A detection method and kit for novel coronavirus N-protein

Abstract

The present invention relates to the technical field of novel coronavirus detection, and in particular to a detection method and kit for novel coronavirus N-protein, incubating the novel coronavirus N-protein standard and performing enzymolysis treatment; using high-performance liquid chromatography tandem triple quadrupole mass spectrometry detection equipment, performing enzymolysis treatment on the recombinant novel coronavirus N-protein standard, taking the standard content as the horizontal coordinate, incubating the novel coronavirus inactivated vaccine sample to be tested, and performing enzymolysis treatment; then the supernatant after enzymolysis is detected, and the concentration of the novel coronavirus N-protein in the inactivated vaccine sample to be tested can be obtained by quantifying the daughter ion peak area of ​​the peptide segment, by using the method. This method effectively avoids interfering factors such as changes in viral protein structure, protein modification and matrix effect that may occur in the novel coronavirus inactivated vaccine sample, and is suitable for the detection of the N-protein content in intermediate products and finished products of the novel coronavirus inactivated vaccine at all stages.

Description

Technical Field

[0001] The present invention relates to the field of novel coronavirus detection technology, and specifically to a method and kit for detecting novel coronavirus N-protein. Background Art

[0002] The detection of the N-protein of the new coronavirus is a detection method used to diagnose the infection of the new coronavirus. It is mainly carried out by detecting the nucleocapsid protein (N protein) in the sample. The detection of the N-protein of the new coronavirus is usually carried out by antigen detection or antibody detection. Antigen detection can quickly screen or exclude the infection of the new coronavirus. It is also called antigen rapid detection, rapid detection and other different terms. Antigen detection can detect the highly expressed nucleocapsid protein, namely N protein, through pharyngeal and nasal swabs. It is generally used for sample detection within 7 days of the onset of symptoms in suspected people during the acute infection period. Antibody detection is to determine whether the person has been infected with the new coronavirus by detecting the new coronavirus and antibodies in the blood of the person being tested.

[0003] However, the existing COVID-19 detection methods still have certain shortcomings during use: Enzyme-linked immunosorbent assay (ELISA) is used to detect the N protein in the novel coronavirus. In the ELISA technique for detecting the N protein, the preparation of specific N protein antibodies is critical, and the antibody preparation process is relatively time-consuming, so it is impossible to establish a highly sensitive specific detection method in a short period of time. Due to its own limitations, ELISA is easily affected by environmental factors during the detection process, so its stability and repeatability are poor. Summary of the invention

[0004] The purpose of the present invention is to provide a method and kit for detecting the N-protein of the new coronavirus, so as to solve the problem that the enzyme-linked immunosorbent assay is used to detect the N protein in the new coronavirus in the above-mentioned background technology. In the technology of detecting the N protein by enzyme-linked immunosorbent assay, it is very critical to prepare specific N protein antibodies. The antibody preparation process is relatively time-consuming, and it is impossible to establish a highly sensitive specific detection method in a short time. Due to its own limitations, the enzyme-linked immunosorbent assay is easily affected by environmental factors during the detection process, so its stability and repeatability are poor.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for detecting the N-protein of a novel coronavirus comprises the following steps: Step 1: incubating and enzymatically treating the N protein standard of the novel coronavirus; using a high performance liquid chromatography-tandem triple quadrupole mass spectrometry analysis and detection device to analyze the N protein standard of the recombinant novel coronavirus after enzymatic treatment, with the content of the standard as the horizontal coordinate; Step 2: Incubate the inactivated new coronavirus vaccine sample to be tested and perform enzymatic hydrolysis; then detect the supernatant after enzymatic hydrolysis, calculate the content of the new coronavirus quantitative peptide segment in the inactivated vaccine sample to be tested by the peak area of ​​the daughter ion of the quantitative peptide segment, and according to the standard curve and the injection volume, finally divide this content by the injection volume to obtain the concentration of the new coronavirus N protein in the inactivated vaccine sample to be tested; Step 3: The process of incubating the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested includes: adding an anionic surfactant to a medium volume of the N protein standard of the new coronavirus or the inactivated vaccine sample to be tested, incubating at 70-90°C for 20-60 minutes, then adding dithiothreitol to a final concentration of 25mmol / L, incubating at 65°C for 60 minutes, and after the temperature drops to room temperature, adding iodoacetamide solution to a final concentration of 50mmol / L, and incubating at room temperature in the dark for 20-60 minutes.

