CN113671183A - Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus past infection detection - Google Patents

Abstract

The invention discloses a diagnostic marker and application thereof in COVID-19 diagnosis and previous infection detection of coronavirus. The diagnostic marker comprises a peptide fragment COVID19-V003, and the amino acid sequence of the peptide fragment COVID19-V003 is as follows: a sequence comprising 5 and more than 5 contiguous amino acids in HADQLTPTWRVYSTGSNV; or the amino acid sequence of the peptide fragment COVID19-V003 is as follows: comprises a sequence formed by substitution or/and deletion or/and addition of 1 to several amino acids in HADQLTPTWRVYSTGSNV. Based on the diagnostic marker, the level of IgG antibody of the anti-peptide segment in human serum is qualitatively detected by an indirect method. The detection kit established based on the invention can be used as an auxiliary means for diagnosing the novel coronavirus pneumonia (COVID-19).

Description

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

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a diagnostic marker and application thereof in COVID-19 diagnosis and previous infection detection of coronavirus, in particular to application of a peptide fragment COVID19-V003 and derivatives thereof in a kit for diagnosing novel coronavirus pneumonia (COVID-19).

Background

The world health organization of the novel coronavirus was named 2019-nCoV at 12 days 1 month in 2020, and then the international committee on viruses named "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" according to taxonomic studies on gene sequencing and the like, and the world health organization named "2019 coronavirus disease (COVID-19)", which is caused by the virus. The world health organization announced that the new coronavirus constitutes an emergent public health event of international concern, 30/1/2020.

SARS-CoV-2 is a novel strain of β -coronavirus, which can infect humans across the species barrier, and can cause infections mainly due to pulmonary diseases by close contact, respiratory droplets, and high-concentration aerosol transmission, and can also induce systemic damage including the nervous system and digestive system, and death in severe cases (Lancet.2020Feb 15; 395(10223): 514-523). Based on the current situation that no effective medicine aiming at SARS-CoV-2 exists and the vaccine is in the clinical verification stage, accurate early diagnosis and timely isolation treatment are the key points for controlling epidemic situation.

By 27 months 3 in 2020, 23 novel coronavirus detection products (15 new coronavirus nucleic acid detection reagents and 8 antibody detection reagents) have been approved by the national drug administration in an emergency to meet the requirements of detection reagents for epidemic prevention and control (http:// www.nmpa.gov.cn /). The 'real-time fluorescence RT-PCR detection of novel coronavirus nucleic acid positive or virus gene sequencing of respiratory tract specimen or blood specimen is highly homologous with the known novel coronavirus' is the gold standard (http:// www.nhc.gov.cn/wjw /) for diagnosis of novel coronavirus pneumonia (COVID-19). Although the nucleic acid detection has the advantages of high sensitivity and strong specificity, the result is not consistent with the COVID-19 imaging performance or is false negative due to the limitations of sampling parts, sample quality and experimental operation, and moreover, the nucleic acid detection has higher requirements on the experimental environment and operators, takes longer time and has high infection risk of the detectors.

As an important effector molecule of the immune system against viruses, specific antibodies in the blood become another basis for diagnosing viral infections. In the novel coronavirus diagnosis and treatment protocol (trial seventh edition) published by the national health committee at 3.3.2020, "serological detection" is formally added as a basis on the basis of original nucleic acid detection and sequencing, i.e., "novel coronavirus-specific IgM antibody and IgG antibody are positive" or "novel coronavirus-specific IgG antibody is converted from negative to positive or is increased by 4 times or more in the recovery period than in the acute period" (http:// www.nhc.gov.cn/wjw /). The novel coronavirus antibody detection kit mainly comprises IgG antibody and IgM antibody detection. Usually, the immune system produces IgM antibodies earlier, which appear within one week after infection, usually indicating acute infection, and the antibody can be used to detect positivity as an indicator of early infection; IgG is produced later but maintained for a relatively longer period of time, and the antibody can be used to detect positivity as a basis for infection and past infection (Clinical and Vaccine Immunology,2004,11(4): 665-. The antibody of the specific anti-virus in the suspected patient serum sample is found by a colloidal gold immunochromatography or a magnetic particle chemiluminescence method, so that the infection condition of the patient is assisted and judged, and the principle is the novel coronavirus antibody detection kit. The antibody detection only needs to take a very small amount of blood samples of patients, has no strict requirements on experimental environment and detection personnel for nucleic acid detection, is simple and easy to operate and short in time, can greatly reduce the infection risk of the detection personnel while improving the detection efficiency, is complementary with the advantages of nucleic acid detection, can reduce the false negative rate, is a high-efficiency guaranteed auxiliary diagnosis means, and provides a convenient and reliable screening means for subsequent primary detection and home detection.

