARS-CoV-2 based on CRISPR-Cas12a
  • Method for identifying D614G mutation in SARS-CoV-2 based on CRISPR-Cas12a
  • Method for identifying D614G mutation in SARS-CoV-2 based on CRISPR-Cas12a

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Embodiment 1

[0033] Example 1. Identification of SARS-CoV-2D614G mutation based on CRISPR-Cas12a

[0034] 1. Find targets containing mutation sites and design target crRNA

[0035] The nucleic acid detection target gene sequence of the CRISPR-Cas12a system needs to have a TTTN / TTN PAM sequence at the 5' end, and select a suitable detection target at the mutation site. After repeated screening and testing, the present invention selects the detection target located at NC_045512.2 (23387bp to 23406bp).

[0036] Design crRNA according to the gene sequence of the target and the CRISPR-Cas12a nucleic acid detection system. The crRNA sequence consists of two parts: the conserved gene sequence at the 5' end (scaffold / repeat part), and the complementary sequence of the target gene sequence at the 3' end. The conserved gene sequence of crRNA is different in different CRISPR-Cas nucleic acid detection systems. Different crRNAs need to be designed for the target. The crRNA sequence can be directly ...

Embodiment 2

[0075] Example 2. SARS-CoV-2D614G mutation identification based on RT-RAA constant temperature amplification and CRISPR-Cas12a

[0076] 1. Design and screening of constant temperature amplification primers for D614G mutation site identification

[0077] (1) Design the constant temperature amplification primers for each detection target

[0078] The design requirements of the RT-RAA constant temperature amplification primers are: the length of the primer is 30-35bp, the 5' end of the primer is an AT base-rich region, and the 3' end of the primer is a CG base-rich region, so as to avoid the formation of hair loss by the primer itself. The clip structure avoids the formation of primer dimers between upstream and downstream primers, and the Tm value of the melting temperature of the primer itself can be ignored. In this experiment, when designing the constant temperature amplification primers, the fragment length of the amplification products should be kept as small as possible w...

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Abstract

The invention provides a crRNA molecule and a method for detecting D614G mutation in SARS-CoV-2 by using CRISPR-Cas12a. The method for detecting D614G mutation in SARS-CoV-2 by using the CRISPR-Cas12a technology through the crRNA is simple, convenient, easy to implement, rapid and specific; when a recombinase polymerase nucleic acid amplification technology is combined, the sensitivity is 100 copies / [mu]L, the sensitivity is extremely high, compared with a common gene sequencing method, the method can achieve the identification of a sample with lower virus content, and is suitable for large-scale screening.

Description

technical field [0001] The invention relates to a method for detecting gene mutation, which belongs to the application field of gene detection. Background technique [0002] Novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus-2, SARS-CoV-2) is a single-stranded positive-sense RNA virus, and its functional coding genes include open reading frame 1ab gene (Open Reading Frame 1ab, ORF1ab), spike protein gene ( Spike protein, S), envelope protein gene (Envelopeprotein, E), membrane protein gene (Membrane, M) and nucleocapsid gene (Nucleocapsid, N). After infecting the human body, it can cause a new type of coronavirus pneumonia (Corona Virus Disease 2019, COVID-19). Patients will have flu-like symptoms such as fever, cough, chest tightness, and fatigue. In severe cases, breathing difficulties, acute respiratory distress syndrome and even death may occur. The source of infection of the new type of coronavirus pneumonia is a person infected with the new coronavirus, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/156C12Q2521/507C12Q2522/101C12Q2521/107C12Q2521/327Y02A50/30
Inventor 宋宏彬刘鸿博邱少富王立贵杜昕颖向莹杨明娟杨超杰刘洪波王辉王琪
Owner 中国人民解放军疾病预防控制中心
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