[0006] As a preferred scheme of the present invention, the steps of enzymatic treatment are as follows: 220 μL of the incubated recombinant novel coronavirus N protein standard or the inactivated vaccine sample to be tested is mixed with NH4HCO3 solution to make the total volume reach 480 μL.

[0007] As a preferred solution of the present invention, 20 μL of trypsin solution is then added, and enzymolysis is carried out overnight at 37° C. After the enzymolysis reaction is completed, 1 μL of formic acid is added to terminate the activity of trypsin, thereby ending the enzymolysis process.

[0008] As a preferred embodiment of the present invention, the mass-to-charge ratio of the parent ion in the quantitative ion pair of the quantitative peptide segment is 563.7853, and the mass-to-charge ratio of the daughter ion is 679.3520 and / or 892.4640.

[0009] As a preferred embodiment of the present invention, the high performance liquid chromatography detection conditions of the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested are as follows: chromatographic column: C18 column, preferably ACQUITY UPLCPEPTIDE BEH C18 (2.1mm×100mm), and the injection volume is 5~10μL.

[0010] As a preferred embodiment of the present invention, the temperature of the chromatographic column is 60°C; the mobile phase composition is: mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; the mobile phase B content changes from 0 to 45% in 0 to 20 minutes for gradient elution, and the flow rate is 0.3 mL / min.

[0011] As a preferred embodiment of the present invention, the mass spectrometry detection conditions of the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested are as follows: ion source parameters: nebulizer flow rate 3.0L / min, dryer flow rate 10.0L / min, interface temperature 300°C, desolvation gas temperature 526°C, electrospray voltage 3.0kV; DL temperature 250°C.

[0012] As a preferred solution of the present invention, the quantification ion pair with a parent ion mass-to-charge ratio of 563.7853 and a daughter ion mass-to-charge ratio of 679.3520 has a Q1Pre deviation (v) of -28.0; CE=-19; and a Q3Pre deviation (v) of -34.0.

[0013] As a preferred solution of the present invention, the quantification ion pair with a parent ion mass-to-charge ratio of 563.7853 and a daughter ion mass-to-charge ratio of 892.4640 has a Q1Pre deviation (v) of -30.0; CE=-21; and a Q3Pre deviation (v) of -26.0.

[0014] As a preferred embodiment of the present invention, a kit for the novel coronavirus N-protein includes an antigen kit.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. In the present invention, by using this method, the quantitative peptide segment and its corresponding quantitative ion are used for quantitative detection of the novel coronavirus N protein in liquid chromatography tandem mass spectrometry, showing good detection linearity, wide linear range, high accuracy, high precision, strong specificity and high sensitivity. This method effectively avoids interfering factors such as changes in viral protein structure, protein modification and matrix effect that may occur in the novel coronavirus inactivated vaccine samples, and is suitable for the detection of N protein content in intermediate products and finished products at all stages of the novel coronavirus inactivated vaccine.

[0016] 2. In the present invention, by introducing specific quantitative peptides and their corresponding quantitative ion pairs into the detection system, the content of the new coronavirus N protein can be accurately determined. On the platform of liquid chromatography-tandem mass spectrometry, these quantitative peptides and ion pairs show excellent performance, ensuring the accuracy and reliability of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure is a schematic diagram of the flow structure of the method for using the present invention. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] For examples, see Figure 1 , the present invention provides a technical solution: A detection method and kit for novel coronavirus N-protein, comprising the following steps: step 1: incubating and enzymolyzing an N protein standard of the novel coronavirus; using a high performance liquid chromatography-tandem triple quadrupole mass spectrometry analysis and detection device to perform enzymolysis on a recombinant novel coronavirus N protein standard, with the standard content as the horizontal coordinate; incubating a novel coronavirus inactivated vaccine sample to be tested and performing enzymolysis; then detecting the supernatant after enzymolysis, calculating the content of the novel coronavirus quantitative peptide segment in the inactivated vaccine sample to be tested by the daughter ion peak area of ​​the quantitative peptide segment, and according to the standard curve and the injection volume, and finally dividing the content by the injection volume to obtain the concentration of the novel coronavirus N protein in the inactivated vaccine sample to be tested; wherein the process of incubating the novel coronavirus N protein standard or the novel coronavirus inactivated vaccine sample to be tested comprises: adding an anionic surfactant to a medium volume of the novel coronavirus N protein standard or the inactivated vaccine sample to be tested, incubating at 70 to 90° C. for 20 to 60 minutes, and then adding dithiothreitol to a final concentration of 25 mmol / L, incubate at 65°C for 60 min, add iodoacetamide solution to a final concentration of 50 mmol / L after the temperature drops to room temperature, and incubate at room temperature in the dark for 20 to 60 min.

