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Primer, probe, reagent, method and kit for normal-temperature and isothermal rapid detection of novel coronavirus SARS-CoV-2

A room temperature isothermal, coronavirus technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as unreported analysis methods

Inactive Publication Date: 2020-09-22
潍坊安普未来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the analytical method of combining test strips with multi-enzyme constant temperature nucleic acid rapid amplification to detect the new coronavirus SARS-CoV-2

Method used

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  • Primer, probe, reagent, method and kit for normal-temperature and isothermal rapid detection of novel coronavirus SARS-CoV-2
  • Primer, probe, reagent, method and kit for normal-temperature and isothermal rapid detection of novel coronavirus SARS-CoV-2
  • Primer, probe, reagent, method and kit for normal-temperature and isothermal rapid detection of novel coronavirus SARS-CoV-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Nucleic acid electrophoresis detection

[0054] This embodiment is used to illustrate the normal temperature and constant temperature detection carried out on the nucleic acid colloidal gold test strip.

[0055] 1. According to the published SARS-CoV-2 gene sequence (accession number NC_045512.2) on NCBI GenBank, based on the design principles of primers and fluorescent probes, according to SARS-CoV-2E (Severe acute respiratory syndrome coronavirus 2) (SEQ ID NO: 4) A pair of primers (SARS-CoV-2-E-F and SARS-CoV-2-E-R) were designed, the sequence of which is shown in Table 1:

[0056] Table I

[0057]

[0058] With reference to the SARS-CoV-2 envelope protein and its 3'UTR and membrane glycoprotein gene sequence (SEQ ID NO: 4) published on GenBank, the inventors of the present application have a clear understanding of the SARS-CoV-2 envelope glycoprotein gene and its function and other information conducted a more in-depth study, and found that the envel...

Embodiment 2

[0064] Example 2: Detection of nucleic acid colloidal gold

[0065] This embodiment is used to illustrate the normal temperature and constant temperature detection carried out on the nucleic acid colloidal gold test strip.

[0066] 1. According to the published SARS-CoV-2 envelope protein and its 3'UTR and membrane glycoprotein gene sequence (SEQ ID NO: 4) on NCBI GenBank, based on the design principles of primers and fluorescent probes, according to SARS-CoV-2 A pair of primers (SARS-CoV-2-E-F and SARS-CoV-2-E-R) and a fluorescent probe (SARS -CoV-2-E-Probe), the sequence is shown in Table 2:

[0067] Table II

[0068]

[0069] 2. The amplification reaction system is:

[0070] Table three

[0071]

[0072]

[0073] *Remarks: The lyophilized powder reagent contains the above-mentioned necessary enzymes and auxiliary components, specifically: polyethylene glycol, 4wt%; Tris, 25mM; sodium acetate, 200mM; dithiothreitol, 8mM; 10mM; creatine phosphate disodium salt, ...

Embodiment 3

[0076] Embodiment three: specificity test

[0077] For testing the specificity of the screened primers and probes, several other coronaviruses were selected for detection with primers SARS-CoV-2-E-F, SARS-CoV-2-E-R and probe SARS-CoV-2-E-Probe , verify whether there will be non-specific amplification, and interpret false positive results. Select MERS-CoV (NCBI accession number NC_019843.3, provided by the China Center for Disease Control for scientific research use, and the sequence corresponds to NCBI) and SARS-CoV (NCBI accession number NC_004718.3, provided by the China Center for Disease Control for scientific research use, Sequences correspond to NCBI) for comparison.

[0078] Experimental procedure: 1) Add 29.4μL A buffer (A buffer: 20mM tris-HCl (pH 8.5, 25°C), 150mM KCl, 5v / v% PEG, 1v / v% TRITON-X100);

[0079] 2) Add 2 μL of upstream primer, 2 μL of downstream primer and 0.6 μL of probe to each reaction tube (the concentration of primer and probe is 10 uM);

[0080...

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Abstract

The invention is suitable for the technical field of detection of novel coronavirus SARS-CoV-2, provides a primer and probe for normal-temperature and isothermal rapid detection of novel coronavirus SARS-CoV-2, and also provides a reagent, method and kit for normal-temperature and isotherma rapid detection of novel coronavirus SARS-CoV-2. The novel coronavirus SARS-CoV-2 can be detected rapidly and specifically in real time under the normal-temperature and isothermal conditions. Under the combined action of the specific primer, specific probe, five engineering enzymes and other chemical components, rapid detection of the target is achieved through nucleic acid colloidal gold test strips. No large-scale equipment is needed, a short detection time, convenient operation and high accuracy areachieved, result interpretation is simple and intuitive, and the primer, probe, reagent, method and kit are especially suitable for on-site detection.

Description

technical field [0001] The invention relates to the technical field of novel coronavirus SARS-CoV-2 detection, in particular to a primer, a probe, a reagent, a method and a kit for rapidly detecting novel coronavirus at room temperature and isothermally. Background technique [0002] Viruses of the genus Coronavirus are RNA viruses with an envelope and a linear single-stranded positive strand genome. They are a large class of viruses that exist widely in nature. [0003] Coronavirus particles are irregular in shape and about 60-220nm in diameter. There are three kinds of glycoproteins on the membrane surface: spike glycoprotein (S, Spike Protein, which is the receptor binding site, cytolysis and main antigenic site); envelope protein (E, Envelope Protein) ; Membrane Glycoprotein (M, Membrane Protein). A few species also have hemagglutinin glycoprotein (HE protein, Haemaglutinin-esterase). The nucleic acid of coronavirus is a non-segmented single-stranded (+) RNA, 27-31kb ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6804C12N15/11C12R1/93
CPCC12Q1/6804C12Q1/701C12Q2527/101C12Q2521/507C12Q2521/107C12Q2521/101C12Q2521/301C12Q2522/101C12Q2563/107C12Q2565/625
Inventor 刘国宪刘琼瑶娄志英宋文凤曹伟
Owner 潍坊安普未来生物科技有限公司
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