The accuracy of antibody detection depends on the selection of antigen sites, and because nucleocapsid (N) proteins of the beta coronavirus are relatively conserved and have strong antigenicity and can induce host immunity to generate high-abundance antibodies, the antibodies are usually selected as the antigen sites for coronavirus diagnosis (Clin Chem 2003 Dec; 49(12): 1989-96; J Microbiol Biotechnol.2008Oct; 18(10):1717-21), but research shows that the antibodies (www.amplion.com) aiming at the N protein can be detected in some lung cancer patients and healthy people, so the N protein is not the most ideal antigen site for detecting novel coronaviruses. The S protein on the surface of SARS-CoV-2 plays an important role in pathogenesis and can stimulate the immune response of human body to generate antibody. The S protein, spike glycoprotein, is located at the outermost layer of SARS-CoV-2, and the binding with human ACE2 (angiotensin converting enzyme 2) is found to be the key for infecting human cells with new coronavirus. The S protein comprises two regions: s1 and S2, wherein S1 comprises mainly a Receptor Binding Domain (RBD) responsible for recognizing the receptors of the cell; s2 is involved in fusion of the virus with the cell membrane (Science 2020Mar 13; 367(6483): 1260-1263). In general, the S protein has functions of binding virus and host cell membrane receptors and fusing membranes, is an important action site of host neutralizing antibodies and a key target point for diagnosis and vaccine development, and has higher specificity compared with the N protein. Therefore, the development of diagnostic reagents for the S protein is the best option from the serological detection point of view.

However, in the preparation of key binding domains such as S protein or S1-RBD, protein expression in correct structure is usually the most difficult step, and viral proteins often have multiple glycosylation sites, further increasing the difficulty of protein expression and purification (Lancet 2020Apr 4; 395(10230): 1101-1102). Therefore, the preparation cost is high and the storage stability is not easy.

If peptides that do not require consideration of the protein structure could be found instead of the complete protein, the preparation of key materials for immunoassays would be greatly simplified and it would be possible to increase specificity without decreasing sensitivity, and therefore, finding peptide fragments that constitute key antigenic sites is a breakthrough to achieve this assumption. The chemical groups that determine the specific structure of the antigen become antigenic determinants, also known as epitopes (Immunology 2014 Aug; 142(4): 526-35). Epitopes are the target structures recognized by immune cells and also the basis for the specificity of the immune response, i.e., the specificity of an antibody is directed to an antigenic epitope rather than the entire antigenic molecule. The analysis of the antigen epitope recognized by IgM and IgG in the serum is not only helpful to reveal the immune reaction in vivo of the COVID-19 rehabilitant and promote the research and development of vaccine, but also can be used as a peptide biomarker, compared with recombinant antigen protein, the antibody can diagnose SARS-CoV-2 more specifically, also solves the problems of difficult expression and purification of the protein in the preparation process, and the preparation cost can be reduced by 1 to 2 orders of magnitude.

Disclosure of Invention

Aiming at the existing technical problems and the need of finding more accurate novel coronavirus (SARS-CoV-2) pneumonia diagnostic markers, the invention provides a diagnostic marker and application thereof in COVID-19 diagnosis and previous infection detection of coronaviruses, in particular to application of a peptide COVID19-V003 (peptide sequence is HADQLTPTWRVYSTGSNV) in a kit for diagnosing the novel coronavirus (SARS-CoV-2) pneumonia, so as to qualitatively detect the level of IgG antibodies resisting the peptide in a human blood sample, and as a means for assisting the diagnosis of the novel coronavirus (SARS-CoV-2) pneumonia, the diagnostic marker is expected to greatly improve the sensitivity and specificity of the diagnosis of the novel coronavirus (SARS-CoV-2) pneumonia and greatly reduce the detection cost of a single sample.

The purpose of the invention is realized by the following technical scheme:

peptide chips are a systematic analytical tool, and their efficient analytical capabilities are not insignificant. The invention tries to cut S1 and S2 parts (the total length is 1273 amino acids, the reference sequence NCBI GenBank: MN908947.3) of S protein of SARS-CoV-2 into 200 small peptides and carries out chemical synthesis, cysteine is added at the N end of each small peptide and is coupled to the surface of Bovine Serum Albumin (BSA), the coupled product is fixed on the surface of a chip and is respectively incubated with serum of a rehabilitee and serum of healthy people, immunoassay is carried out aiming at IgG in the serum, and finally small peptides which can distinguish and have higher distinguishing capability than full-length S protein are screened out, so as to obtain the effective novel diagnostic marker of coronavirus pneumonia peptide.

In a first aspect, the present invention provides a diagnostic marker of codv-19, wherein the diagnostic marker comprises peptide fragments codv 19-V003, and the amino acid sequence of peptide fragments codv 19-V003 is: a sequence comprising 5 and more than 5 contiguous amino acids in HADQLTPTWRVYSTGSNV; or

The amino acid sequence of the peptide fragment COVID19-V003 is as follows: comprises a sequence formed by substitution or/and deletion or/and addition of 1 to several amino acids in HADQLTPTWRVYSTGSNV.

Preferably, the amino acid sequence of the peptide fragment is HADQLTPTWRVYSTGSNV, or a sequence comprising deletion or mutation of 1 or several amino acids in the amino acid sequence HADQLTPTWRVYSTGSNV.

Preferably, the peptide fragment comprises: a sequence HADQLTPTWRVY or PTWRVYSTGSNV comprising 6 contiguous amino acids of HADQLTPTWRVYSTGSNV.

The peptide fragment COVID19-V003 of the invention can be prepared by a method including but not limited to chemical synthesis, recombinant expression or other methods, and preferably by chemical synthesis.