[0020] Among them, the quantitative detection of the new coronavirus N protein showed good detection linearity, a wide linear range, high accuracy, high precision, strong specificity and high sensitivity.

[0021] In this embodiment, according to Figure 1As shown, 220 μL of the incubated recombinant new coronavirus N protein standard or the inactivated vaccine sample to be tested was mixed with NH4HCO3 solution to a total volume of 480 μL, and then 20 μL of trypsin solution was added, and enzymatic hydrolysis was carried out overnight at 37°C. After the enzymatic hydrolysis reaction was completed, 1 μL of formic acid was added to terminate the activity of trypsin, thereby ending the enzymatic hydrolysis process. Subsequently, 20 μL of trypsin solution was added, and enzymatic hydrolysis was carried out overnight at 37°C. After the enzymatic hydrolysis reaction was completed, 1 μL of formic acid was added to terminate the activity of trypsin, thereby ending the enzymatic hydrolysis process. The mass-to-charge ratio of the parent ion in the quantitative ion pair of the quantitative peptide segment was 563.7853, and the mass-to-charge ratio of the daughter ion was 679.3520 and / or 892.4640. The high-performance liquid chromatography detection conditions of the new coronavirus N protein standard or the inactivated vaccine sample of the new coronavirus to be tested are as follows: Chromatographic column: C18 column, preferably ACQUITY UPLC PEPTIDE BEH C18 (2.1mm×100mm), injection volume of 5~10μL, column temperature of 60℃; mobile phase composition: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile solution containing 0.1% formic acid; mobile phase B is gradient eluted from 0 to 45% in 0-20min, flow rate is 0.3mL / min, and the mass spectrometry detection conditions of the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested are as follows: ion source parameters: nebulizer flow rate 3.0L / min, dryer flow rate 10.0L / min, interface temperature 300℃, desolvation gas temperature 526℃, electrospray voltage 3.0kV; DL temperature 250℃, parent ion mass-to-charge ratio 563 .7853, product ion mass-to-charge ratio of 679.3520, the Q1Pre deviation (v) of the quantification ion pair is -28.0; CE=-19; Q3Pre deviation (v) -34.0, the Q1Pre deviation (v) of the quantification ion pair with a parent ion mass-to-charge ratio of 563 .7853 and a product ion mass-to-charge ratio of 892.4640 is -30.0; CE=-21; Q3Pre deviation (v) -26.0, including the antigen kit.

[0022] Among them, the content of the new coronavirus N protein can be accurately determined. On the liquid chromatography-tandem mass spectrometry platform, these quantitative peptides and ion pairs have demonstrated excellent performance, ensuring the accuracy and reliability of the test results.

[0023] The workflow of the present invention is as follows: when using the detection method and kit for the novel coronavirus N-protein designed by the present invention, first check the equipment to be used, incubate and enzymatically hydrolyze the novel coronavirus N protein standard; use a high performance liquid chromatography-tandem triple quadrupole mass spectrometry detection device to perform enzymatic hydrolysis on the recombinant novel coronavirus N protein standard, use the standard content as the horizontal coordinate, incubate the novel coronavirus inactivated vaccine sample to be tested, and perform enzymatic hydrolysis; then detect the supernatant after enzymatic hydrolysis, and quantify the sub-ion peak area of ​​the peptide segment, and then According to the standard curve and the injection volume, the content of the quantitative peptide of the new coronavirus in the inactivated vaccine sample to be tested is calculated, and finally the content is divided by the injection volume to obtain the concentration of the new coronavirus N protein in the inactivated vaccine sample to be tested. The process of incubating the new coronavirus N protein standard or the inactivated vaccine sample to be tested includes: adding an anionic surfactant to a medium volume of the new coronavirus N protein standard or the inactivated vaccine sample to be tested, incubating at 70-90°C for 20-60 minutes, and then adding dithiothreitol to a final concentration of 25mmol / L, incubated at 65°C for 60 min, and after the temperature dropped to room temperature, iodoacetamide solution was added to a final concentration of 50 mmol / L, and incubated at room temperature in the dark for 20-60 min. The quantitative peptides and their corresponding quantitative ions were used for quantitative detection of the novel coronavirus N protein in liquid chromatography-tandem mass spectrometry, showing good detection linearity, wide linear range, high accuracy, high precision, strong specificity and high sensitivity. This method effectively avoids interfering factors such as viral protein structure changes, protein modifications and matrix effects that may occur in the inactivated new coronavirus vaccine samples. It is suitable for the detection of N protein content in intermediate products and finished products at all stages of the inactivated new coronavirus vaccine.

