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CN-113671190-B - Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus past infection detection

CN113671190BCN 113671190 BCN113671190 BCN 113671190BCN-113671190-B

Abstract

The invention discloses a diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus past infection detection. The diagnostic marker comprises a peptide fragment COVID-V004, wherein the amino acid sequence of the peptide fragment COVID-V004 is a sequence containing 5 or more than 5 continuous amino acids in NRALTGIAVEQDKNTQEV, or the amino acid sequence of the peptide fragment COVID-V004 is a sequence containing substitution or/and deletion or/and addition of 1 to several amino acids in NRALTGIAVEQDKNTQEV. The diagnostic markers based on the invention are used for qualitatively detecting the level of anti-peptide IgG antibodies in human serum by indirect method. The detection kit established based on the invention can be used as an auxiliary means for diagnosing novel coronavirus pneumonia (COVID-19).

Inventors

  • TAO SHENGCE
  • LI YANG
  • LAI DANYUN
  • JIANG HEWEI
  • ZHANG HAINAN
  • QI HUAN

Assignees

  • 上海交通大学
  • 上海交通大学

Dates

Publication Date
20260421
Application Date
20200515
Priority Date
20200515

Claims (8)

  1. 1. A diagnostic marker of COVID-19, wherein the diagnostic marker is peptide fragment COVID-V004, and the amino acid sequence of peptide fragment COVID-V004 is NRALTGIAVEQD or IAVEQDKNTQEV.
  2. 2. A diagnostic kit for COVID-19 comprising the diagnostic marker of claim 1.
  3. 3. A test kit for detecting a past infection with SARS-CoV-2 virus comprising the diagnostic marker of claim 1.
  4. 4. A diagnostic kit according to claim 2 or 3 wherein the diagnostic marker is coupled to BSA via SMCC to form an SMCC-BSA-peptide coupled product.
  5. 5. A diagnostic kit according to claim 2 or 3, wherein the kit further comprises a standard, a coating buffer, a blocking solution, a sample diluent, a stop solution, an enzyme-labeled reagent, an enzyme substrate solution and a wash solution.
  6. 6. The diagnostic kit according to claim 5, wherein the standard comprises a standard serum 1 having a concentration of IgG antibody against a diagnostic marker (peptide fragment COVID-V004) of 0U/mL and a standard serum 2 having a concentration of IgG antibody against a diagnostic marker (peptide fragment COVID-V004) of 100U/mL, wherein the standard serum 1 is normal human serum and the standard serum 2 is serum positive for COVID19-V004 antibodies; The antigen of the peptide COVID-V004 is diluted by a coating buffer solution, wherein the coating buffer solution is carbonate buffer solution with the pH value of 9.6+/-0.05 and 0.05+/-0.005M, namely 1.59 g Na 2 CO 3 ,2.93 gNaHCO 3 is contained in each 1L solution; The blocking solution is phosphate-NaCl buffer (PBS) solution containing 3% bovine serum albumin (0.01+ -0.005M) and pH 7.4+ -0.05, namely 5g Bovine Serum Albumin (BSA), 8 g NaCl,0.2 g KH 2 PO 4 ,2.9 g Na 2 HPO 4 ·12H 2 O and 0.2 g KCl are contained in each 1L.
  7. 7. The diagnostic kit according to claim 5, wherein the enzyme substrate solution comprises a solution of a color reagent A of 500 mL containing 13.6: 13.6 g sodium acetate, 1.6 g citric acid and 0.3: 0.3 mL 30% hydrogen peroxide, and a solution of a color reagent B of 500 mL containing TMB 350 mg,DMSO 20 mL and citric acid H 2 O5.1: 5.1 g.
  8. 8. The diagnostic kit of claim 5, wherein the standard and the serum sample to be tested are diluted with a sample diluent of 0.01M pH 7.4 phosphate-NaCl buffer (PBS); The washing solution used in the washing is 0.01+ -0.005M, pH 7.4+ -0.05 phosphate-NaCl buffer (PBST) containing 0.05% Tween-20, i.e. 1 liter of solution 8 g NaCl,0.2 g KH 2 PO 4 ,2.9 g Na 2 HPO 4 ·12H 2 O,0.2 g KCl,0.5 mL Tween-20; The stop solution is 2+/-0.1M H 2 SO 4 solution; the enzyme-labeled reagent is an enzyme-labeled reagent containing an anti-Human IgG antibody marked by horseradish peroxidase.