The invention is used for diagnosing whether the patient is COVID-19 or/and the previous infection of SARS-CoV-2 virus by detecting antibodies (including IgM, IgG and IgA, preferably IgG type antibodies) of peptide fragments COVID19-V003 in body fluid of the patient.

The samples tested include, but are not limited to, whole blood, serum, plasma, interstitial fluid, urine, and alveolar lavage, preferably serum or plasma samples;

the patient with COVID-19 is in a disease state caused by SARS-CoV-2 virus infection;

the previous infection of the SARS-CoV-2 virus is a previous infected person who recovers after being infected by the SARS-CoV-2 virus and has no obvious disease symptoms or/and no symptoms at present. Past infected persons show prior infection, but relatively good immune clearance occurs in the body, and protective antibodies are produced by themselves. However, it is not excluded that the virus still remains in the body and remains activated, and that the virus may be infected.

People who have been infected with new coronavirus but are not found due to no symptoms or slight symptoms can be found by detecting the new coronavirus antibody, on one hand, the diagnosis effect is improved by complementary verification with nucleic acid detection, and on the other hand, in the process of vaccine development, antibody detection can help researchers know which people are infected and recovered.

The antibody detection method adopted by the invention comprises but is not limited to enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, liquid phase chip and protein chip technology. The presented specific numerical values are greatly different according to different detection methods, but the change trend is not influenced.

The specific detection method comprises the steps of directly fixing the peptide fragment on a solid phase carrier (or microbead), then incubating with a sample to be detected, and detecting by using an enzyme-labeled or fluorescence-labeled secondary antibody;

or coupling the peptide segment to a protein (such as BSA, KLH and the like) carrier (or microbead), then incubating with a sample to be detected, and detecting by using an enzyme-labeled or fluorescent-labeled secondary antibody.

In a second aspect, the present invention provides a diagnostic kit for diagnosing COVID-19, comprising the diagnostic markers described above.

Preferably, the diagnostic marker is coupled to BSA via cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to form an SMCC-BSA-diagnostic marker conjugate product.

Preferably, the kit further comprises a standard substance, a coating buffer solution, a blocking solution, a sample diluent, a stop solution, an enzyme labeling reagent, an enzyme substrate solution and a washing solution.

Preferably, the standard comprises standard serum 1 with a concentration of 0U/mL of IgG antibodies against the diagnostic marker (peptide fragments COVID19-V003) and standard serum 2 with a concentration of 100U/mL of IgG antibodies against the diagnostic marker (peptide fragments COVID 19-V003); the standard serum 1 is normal human serum, and the standard serum 2 is serum with positive COVID19-V003 antibody;

the peptide fragment COVID19-V003 antigen is diluted by a coating buffer solution which is a carbonate buffer solution with the concentration of 0.05 plus or minus 0.005M, pH 9.6.6 plus or minus 0.05, namely 1.59g of Na is contained in each 1L of solution₂CO₃，2.93g NaHCO₃；

The confining liquid is phosphate-NaCl buffer solution (PBS) containing 3% Bovine Serum Albumin (BSA) and 0.01 + -0.005M, pH 7.4.4 + -0.05, i.e. each 1L contains 5g Bovine Serum Albumin (BSA), 8g NaCl, and 0.2g KH₂PO₄， 2.9g Na₂HPO₄·12H₂O，0.2g KCl。

Preferably, the enzyme substrate solution comprises: color-developing agent A: 500mL of solutionThe solution contains 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30% hydrogen peroxide; and a color developing agent B: 500mL of solution contains 350mg of TMB, 20mL of DMSO, and citric acid & H₂O 5.1g。

Preferably, the standard and the serum sample to be detected are diluted by a sample diluent, wherein the sample diluent is 0.01M phosphate-NaCl buffer solution (PBS) with the pH value of 7.4;

the washing solution adopted by the washing is 0.01 plus or minus 0.005M, pH 7.4.4 plus or minus 0.05 phosphate-NaCl buffer solution (PBST) containing 0.05 percent Tween-20, namely, 8g of NaCl and 0.2g of KH are contained in each 1 liter of solution₂PO₄，2.9g Na₂HPO₄·12H₂O， 0.2g KCl，0.5mL Tween-20；

The stop solution is 2 +/-0.1M H₂SO₄A solution;

the enzyme-labeled reagent is an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase.

Preferably, each reagent employed in the kit further comprises a preservative to facilitate preservation.

In a third aspect, the present invention provides a detection kit for detecting a past infection of SARS-CoV-2 virus, comprising the aforementioned diagnostic marker.

Preferably, the diagnostic marker is coupled to BSA via cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to form an SMCC-BSA-diagnostic marker conjugate product.

Preferably, the kit further comprises a standard substance, a coating buffer solution, a blocking solution, a sample diluent, a stop solution, an enzyme labeling reagent, an enzyme substrate solution and a washing solution.

