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Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus previous infection detection

A COVID-19, COVID19-V001 technology, applied in the field of biomedicine, can solve the problems of difficult expression of protein structure, difficulty in stable storage, and increased difficulty in protein purification, so as to improve the effect of diagnosis

Inactive Publication Date: 2020-11-27
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, when preparing key binding domains such as S protein or S1-RBD, it is usually the most difficult step to express the protein with the correct structure, and viral proteins often have multiple glycosylation sites, which increases the efficiency of protein expression and purification. Difficulty (Lancet2020Apr 4;395(10230):1101-1102)
Therefore, the preparation cost is high and it is not easy to store stably

Method used

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  • Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus previous infection detection
  • Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus previous infection detection
  • Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus previous infection detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Detection of serum from recovered patients with COVID-19 in the form of a small peptide chip

[0071] 1. Peptide processing and coupling

[0072] 1.1 Preparation of samples: According to the condition that each peptide is 12aa in length and there is an overlapping length of 6aa between every two peptides, there are 220 peptides in S protein. Among them, the S1 region contains 118 peptides, and the S2 region contains 102 peptides, including the peptide segment FKEELDKYFKNH in the present invention, which was finally synthesized and purified by Jill Biochemical (Shanghai) Co., Ltd., and a Cys couple was added to the N-terminus of each purified peptide. Linked to BSA, 197 successful co-couplings (coupling products).

[0073] The specific coupling steps are as follows:

[0074] ① Dissolve 10mg of BSA in 1mL of PBS Buffer at a concentration of 10mg / mL.

[0075] ② Dissolve 10 μL SMCC (weigh 1 mg SMCC and dissolve in 10 μL DMSO) in the BSA solution, and place it...

Embodiment 2

[0148] Example 2 Verification of a large number of serum samples by small peptide chips

[0149] 1. Peptide processing and coupling

[0150] This step is the same as in Example 1

[0151] 2. Incubation of chip and serum

[0152] 2.1 Preparation of required reagents

[0153] Blocking solution: 3g BSA, add 100mL 1x PBS solution (diluted from 10x PBS), mix well.

[0154] Incubation solution: 1x PBST solution (0.1% Tween20).

[0155] Washing solution: 1x PBST.

[0156] The 10x PBS (1L) formulation was prepared using the components shown in Table 1 in Example 1.

[0157] 2.2 Serum experiment

[0158] a. Block the chip: Prepare 30 mL of blocking solution (3% BSA in PBSbuffer) in a chip box that can hold 4 chips. Take the chip prepared in step 1 from -80°C to 4°C and rewarm at room temperature. After the chip enters the blocking solution, quickly shake the chip in parallel and reverse it in the blocking solution. Place the sealing box on a side swing shaker for 20- At 30rpm...

Embodiment 3

[0199] Example 3 By performing site mutation on the candidate peptide COVID19-V001

[0200] 1. Peptide processing and coupling

[0201] The last 7 amino acids (DKYFKNH) of the candidate peptide COVID19-V001 obtained in Examples 1 and 2 were mutated into alanine (A) one by one to generate 7 mutant types (the last 7 amino acid sequences after mutation were AKYFKNH, AKYFKNH, DAYFKNH, DKAFKNH, DKYAKNH, DKYFANH, DKYFKAH, DKYFKNA), 7 mutant types and 1 wild type were coupled to BSA (the coupling method was the same as in Example 1).

[0202] 2. Preparation of the chip: the method is the same as that of the chip in Example 1

[0203] 3. Incubation of chip and serum

[0204] 3.1 Preparation of required reagents

[0205] Blocking solution: 3g BSA, add 100mL 1x PBS solution (diluted from 10x PBS), mix well.

[0206] Incubation solution: 1x PBST solution (0.1% Tween20).

[0207] Washing solution: 1x PBST.

[0208] The 10x PBS (1L) formulation was prepared using the components sho...

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Abstract

The invention discloses a diagnostic marker and application of the diagnostic marker in COVID-19 diagnosis and coronavirus previous infection detection. The diagnostic marker comprises a peptide fragment COVID19-V001, the amino acid sequence of the peptide fragment COVID19-V001 is a sequence containing five or more continuous amino acids in FKEELDKYFKNH, or the amino acid sequence of the peptide fragment COVID19-V001 is a sequence formed by substituting or / and deleting or / and adding one or more amino acids in FKEELDKYFKNH. Based on the diagnostic marker provided by the invention, an indirect method is adopted to qualitatively detect the level of the IgG antibody of the anti-peptide fragment in human serum. A detection kit established on the basis of the diagnostic marker can be used as anauxiliary means for diagnosing the coronavirus disease 2019 (COVID-19).

Description

technical field [0001] The invention belongs to the field of biomedicine technology, and specifically relates to a diagnostic marker and its application in the diagnosis of COVID-19 and the detection of previous coronavirus infection, especially a peptide COVID19-V001 and its derivatives in the diagnosis of new coronavirus Application in pneumonia (COVID-19) kit. Background technique [0002] SARS-CoV-2 is a new strain of β-coronavirus, which can cross the species barrier and infect humans. It can be transmitted through close contact, respiratory droplets, and high-concentration aerosols, causing infectious diseases mainly involving lung lesions. , can also induce systemic damage including the nervous system and digestive system, and severe cases can lead to death (Lancet. 2020 Feb 15; 395(10223): 514-523). Based on the current situation that there is no effective drug against SARS-CoV-2 and the vaccine is still in the clinical verification stage, accurate early diagnosis a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/569G01N33/543
CPCG01N33/6854G01N33/581G01N33/56983G01N33/54306G01N2333/165G01N2469/20
Inventor 陶生策李阳赖丹昀江何伟张海南祁环
Owner SHANGHAI JIAO TONG UNIV
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