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 biological medicines, in particular relates to a diagnosis marker and application thereof in COVID-19 diagnosis and coronavirus past infection detection, and particularly relates to application of a peptide COVID-V004 and derivatives thereof in a kit for diagnosing novel coronavirus pneumonia (COVID-19). Background The new coronavirus was named 2019-nCoV by the world health organization on 1 month 12 of 2020, and then the international commission on viruses was named "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" according to taxonomic study on gene sequencing and the like, and the disease caused by the virus was named "2019 coronavirus disease (COVID-19)" by the world health organization. The world health organization announced that the novel coronaviruses constituted an international sudden public health event of interest on day 1 and 30 of 2020. SARS-CoV-2 is a novel strain of beta coronavirus, which can cross species barrier to infect human beings, can cause infectious diseases mainly including pulmonary lesions through close contact, respiratory tract droplets and high-concentration aerosol transmission, can also induce systemic injury including nervous system and digestive system, and can cause 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 at present and the vaccine is in clinical verification stage, accurate early diagnosis and timely isolation treatment are key to controlling epidemic situation. By the 3 rd month and 27 th year of 2020, 23 novel coronavirus detection products (15 novel coronavirus nucleic acid detection reagents and 8 antibody detection reagents) have been urgently approved by the national drug administration to meet the requirements of detection reagents for epidemic prevention and control (http:// www.nmpa.gov.cn /). The "real-time fluorescent RT-PCR detection of novel coronavirus nucleic acid positives or viral gene sequencing of respiratory tract specimens or blood specimens is highly homologous to known novel coronaviruses" is the gold standard for diagnosis of novel coronavirus pneumonia (COVID-19) (http:// www.nhc.gov.cn/wjw /). Although the nucleic acid detection has the advantages of high sensitivity and strong specificity, the result is easy to be limited by a sampling part, sample quality and experimental operation, so that the imaging performance of the nucleic acid detection is inconsistent with COVID-19 or is false negative, and furthermore, the nucleic acid detection has higher requirements on experimental environment and operators, longer time consumption and high infection risk of detection personnel. As an important effector molecule of the immune system against viruses, specific antibodies in the blood become another basis for diagnosing viral infections. The national health committee in 3 months 3 of 2020 release "new coronavirus diagnosis and treatment scheme (seventh edition of trial) in the officially on the basis of original nucleic acid detection and sequencing, increased" serological detection ", namely" new coronavirus specific IgM antibodies and IgG antibodies positive "or" new coronavirus specific IgG antibodies from negative to positive or recovery phase more than 4 times of acute phase rise "can also be diagnosed (http:// www.nhc.gov.cn/wjw /). The detection kit of the novel coronavirus antibody mainly comprises IgG antibody and IgM antibody detection. Typically, igM antibodies are produced by the immune system earlier, within one week after infection, usually indicating an acute infection, and positive detection can be used as an indicator of early infection, while IgG is produced later, but for a relatively longer period of time, and positive detection can be used as a basis for infection and past infection (CLINICAL AND VACCINE Immunology,2004,11 (4): 665-668). The specific antiviral antibody in the suspected patient serum sample is found by a colloidal gold immunochromatography method or a magnetic particle chemiluminescence method, so that the infection condition of the patient can be judged, and the principle of the novel coronavirus antibody detection kit is adopted. The antibody detection only needs to take a very small amount of blood sample of a patient, has no strict requirement on the experiment 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 detection personnel while improving the detection efficiency, complements the advantages of the nucleic acid detection, can reduce the false negative rate, is an auxiliary diagnosis means with high efficiency and effective guarantee, and provides a convenient and reliable screening means for subsequent basic layer