Preferably, the standard comprises standard serum 1 with a concentration of 0U/mL of IgG antibodies against the diagnostic marker (peptide fragments COVID19-V003) and standard serum 2 with a concentration of 100U/mL of IgG antibodies against the diagnostic marker (peptide fragments COVID 19-V003); the standard serum 1 is normal human serum, and the standard serum 2 is serum with positive COVID19-V003 antibody;

the peptide fragment COVID19-V003 antigen is diluted by adopting a coating buffer solution which is 0.05 plus or minus 0.005M, pH 9.6.6 plus or minus 0.05 carbonate buffer solution, namely, every 1L of solutionThe solution contained 1.59g of Na₂CO₃，2.93g NaHCO₃；

The confining liquid is phosphate-NaCl buffer solution (PBS) containing 3% Bovine Serum Albumin (BSA) and 0.01 + -0.005M, pH 7.4.4 + -0.05, i.e. each 1L contains 5g Bovine Serum Albumin (BSA), 8g NaCl, and 0.2g KH₂PO₄， 2.9g Na₂HPO₄·12H₂O，0.2g KCl。

Preferably, the enzyme substrate solution comprises: color-developing agent A: 500mL of solution contains 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30% hydrogen peroxide; and a color developing agent B: 500mL of solution contains 350mg of TMB, 20mL of DMSO, and citric acid & H₂O 5.1g。

Preferably, the standard and the serum sample to be detected are diluted by a sample diluent, wherein the sample diluent is 0.01M phosphate-NaCl buffer solution (PBS) with the pH value of 7.4;

the washing solution adopted by the washing is 0.01 plus or minus 0.005M, pH 7.4.4 plus or minus 0.05 phosphate-NaCl buffer solution (PBST) containing 0.05 percent Tween-20, namely, 8g of NaCl and 0.2g of KH are contained in each 1 liter of solution₂PO₄，2.9g Na₂HPO₄·12H₂O， 0.2g KCl，0.5mL Tween-20；

The stop solution is 2 +/-0.1M H₂SO₄A solution;

the enzyme-labeled reagent is an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase.

Preferably, each reagent employed in the kit further comprises a preservative to facilitate preservation.

In a fourth aspect, the invention provides a method for qualitatively detecting an IgG antibody against peptide codv 19-V003 in human serum, comprising the following steps:

A. coupling the diagnostic marker peptide fragment COVID19-V003 to BSA through SMCC;

B. diluting the coupled peptide segment, coating the peptide segment in micropores on an enzyme label plate to prepare a solid phase antigen, and adding a confining liquid;

C. diluting the standard substance and a serum sample to be detected, adding the diluted standard substance and the serum sample to be detected into respective antigen measuring holes, incubating, and adding an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase into each hole to form a COVID 19-V003-antibody-enzyme-labeled secondary antibody compound;

D. after the treatment of step C, thoroughly washing, adding an enzyme substrate solution for color development, adding a stop solution to stop the reaction, and passing through OD₄₅₀The value was obtained as the level of IgG antibodies against the peptide COVID19-V003 in the sample.

Preferably, in step a, the step of coupling the peptide COVID19-V003 to BSA via SMCC specifically comprises:

a1, adding cyclohexane-1-carboxylic acid succinimide ester (SMCC) into buffer solution PBS containing BSA, uniformly mixing, and reacting at 25 ℃ for 1h to obtain a BSA-SMCC solution;

a2, adding BSA-SMCC solution into the peptide COVID19-V003 solution, mixing uniformly, standing at 25 ℃ for 4-6 hours to obtain the coupling product BSA-SMCC-peptide COVID 19-V003.

More preferably, in step a1, the mass ratio of SMCC to BSA is 1: 5;

the concentration of the BSA-SMCC solution is 4 mg/mL.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention develops a set of technology for rapidly obtaining serum markers of diseases by utilizing the advantages of high throughput and rapid analysis of peptide chips. 55 parts of serum of a novel coronavirus (SARS-CoV-2) pneumonia rehabilitator and 18 parts of serum of a healthy person are analyzed, the IgG reactivity difference of the rehabilitator and the serum of the healthy person is compared in a short time, and a serum marker, namely a peptide fragment COVID19-V003 is screened out, and the peptide fragment is expected to be used for assisting in specifically diagnosing the novel coronavirus (SARS-CoV-2) pneumonia.

2. The specificity of the biomarker provided by the invention is 72.22%, and the sensitivity is 82.27%.

3. The invention provides a sensitive, safe, reliable and easy-to-operate commercialized kit, which can qualitatively determine the antibody level of the anti-peptide fragment COVID19-V003 in human blood, and is used for specifically diagnosing novel coronavirus pneumonia (COVID-19) or detecting the past infection of SARS-CoV-2 virus.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a quality control chart of an S protein peptide chip according to example 1 of the present invention;

FIG. 2 is a graph showing the analysis of the diagnostic ability of the peptide HADQLTPTWRVY at the verification stage in example 1 of the present invention; wherein FIG. 2a is a ROC plot; FIG. 2b is a scatter plot;

FIG. 3 is a graph showing the analysis of the diagnostic ability of the peptide PTWRVYSTGSNV at the verification stage in example 2 of the present invention; wherein FIG. 3a is a ROC plot; FIG. 3b is a scatter plot.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

EXAMPLE 1 detection of COVID-19 convalescent patient sera in the form of a small peptide chip

1. Processing and coupling of peptides

1.1 preparation of samples: the S protein was specifically truncated for a total of 220 peptides, based on the condition that each peptide was 12aa in length with an overlap length of 6aa between each two peptides. Wherein the S1 region contains 118 peptides, the S2 region contains 102 peptides including peptide fragments HADQLTPTWRVY and PTWRVYSTGSNV of the present invention, and the peptides are finally synthesized and purified by Gill Biochemical (Shanghai) Co., Ltd, and coupled to BSA with Cys added to the N-terminus of each purified peptide, and co-coupled successfully in 197 (coupled products).

