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Detection kit of IgM/IgG antibodies of novel coronavirus (SARS-CoV-2)

A sars-cov-2 and detection test paper technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems that the detection results are greatly affected by the RNA extraction effect, quality and aerosol pollution, and cannot meet the detection requirements, etc. Achieve short detection time, improve reaction sensitivity, and reduce missed detection

Active Publication Date: 2020-05-22
北京新创生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, clinical diagnosis is to detect the specific nucleic acid sequence of SARS-CoV-2 in the blood of patients and suspected patients through ordinary real-time quantitative fluorescent PCR nucleic acid detection reagents. There are restrictions on the length of time and the need for centralized inspection, etc. At the same time, the test results are greatly affected by the RNA extraction effect, and the false negatives are high
The detection results of this method are also affected by the quality of PCR reagents and aerosol pollution
At present, the existing technology cannot meet the current rapid growth of a large number of suspected patients, asymptomatic infections and other screening and diagnosis testing needs

Method used

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  • Detection kit of IgM/IgG antibodies of novel coronavirus (SARS-CoV-2)
  • Detection kit of IgM/IgG antibodies of novel coronavirus (SARS-CoV-2)
  • Detection kit of IgM/IgG antibodies of novel coronavirus (SARS-CoV-2)

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Experimental program
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preparation example Construction

[0096] In a preferred embodiment, the preparation method of the binding pad gold-labeled protein comprises: mixing and reacting the colloidal gold solution with the mouse anti-label antibody to obtain the mouse anti-label antibody-colloidal gold cross-linked complex, and then mixing and reacting with nCOVAg01, Obtain labeled antigen 1; mix and react the colloidal gold solution with rabbit IgG to obtain labeled antibody 2; mix labeled antigen 1 and labeled antigen 2 at a molar ratio of 2-4:7 to obtain gold-labeled protein.

[0097] In a more preferred embodiment, nCOVAg01 is a protein composed of the amino acid sequence described in SEQ ID NO.9, and the mouse anti-Trx antibody is first labeled with colloidal gold to obtain a mouse anti-Trx antibody-colloidal gold complex, and then nCoVAg01 is labeled with mouse On the anti-Trx antibody-colloidal gold complex, the labeled antigen 1 is obtained; the colloidal gold solution is mixed with rabbit IgG to obtain the labeled antibody 2;...

Embodiment 1

[0101] Example 1 Antigen Sequence Design

[0102]According to the known complete gene sequence of SARS-CoV-2 coronavirus NC_045512.2, the recombinant expression antigens, nCoVAg01 and nCoVAg02, were designed. Among them, the antigenic structure of nCOVAg01 is Trx+N+S+hydrophilic sequence+His, and the antigenic structure of nCOVAg02 is His+N+S+His. Specifically, the nCOVAg01 antigen is a protein composed of the amino acid sequence described in SEQ ID NO.9; nCOVAg02 is a protein composed of the amino acid sequence described in SEQ ID NO.10.

Embodiment 2

[0103] Embodiment 2 antigen construction

[0104] Three sequences of whole gene synthesis N01 (SEQ ID NO.11), S01 (SEQ ID NO.12) and S02 (SEQ ID NO.13) were used to construct the antigen.

[0105] N01-pUC plasmid was digested with NcoI and NheI to obtain fragment 1, S01-pUC plasmid was digested with NheI and XhoI to fragment 2, and fragment 1 and fragment 2 were connected to pET30a-Trx vector backbone (NcoI and XhoI) to transform Escherichia coli DH5α, positive clones were screened, and the nCoVAg01 plasmid was identified by sequencing.

[0106] The N01-pUC plasmid was digested with NdeI and NheI to obtain fragment 3, the S02-pUC plasmid was digested with NheI and XhoI to obtain fragment 4, and fragment 3 and fragment 4 were connected to the pET28a vector backbone (NdeI and XhoI) to transform Escherichia coli DH5α, Positive clones were screened, and the nCoVAg02 plasmid was identified by sequencing.

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Abstract

The invention provides a detection kit of IgM / IgG antibodies of novel coronavirus (SARS-CoV-2), and relates to the technical field of biology. According to the detection kit of IgM / IgG antibodies of novel coronavirus (SARS-CoV-2), the IgM / IgG antibodies of novel coronavirus (SARS-CoV-2) are detected through adoption of a colloidal gold capture method, a colloidal gold indirect method and a colloidal gold double antigen sandwich method, wherein adopted antigens are highly active recombinant antigens which are obtained through fusion expression of N protein fragments and S protein dominant epitope fragments, and colloidal gold is labeled indirectly, so that steric hindrance is reduced, and the activity, reaction consistency and accuracy of the antigens are increased. Since a sample pad is pretreated, the interference of complex components in a blood sample to a reaction can be reduced greatly, the detection kit has no requirements for instruments during detection, simple and flexible operation, direct and manual interpretation of results and a short test time, the results can be interpreted in only 15 minutes, and the test results are accurate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel coronavirus (SARS-CoV-2) IgM / IgG antibody detection kit. Background technique [0002] Since the outbreak of pneumonia caused by the novel coronavirus (SARS-CoV-2), there has been no immunological detection reagent for the novel coronavirus in the market. At present, clinical diagnosis is to detect the specific nucleic acid sequence of SARS-CoV-2 in the blood of patients and suspected patients through ordinary real-time quantitative fluorescent PCR nucleic acid detection reagents. The length of time and the need for centralized inspection are limited. At the same time, the test results are greatly affected by the RNA extraction effect, and the false negatives are high. The detection results of this method are also affected by the quality of PCR reagents and aerosol pollution. At present, the existing technology cannot meet the current rapidly growing detection needs for scr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62G01N33/577G01N33/569G01N33/558G01N33/531
CPCC07K14/005C07K2319/00C12N2770/20022G01N33/531G01N33/558G01N33/56983G01N33/577
Inventor 孟婷婷刘兴旺姜化冰景滢滢田仓
Owner 北京新创生物工程有限公司
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