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Novel coronavirus pneumonia (COVID-19) serological diagnosis kit

A technology of COVID-19 and diagnostic kits, which is applied in the field of serological diagnostic kits for novel coronavirus pneumonia, can solve the problems of very large differences in antibody levels and undetectable levels, so as to improve detection accuracy, improve detection sensitivity, and guarantee Biosecurity Effects

Active Publication Date: 2020-06-05
浙江诺迦生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used method for the detection of this indicator is colloidal gold immunochromatography test paper, but due to individual differences, the level of antibodies produced by patients will vary greatly, and some levels will be very low, which cannot be detected with traditional colloidal gold test paper

Method used

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  • Novel coronavirus pneumonia (COVID-19) serological diagnosis kit
  • Novel coronavirus pneumonia (COVID-19) serological diagnosis kit
  • Novel coronavirus pneumonia (COVID-19) serological diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 pCDH-nCoVSmFc.copGFP plasmid construction

[0041] The C-terminal sequence of the outer part of the novel coronavirus spike protein is connected to the protein site-directed biotinylation sequence, a specific protease cleavage site tag and a purification tag to form an artificially designed sequence, wherein the outer part of the new coronavirus spike protein is NCBI Reference Sequence: 1-1213aa part of YP_009724390.1; the protein site-directed biotinylation sequence is biotin protein ligase BirA, its recognition site is GSGGSGGSAGGGLNDIFEAQKIEW, the specific protease is EK enzyme, and its cleavage site label is Flag Label DYKDDDDK, the purification label is mouse IgG Fc fragment (mFc).

[0042] Finally, the nucleic acid sequence of the gene-designed nCoVS-Flag-mFc fusion protein is obtained, and the host (human) codon-optimized sequence is shown in SEQ ID NO: 1, and EcoR I and Not I restriction endonucleases are respectively designed at both ends to express ...

Embodiment 2

[0049] Example 2 Establishment of nCoVSmFc.copGFP / 293 stably transfected cell line

[0050] The pCDH-nCoVSmFc.copGFP plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into the lentiviral packaging line cell 293V to prepare nCoVSmFc.copGFP lentivirus, and transfected into HEK293 cells. The clones were picked under a fluorescent microscope to establish nCoVSmFc.copGFP / 293 stable transfected cell lines. Specific steps are as follows:

[0051] 1) The day before the experiment, five 15cm dishes were plated to ensure that the cells reached 70%-80% confluence before transfection.

[0052] 2) 1-2 hours before transfection, replace the medium in the dish with DMEM medium without serum and without double antibodies.

[0053] 3) Prepare a 15mL centrifuge tube, add 5mL 1×HBS, then add 100μg pCDH-nCoVSmFc.copGFP plasmid, 100μg PH1 / PH2 mixed plasmid (PH1:PH2=3:1, mass ratio), and mix gently .

[0054]4) Add 4mL PEI working solution (10μM), mix gently, and incubate at 37°C for 20...

Embodiment 3

[0065] Embodiment 3S-Biotin protein preparation

[0066] Use protein-free 293 cell culture medium to amplify and culture nCoVSmFc.copGFP / 293 stably transfected cells, collect supernatant culture fluid, and use protein A / G to purify nCoVSmFc fusion protein, use EK enzyme to cut nCoVS from the column, pass through biotin protein The ligase couples biotin to the (GSGGSGGSAGGGLNDIFEAQKIEW) site of nCoVS to obtain S-Biotin protein. Specific steps are as follows:

[0067] 1) Expand nCoVSmFc.copGFP / 293 stably transfected cells to a five-layer cell factory, and culture them in HektorHEK293 cell culture medium without proteins and peptides;

[0068] 2) Collect the culture medium, centrifuge at 1200g, 4°C for 20min, take the supernatant, and use saturated ammonium sulfate method to precipitate protein;

[0069] 3) Precipitated protein was reconstituted with PBS (pH7.4) of 1 / 10-1 / 100 volume of the original solution, desalted, concentrated, and replaced with binding / washing buffer (0.5M...

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Abstract

The invention discloses a novel coronavirus pneumonia (COVID-19) serological diagnosis kit. The kit comprises an S-IgM / IgG test strip and an N-IgM / IgG test strip, the double-antigen quadruple detection kit can be used for simultaneously detecting four indexes of an IgM / IgG antibody for resisting novel coronavirus spinous process protein S and an IgM / IgG antibody for resisting novel coronavirus nucleocapsid protein N in serum of a patient suffering from novel coronavirus pneumonia COVID-19. According to the kit, the detection sensitivity is improved through quantum dot fluorescence labeling andmultistage coupling amplification signals, the detection accuracy is improved through double-antigen quadruple detection, and the biological safety in the detection process is guaranteed by establishing a virus inactivation system. The kit is suitable for whole blood, plasma and serum detection, and can be applied to novel COVID-19 serological diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a serological diagnostic kit for novel coronavirus pneumonia (COVID-19). Background technique [0002] Coronaviruses belong to the genus Coronavirus of the family Coronaviridae in systematic classification. Viruses of the genus Coronavirus are positive-sense single-stranded RNA viruses with an envelope (envelope), with a diameter of about 80-120nm. Pangolins, dogs, wolves, chickens, cattle, snakes, birds and other vertebrates. The 2019 novel coronavirus (SARS-CoV-19, formerly known as 2019-nCoV) is the seventh known coronavirus that can infect humans, and the remaining six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. When a new outbreak occurs, such as 2019 novel coronavirus pneumonia (COVID-19), the early detection of new viruses generally adopts the method of nucleic acid detection, because the genome of the virus can be deciphered quickly, and then spe...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N33/543C12N15/867C12N15/65C12N15/50C07K14/165
CPCG01N33/56983G01N33/558G01N33/54313C07K14/005C12N15/86C12N15/65C12N2800/22C12N2740/15043C12N2770/20022Y02A50/30
Inventor 范春雷朱晓进程向荣
Owner 浙江诺迦生物科技有限公司
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