The specific coupling steps are as follows:

taken 10mg BSA to be dissolved in 1mL PBS Buffer, and the concentration is 10 mg/mL.

② 10 microliter of SMCC (1 mg of SMCC is weighed and dissolved in 10 microliter of DMSO) is dissolved in BSA solution and is placed for 1 hour at 25 ℃.

③ transfer the activated BSA-SMCC solution to a dialysis bag and dialyze overnight against 1 XPBS Buffer at 4 ℃. During which the Buffer is changed twice.

And fourthly, diluting the dialyzed BSA-SMCC solution to 4 mg/mL.

Fifthly, taking 1mg of the synthesized peptide in each Eppendorf tube.

Sixthly, adding 10 mu L DMSO to dissolve the peptide, adding 200 mu L1 XPBS to resuspend, and adjusting the pH value to be 7-7.5.

Seventhly, 200 mu L of activated BSA-SMCC solution is added into the peptide. The mixture is placed at 25 ℃ to react for 4 to 6 hours.

Dissolving the coupled product in ddH ₂0, using PBS containing 10% glycerol and 0.01% SDS to make the volume of the solution to be 0.9 mg/mL,0.3mg/mL and 0.1mg/mL to determine the optimal reaction concentration in the screening process (to prevent the final statistics from being influenced by the conditions of high signal and the like).

Additional control samples were added: region S1, region S2, region S-RBD, respectively, 3 concentration gradients per control were prepared; and other control samples: BSA (bovine serum albumin), IgG standard, IgM standard, Cy3 fluorescent secondary antibody, Cy5 fluorescent secondary antibody, PBS buffer. The above controls were set up to ensure the correctness of the subsequent experimental protocol. For example, the region S1 was used to demonstrate that the serum of the former infected persons contained antibodies against the S protein, BSA was used as a negative control without conjugate peptide, IgG and IgM standards were used as reference standards for both serum heavy IgG and IgM channels during chip scanning, and Cy3 and Cy5 fluorescent secondary antibodies were used to localize the entire array during data extraction.

1.2 point-system chip: and (3) spotting each sample prepared in the step 1.1 by using an ink-jet type spotting instrument ArrayJet Marathon, standing at 4 ℃ for overnight fixation, and storing at-80 ℃ after fixation.

1.3 chip quality inspection: in order to detect the quality of a chip, namely whether the chip has common problems such as sample leakage points, tailing and the like, the quality of the chip is detected by aiming at BSA (bovine serum albumin) in a coupling product. Firstly, a chip is taken out from minus 80 ℃, moved to a refrigerator with 4 ℃ for rewarming for 1 hour, then placed at room temperature for rewarming for 1 hour, and the chip box is sealed in the whole process. In a protein-free blocking solution (QuickBlock)^TMWestern blocking liquid, available from Shanghai Biyuntian Biotech LimitedSi) for 3 hours, washed clean with 1x PBST, incubated with rabbit anti-BSA polyclonal antibody (purchased from shanghai bio-engineering gmbh, diluted 6 μ L in 1x PBST at a ratio of 1: 5000) for 1 hour at 4 ℃, washed clean with 1x PBST, incubated with Cy5 fluorescent secondary antibody (according to 1: diluted in 1 XPBST at a ratio of 5000), cleaned with 1 XPBST and dried, according to the operating specifications and instructions of the scanner (Genepix4200A), set the parameters: 635nm, Power 100%, PMT value 550; 532nm, Power 100%, PMT value 550. The scanning results are shown in FIG. 1, and no signal was detected in 9 conjugated products, which may be due to the low purity of the sample after synthesis and purification and the poor solubility during dissolution, and the remaining conjugated products (including peptide fragments of candidate peptides COVID19-V003 and amino acid sequences HADQLTPTWRVY and PTWRVYSTGSNV) and the control showed no signal abnormality. The result shows that the phenomena of missing dots, tailing and the like do not occur in the chip dotting process, and the quality of the chip is enough to ensure the normal operation of subsequent screening.

2. Incubation of chips with serum

2.1 preparation of the required reagents

Sealing liquid: 3g BSA, 100mL 1x PBS solution (diluted with 10x PBS), mixed.

Incubation liquid: 1x PBST solution (0.1% Tween 20).

Cleaning solution: 1x PBST.

The 10 × PBS (1L) formulation is shown in Table 1 below.

