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Primer for amplifying novel coronavirus and application of primer

A coronavirus, a new type of technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problem of no novel coronavirus nucleic acid loop-mediated isothermal amplification primer detection technology, etc. , to achieve the effect that is easy to obtain, highly conservative, and easy to quantify

Inactive Publication Date: 2020-06-12
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no reports on primers and detection techniques for nucleic acid loop-mediated isothermal amplification of novel coronavirus

Method used

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  • Primer for amplifying novel coronavirus and application of primer
  • Primer for amplifying novel coronavirus and application of primer
  • Primer for amplifying novel coronavirus and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of a kit for detecting novel coronavirus

[0056] Step 1: Prepare Positive Control Standard

[0057] Entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize the N sequence of the nucleocapsid encoding gene of the new coronavirus reference strain (GenBank accession number: NC_045512.2, sequence from 28274 to 29533 in the genome). KpnI and BamHI restriction enzyme sites were introduced into the 5'-end and 3'-end of the gene respectively, and it was named SARS-CoV-2N gene.

[0058] Synthetic SARS-CoV-2N gene and The -3Z (Promega) plasmid vector was double digested with Kpn I and BamHI to obtain a linear vector with cohesive ends and an exogenous gene. The specific enzyme digestion reaction system is as follows:

[0059]

[0060]The enzyme digestion reaction was carried out at 37°C for 30 minutes. After the reaction, the linearized plasmid vector and the foreign gene were recovered with the Cycle-Pure PCR Product Recovery Kit (Omega Bi...

Embodiment 2

[0080] Verify the sensitivity of the detection results of the novel coronavirus loop-mediated isothermal amplification (RT-LAMP) using the primers described in Example 1 of the present invention. Include the following steps:

[0081] Dilute the RNA template: take the RNA prepared by in vitro transcription as the template (i.e. the positive control standard prepared in step 1 of the embodiment), and after quantification, dilute the RNA copy number concentration to 10 with sterile enzyme-free deionized water. 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 5copies / μL and 10 0 copy / μL, and take the negative control as 0copy / μL template RNA.

[0082] Establishment of detection system

[0083] In a 0.2ml micro-reaction tube (Haijibio), add the reagents in the doses described in the table below to establish a loop-mediated isothermal amplification reaction system:

[0084]

[0085] After the reaction system is prepared, gently mix the liquid in the micro reaction tube, and c...

Embodiment 3

[0089] Verify the specificity of the detection results of the novel coronavirus loop-mediated isothermal amplification (RT-LAMP) using the primers described in Example 1 of the present invention. Including the following steps: Select the inactivated severe respiratory syndrome virus (SARS-CoV), coronavirus 229E, coronavirus OC43, H1 subtype influenza A virus (Influenza H1) in the NATtrol Respiratory Validation Panel 3 kit (ZeptoMetrix) Influenza H3, Influenza B, and Respiratory Syncytial Virus A (RSV-A) are 7 common respiratory virus controls for specific detection of the tested virus. Use the virus DNA / RNA extraction kit (Novizem Biotechnology Co., Ltd.) to extract the RNA of the above viruses, and use it together with the SARS-CoV-2 N gene transcript prepared in this study as the sample to be tested, and establish a loop-mediated isothermal amplification method. Increased response. React in a water bath at 60°C for 30 minutes. After the reaction is over, judge the reaction ...

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Abstract

The invention relates to a primer for amplifying novel coronavirus and an application thereof. The primer comprises at least one oligonucleotide primer capable of recognizing a specific region on a novel coronavirus N gene, and the sequence range of the specific region on the novel coronavirus N gene comprises a region sequence of a novel coronavirus reference strain genome 28778-28971. By adopting the primer, novel coronavirus nucleic acid can be rapidly detected from RNA extracted from samples such as blood, saliva, sputum and air; the method has the advantages of simplicity, convenience, quickness and no need of training of large instruments and professionals, can be used as a primary screening and environment detection method for novel coronavirus infected suspected pneumonia cases, and can also be used as an important reference for clinical diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel coronavirus amplification primer and its application. Background technique [0002] At present, in the absence of effective therapeutic drugs and vaccines for Corona Virus Disease 2019 (COVID-19), accurate diagnosis and isolation of patients are still the most effective prevention and control measures to prevent the spread of the epidemic. Therefore, rapid, specific, and sensitive pathogen detection is crucial for the scientific prevention and control of novel coronavirus pneumonia. [0003] The clinical diagnosis of COVID-19 is mainly based on epidemiological history, clinical manifestations and some auxiliary examinations, such as nucleic acid testing, CT scanning, immune recognition techniques (IgM / IgG point-of-care detection (POCT), enzyme-linked immunosorbent assay (ELISA)) and blood cultures. In the field of nucleic acid detection technology, two commonly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6806C12N15/11
CPCC12Q1/701C12Q1/6844C12Q1/6806C12Q2531/119C12Q2521/107Y02A50/30
Inventor 朱洪伟张兴晓张建龙姜琳琳黄清荣于馨陈国忠
Owner LUDONG UNIVERSITY
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