[0024] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the N-protein of the new coronavirus, comprising the following steps: Step 1: Incubate the N protein standard of the new coronavirus and perform enzymatic hydrolysis; use high performance liquid chromatography tandem triple quadrupole mass spectrometry to analyze and detect the N protein standard of the recombinant new coronavirus after enzymatic hydrolysis, with the content of the standard as the horizontal axis; Step 2: Incubate the inactivated new coronavirus vaccine sample to be tested and perform enzymatic hydrolysis; then detect the supernatant after enzymatic hydrolysis, calculate the content of the new coronavirus quantitative peptide segment in the inactivated vaccine sample to be tested by the peak area of ​​the daughter ion of the quantitative peptide segment, and according to the standard curve and the injection volume, finally divide this content by the injection volume to obtain the concentration of the new coronavirus N protein in the inactivated vaccine sample to be tested; Step 3: The process of incubating the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested includes: adding an anionic surfactant to a medium volume of the N protein standard of the new coronavirus or the inactivated vaccine sample to be tested, incubating at 70-90°C for 20-60 minutes, then adding dithiothreitol to a final concentration of 25mmol / L, incubating at 65°C for 60 minutes, and after the temperature drops to room temperature, adding iodoacetamide solution to a final concentration of 50mmol / L, and incubating at room temperature in the dark for 20-60 minutes.

2. A method for detecting the novel coronavirus N-protein according to claim 1, characterized in that: The steps of enzymatic hydrolysis are as follows: Mix 220 μL of the incubated recombinant novel coronavirus N protein standard or the inactivated vaccine sample to be tested with NH4HCO3 solution to make the total volume reach 480 μL.

3. A method and kit for detecting the N-protein of a novel coronavirus according to claim 2, characterized in that: Then, 20 μL of trypsin solution was added and enzymatic hydrolysis was carried out at 37°C overnight. After the enzymatic hydrolysis reaction was completed, 1 μL of formic acid was added to terminate the activity of trypsin, thereby ending the enzymatic hydrolysis process.

4. A method for detecting the novel coronavirus N-protein according to claim 1, characterized in that: In the quantitative ion pair of the quantitative peptide segment, the mass-to-charge ratio of the parent ion is 563.7853, and the mass-to-charge ratio of the daughter ion is 679.3520 and / or 892.4640.

5. A method for detecting the novel coronavirus N-protein according to claim 1, characterized in that: The high performance liquid chromatography detection conditions of the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested are as follows: Chromatographic column: C18 column, preferably ACQUITY UPLC PEPTIDE BEH C18 (2.1mm×100mm), injection volume is 5~10μL.

6. A method for detecting the novel coronavirus N-protein according to claim 5, characterized in that: The temperature of the chromatographic column is 60°C; the mobile phase composition is: mobile phase A is a 1% formic acid aqueous solution, and mobile phase B is an acetonitrile solution containing 1% formic acid; the mobile phase B content changes from 0 to 45% in 0 to 20 minutes for gradient elution, and the flow rate is 0.3 mL / min.

7. A method for detecting the novel coronavirus N-protein according to claim 1, characterized in that: The mass spectrometry detection conditions of the N protein standard of the new coronavirus or the inactivated vaccine sample of the new coronavirus to be tested are as follows: ion source parameters: nebulizer flow rate 0 L / min, dryer flow rate 10 L / min, interface temperature 300°C, desolvation gas temperature 526°C, electrospray voltage 3.0 kV; DL temperature 250°C.

8. A method for detecting the novel coronavirus N-protein according to claim 1, characterized in that: The quantitative ion pair with a parent ion mass-to-charge ratio of 567853 and a product ion mass-to-charge ratio of 679.3520 has a Q1Pre deviation (v) of -28.0; CE=-19; and a Q3Pre deviation (v) of -34.

0.

9. A method for detecting the novel coronavirus N-protein according to claim 1, characterized in that: The quantitative ion pair with a parent ion mass-to-charge ratio of 567853 and a daughter ion mass-to-charge ratio of 892.4640 has a Q1Pre deviation (v) of -30.0; CE=-21; and Q3Pre deviation (v) of -26.

0.

10. A kit for detecting the N-protein of a novel coronavirus, suitable for use in a method for detecting the N-protein of a novel coronavirus as described in any one of claims 1 to 9 above, characterized in that: Includes antigen kit.

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