TABLE 1

2.2 serum experiments

a. Sealing the chip: in a chip cassette in which 4 chips can be placed, 30mL of a blocking solution (3% BSA in PBS buffer) was prepared. Taking the chip prepared in the step 1.2 out of the temperature of minus 80 ℃ to 4 ℃ and rewarming at room temperature, quickly and parallelly shaking the chip after the chip enters the sealing liquid, reversely placing the chip in the sealing liquid, and placing the sealing box in a side-swinging shaking table at 20-30rpm for 3 hours at room temperature. Pouring and discarding the sealing liquid respectivelyUsing 1 XPBS, 0.2 XPBS (1 XPBS diluted 5 times ddH)₂In O) and ddH₂O cleaning for 1 time and 5 min/time; and then centrifugally dried. And (5) mounting a fence for later use.

b. Sample incubation: serum samples (27 of the former infected persons, 9 of the vs healthy persons) were taken out at-80 ℃, thawed on ice, centrifuged at 4 ℃ for 20min (12000rpm) after complete thawing, and the supernatant was taken as a sample for sample detection. The samples were diluted with the incubation solution (1% BSA in PBST) (dilution ratio 1: 20) and added to the chip of step a (addition 200. mu.L volume) and then placed in a wet box and reacted overnight at 4 ℃ on a side-shaking shaker at 20-30 rpm.

c. Cleaning: the pens were kept mounted on the chips, and the reaction solution was aspirated by a line gun, and each well was washed 3 times one by one, 300. mu.L of PBST each time (about 11min for each chip). Washing the chip once by using PBST, removing the fence, placing the chip into a chip cleaning box with 30mL of cleaning solution, violently shaking for 10-15 times, replacing the cleaning solution, and violently shaking for 10-15 times again; then 20-25mL of cleaning solution is replaced, and the cleaning solution is placed on a horizontal shaker for 3 times at 100-110rpm for 10min each time.

d. Incubation with fluorescently labeled IgG/IgM secondary antibody: secondary antibody dilutions (1: 1000, 1% BSA in PBST) were prepared in advance. The volume of the secondary antibody diluent is determined by the number of chips. If a chip is available, a special chip incubation box can be used, and the chip incubation box is configured according to the volume of 3 mL; if 3-4 sheets, a washing box can be placed to prepare a volume of 15 ml. And d, adding the secondary antibody diluent into the chip cleaned in the step c, and incubating for 1h at room temperature in a dark place under the condition of a side shaking table at 20-30 rpm.

e. Cleaning: placing in a chip cleaning box with 30ml of cleaning solution (PBST), shaking vigorously for 10-15 times, replacing the cleaning solution, and shaking vigorously for 10-15 times again; then 20-25mL of cleaning solution is replaced, and the cleaning solution is placed on a horizontal shaker for 3 times at 100-110rpm for 10min each time. And the washing is carried out in a dark place.

f. After completion of step e with ddH₂O wash 5min x 2 times and rinse again for 10 s.

g. And (3) drying: and f, placing the chip treated in the step f in a chip dryer, and centrifugally drying.

h. Scanning: according to the operation specification and the use specification of the scanner (Genepix4200A), the parameters are set as follows: 635nm, Power 100%, PMT value 550; 532nm, Power 100%, PMT value 550.

i. Data extraction: the corresponding GAL file is opened, the chip image and each array of GAL files are aligned integrally, the automatic alignment button is pressed, the data is extracted, and the GPR file is saved. And performing primary processing on the extracted data through Excel and R languages.

j. And (3) data analysis: after signal values of different samples corresponding to each extracted peptide are normalized and logarithmized, a ROC curve graph and a scatter diagram are obtained by utilizing Graphpad prism 6.0, and diagnostic power is evaluated according to the AUC (area under the curve) in the ROC curve and the difference significance analysis between two groups, so that two candidate peptides COVID19-V003 (amino acid sequences are HADQLTPTWRVY and PTWRVYSTGSNV respectively) are obtained, the AUC of the two candidate peptides reaches more than 0.8 in the discovery stage, the P-value for distinguishing an infected person from a healthy control is lower than 0.0001, and the peptide has the potential as a diagnostic marker compared with other peptides.

2.3 ELISA validation of candidate peptides

a. Independent sample validation of candidate peptides analyzed by the chip experiment was performed (28 cases of previous infected persons 9 cases of vs healthy persons), and two candidate peptides COVID19-V003 (amino acid sequences: HADQLTPTWRVY and PTWRVYSTGSNV, respectively) were synthesized and purified by Gill Biochemical (Shanghai) Co., Ltd. and coupled to BSA with Cys added to the N-terminus to obtain a coupled product.

The preparation method of various buffers and reagents comprises the following steps:

sample diluent: pH 7.4PBS solution, the composition is shown in Table 2 below.

TABLE 2

Washing liquid: PBST solution pH 7.4, composition as shown in table 3 below.

TABLE 3

Sealing liquid: 3% BSA in PBS at pH 7.4, with the composition shown in Table 4 below.

TABLE 4

Enzyme substrate solution: color-developer A and color-developer B (ready for use) were prepared in the following tables 5 and 6.

TABLE 5

TABLE 6

Stopping liquid: 2mol/L H₂SO₄The composition of the solution (concentrated sulfuric acid was slowly added dropwise to distilled water during the preparation, and mixed uniformly) is shown in table 7 below.

TABLE 7

b. Coating: diluting the coupling product obtained in the step a to 1 mu g/mL by using PBS, adding the diluted coupling product into a 96-well enzyme label plate, wherein each well is 100 mu L, and coating the coupling product for 2 hours at 37 ℃ or overnight at 4 ℃; the plate was washed with washing liquid 1 time and spin-dried.

c. And (3) sealing: adding 200 mu L of confining liquid into the 96-hole enzyme label plate treated in the step b, and preserving the temperature for 2 hours at room temperature; the plate was then washed 1 time with washing solution and spun dry.

d. Incubation with serum: the standard (the concentration of IgG antibody against peptide COVID19-V003 is 0U/mL standard serum 1 and the concentration of IgG antibody against peptide COVID19-V003 is 100U/mL standard serum 2; the standard serum 1 is normal human serum and the standard serum 2 is serum positive to antibody COVID19-V003) and the serum sample to be tested were diluted to 100. mu.L with a sample buffer at a ratio of 1:100, and added to respective antigen assay well plates. And d, paying attention to no air bubbles, adding the diluted serum sample to be detected to the bottom of the 96-hole enzyme label plate hole processed in the step c during sample adding, keeping the hole wall as untouched as much as possible, slightly shaking and uniformly mixing, and covering or laminating a film on the enzyme label plate. Then placing the enzyme label plate at 37 ℃ for reaction for 60 minutes, completely throwing off liquid in the hole, and washing for 6 times.

e. Adding an enzyme: and d, adding 100 mu L of enzyme labeling reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase into each hole of the enzyme labeling plate treated in the step d, and forming a peptide fragment-antibody-enzyme labeling secondary antibody compound at 37 ℃ for 60 minutes. And (5) completely throwing off the liquid in the holes, and patting the liquid for 6 times with the upper washing plate.

f. Color development: and e, after the mixture is dried, 50 mu L of color developing agent A is firstly dripped into each hole, 50 mu L of color developing agent B is then added, the mixture is gently shaken and uniformly mixed, and the mixture is shaded at 37 ℃ for developing for 15 minutes.

g. And (4) terminating: after the color development, 100. mu.L of a stop solution was added to each well in order to stop the reaction. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. The stop solution should be added as soon as the substrate reaction time is reached.

h. And (4) judging a result:

1) the optical density (OD value) of each well was measured sequentially at a wavelength of 450nm using an enzyme-linked analyzer.

Unit value (U/mL) ═ (a450< test serum sample > -a450< standard serum 1>)/(a450< standard serum 2> -a450< standard serum 1>) × 100

A450 is an abbreviation for absorbance at 450 nm.

The antibodies such as the current peptide and the like have no international standard, so that the detection result is calibrated by using relative units.

2) Determination of the value of the anti-peptide COVID19-V003 in serum

The unit value is more than or equal to 100U/mL: can be used for preliminary diagnosis of patients with novel coronavirus pneumonia (COVID-19)

Unit value < 100U/mL: the patient could not be diagnosed as a new coronavirus) pneumonia (COVID-19) patient

3) Quality control

Each test result must meet the following criteria:

a450 of standard serum 1: less than or equal to 0.100

A450 of standard serum 2: not less than 0.700

If the above criteria are not met, the result is deemed invalid and must be retested.

i. Interpretation of test results

This example establishes the above reference values by ROC analysis of 18 healthy human sera, 55 COVID-19 rehabilitative sera.

Specificity and sensitivity detection:

the diagnostic kit of the present invention (peptide COVID19-V003 (amino acid sequence: HADQLTPTWRVY) as a diagnostic marker) was tested for specificity and sensitivity using 55 parts of COVID-19 rehabilitative serum and 18 parts of control serum (healthy human serum). Detecting the light absorption value OD₄₅₀Then, a ROC curve and a scattergram were obtained using Graphpad prism 6.0 (the results are shown in FIG. 2, FIG. 2 shows the results of verification of candidate peptide COVID19-V003 (amino acid sequence: HADQLTPTWRVY) by ELISA, in which FIG. 2a is a ROC plot with 1-specificity on the abscissa and sensitivity on the ordinate, and AUC reached 0.8747; FIG. 2b is a scattergram with p-value between two sera of less than 0.0001). The specificity of the diagnosis kit for assisting in diagnosing the novel coronavirus pneumonia (COVID-19) is 83.33%, the sensitivity is 87.27%, and the AUC is 0.8747, so that the diagnosis indexes of the novel coronavirus pneumonia (COVID-19) in the prior art are improved.

The diagnostic kit of the present invention (peptide COVID19-V003 (amino acid sequence: PTWRVYSTGSNV) as a diagnostic marker) was tested for specificity and sensitivity using 55 parts of COVID-19 rehabilitative serum and 18 parts of control serum (healthy human serum). Detecting the light absorption value OD₄₅₀Then, a ROC curve and a scattergram were obtained using Graphpad prism 6.0 (the results are shown in FIG. 3, FIG. 3 shows the results of verification of candidate peptide COVID19-V003 (amino acid sequence: PTWRVYSTGSNV) by ELISA, which is a peptide of CoVID19-V003 (amino acid sequence: PTWRVYSTGSNV)FIG. 3a is a ROC plot with 1-specificity on the abscissa and sensitivity on the ordinate, and an AUC of 0.8692; FIG. 3b is a scatter plot with a p-value of less than 0.0001 between two sera). The specificity of the diagnosis kit for assisting in diagnosing the novel coronavirus pneumonia (COVID-19) is 72.22%, the sensitivity is 87.27%, and AUC is 0.8692, so that the diagnosis indexes of the novel coronavirus pneumonia (COVID-19) in the prior art are improved.

Example 2

1. Processing and coupling of peptides

The peptide COVID19-V003 (amino acid sequence: HADQLTPTWRVYSTGSNV) was synthesized and purified by Gill Biochemical (Shanghai) Co., Ltd, and coupled to BSA with Cys added to the N-terminus to obtain a coupled product. The specific coupling procedure was the same as in example 1.

2. ELISA validation peptides

The procedure was carried out in the same manner as in example 1 (step 2.3).

The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.

Claims (10)

1. The diagnostic marker of COVID-19, which comprises peptide fragments COVID19-V003, wherein the amino acid sequence of the peptide fragments COVID19-V003 is as follows: a sequence comprising 5 and more than 5 contiguous amino acids in HADQLTPTWRVYSTGSNV; or

The amino acid sequence of the peptide fragment COVID19-V003 is as follows: comprises a sequence formed by substitution or/and deletion or/and addition of 1 to several amino acids in HADQLTPTWRVYSTGSNV.

2. The diagnostic marker of COVID-19 as claimed in claim 1, wherein the amino acid sequence of the peptide fragment is HADQLTPTWRVYSTGSNV or a sequence comprising deletion or mutation of 1 or several amino acids in the amino acid sequence HADQLTPTWRVYSTGSNV.

3. A diagnostic kit for codv-19 comprising a diagnostic marker according to any of claims 1 to 2.

4. A test kit for detecting a past infection with a SARS-CoV-2 virus, comprising the diagnostic marker of any one of claims 1-2.

5. The diagnostic kit of claim 3 or 4, wherein the diagnostic marker is conjugated to BSA via SMCC to form an SMCC-BSA-peptide conjugate product.

6. The diagnostic kit of claim 3 or 4, wherein the kit further comprises a standard, a coating buffer, a blocking solution, a sample diluent, a stop solution, an enzyme labeling reagent, an enzyme substrate solution and a washing solution.

7. The diagnostic kit of claim 3 or 4, wherein the standard comprises standard serum 1 at a concentration of 0U/mL of IgG antibodies against the diagnostic marker (peptide fragments COVID19-V003) and standard serum 2 at a concentration of 100U/mL of IgG antibodies against the diagnostic marker (peptide fragments COVID 19-V003); the standard serum 1 is normal human serum, and the standard serum 2 is serum with positive COVID19-V003 antibody;

the peptide fragment COVID19-V003 antigen is diluted by a coating buffer solution which is a carbonate buffer solution with the concentration of 0.05 plus or minus 0.005M, pH 9.6.6 plus or minus 0.05, namely 1.59g of Na is contained in each 1L of solution₂CO₃，2.93g NaHCO₃；

The confining liquid is phosphate-NaCl buffer solution (PBS) containing 3% Bovine Serum Albumin (BSA) and 0.01 + -0.005M, pH 7.4.4 + -0.05, i.e. each 1L contains 5g Bovine Serum Albumin (BSA), 8g NaCl, and 0.2g KH₂PO₄，2.9g Na₂HPO₄·12H₂O，0.2g KCl。

8. The diagnostic kit of claim 3 or 4, wherein the enzyme substrate solution comprises: color-developing agent A: 500mL of solution contains 13.6g of sodium acetate, 1.6g of citric acid and 0.3mL of 30% hydrogen peroxide; and a color developing agent B: 500mL of solution contains 350mg of TMB, 20mL of DMSO, and citric acid & H₂O 5.1g。

9. The diagnostic kit of claim 3 or 4, wherein the standard and the serum sample to be tested are diluted with a sample diluent, which is 0.01M phosphate-NaCl buffer (PBS) with pH 7.4;

the washing solution adopted by the washing is 0.01 plus or minus 0.005M, pH 7.4.4 plus or minus 0.05 phosphate-NaCl buffer solution (PBST) containing 0.05 percent Tween-20, namely, 8g of NaCl and 0.2g of KH are contained in each 1 liter of solution₂PO₄，2.9g Na₂HPO₄·12H₂O，0.2g KCl，0.5mL Tween-20；

The stop solution is 2 +/-0.1M H₂SO₄A solution;

the enzyme-labeled reagent is an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase.

10. A method for qualitatively detecting IgG antibody of anti-peptide COVID19-V003 in human serum is characterized by comprising the following steps:

A. coupling the diagnostic marker peptide fragment COVID19-V003 of claim 1 to BSA via SMCC;

B. diluting the coupled peptide segment COVID19-V003, coating the peptide segment COVID19-V003 in micropores on an enzyme label plate to prepare a solid phase antigen, and adding a confining liquid;

C. diluting the standard substance and a serum sample to be detected, adding the diluted standard substance and the serum sample to be detected into respective antigen measuring holes, incubating, and adding an enzyme-labeled reagent containing an anti-Human IgG antibody labeled by horseradish peroxidase into each hole to form a COVID 19-V003-antibody-enzyme-labeled secondary antibody compound;

D. after the treatment of step C, thoroughly washing, adding an enzyme substrate solution for color development, adding a stop solution to stop the reaction, and passing through OD₄₅₀The value was obtained as the level of IgG antibodies against the peptide COVID19-V003 in the sample.

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