CN113325172A

CN113325172A - Novel coronavirus detection kit

Info

Publication number: CN113325172A
Application number: CN202010126712.3A
Authority: CN
Inventors: 程方明; 马腾腾; 钱纯亘; 徐天旺; 胡鹍辉
Original assignee: Shenzhen Yhlo Biotech Co Ltd
Current assignee: Shenzhen Yhlo Biotech Co Ltd
Priority date: 2020-02-28
Filing date: 2020-02-28
Publication date: 2021-08-31
Anticipated expiration: 2040-02-28
Also published as: CN113325172B

Abstract

The invention relates to a novel coronavirus detection kit, which comprises an antigen component and a luminescent component; the antigen component contains a novel coronavirus protein-magnetic bead complex, wherein the novel coronavirus protein-magnetic bead complex comprises an S1 protein-magnetic bead complex, an S2 protein-magnetic bead complex, an E protein-magnetic bead complex, an M protein-magnetic bead complex and an N protein-magnetic bead complex; the luminescent component contains anti-human IgG antibody, and the anti-human IgG antibody is marked with a luminescent marker. The novel coronavirus detection kit disclosed by the invention is based on a two-step indirect immunoassay principle of direct chemiluminescence, the concentration of the novel coronavirus IgG antibody in a sample to be detected can be rapidly judged by using a corresponding chemiluminescence determinator, and all novel coronavirus antigens which can possibly generate the antibody are coated on the surface of magnetic beads, so that the omission caused by different antibodies generated in vivo of individual patients is avoided to the greatest extent.

Description

Novel coronavirus detection kit

Technical Field

The invention relates to the technical field of virus detection, in particular to a novel coronavirus detection kit.

Background

The novel coronavirus (SARS-CoV-2, also known as 2019-nCoV, HCoV-19) belongs to coronavirus of beta genus, the particle is circular or elliptical, the diameter is 60 nm-140 nm, the gene characteristic is obviously different from SARS, and the coronavirus has higher infection capacity compared with SARS. Based on epidemiological investigation, the incubation period of COVID-19 is 1-14 days, most of which are 3-7 days, symptoms mainly comprise fever, dry cough and hypodynamia, severe patients mostly have dyspnea and/or hypoxemia after one week of morbidity, and severe patients can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, coagulation dysfunction, multiple organ failure and the like. Therefore, how to quickly diagnose suspected cases and how to effectively monitor the disease process will set the focus of epidemic situation fighting for treating COVID-19.

According to the protocol for diagnosis and treatment of novel coronavirus pneumonia (trial sixth edition), the method for diagnosing COVID-19 is to detect the positivity of novel coronavirus nucleic acid by real-time fluorescence RT-PCR or to determine the high homology of virus gene sequencing and the known novel coronavirus. However, the problem of missed detection of nucleic acid detection reagents is exposed again until the epidemic situation develops, and the positive detection rate is only 30% -50%, which is mainly caused by the limitation of the detection mode. Nucleic acid detection is generally performed by collecting nasopharyngeal swabs, sputum, blood and feces of suspected patients, extracting RNA fragments in the sputum, blood and feces, and performing fluorescence RT-PCR, wherein a large amount of human cells and bacteria are mixed in the sampling process, so that the abundance of RNA to be detected is low, a detection system cannot detect the RNA, and false negative is generated finally. Meanwhile, except for blood drawing, other sampling modes can cause the sampling personnel to be exposed to the environment possibly containing pathogens, and have potential infection risks. In addition, the preparation process of the nucleic acid detection sample needs many steps and long operation time, and has high requirements on the technical level of detection personnel.

Disclosure of Invention

Accordingly, there is a need for a novel coronavirus detection kit with high detection sensitivity, high detection speed and low omission factor.

A novel coronavirus detection kit comprises an antigen component and a luminescent component; the antigen component contains novel coronavirus protein-magnetic bead complexes, wherein the novel coronavirus protein-magnetic bead complexes comprise S1 protein-magnetic bead complexes, S2 protein-magnetic bead complexes, E protein-magnetic bead complexes, M protein-magnetic bead complexes and N protein-magnetic bead complexes; the luminescent component contains an anti-human IgG antibody, and the anti-human IgG antibody is marked with a luminescent marker.

In one embodiment, the molar ratio of the S1 protein, the S2 protein, the E protein, the M protein and the N protein in the antigen component is (2-5): 1-5): 2-5: (0.1-3): 0.1-3.

In one embodiment, the antigen component has a total concentration of the novel coronavirus protein-magnetic bead complexes of 0.1mg/mL to 0.5 mg/mL.

In one embodiment, the anti-human IgG antibody is a murine anti-human IgG antibody.

In one embodiment, the Fc fragment of the anti-human IgG antibody is derived from a species other than mouse.

In one embodiment, the species other than murine is selected from any of the following species: horses, pigs, rabbits, dogs and sheep.

In one embodiment, the source of the Fc fragment of the anti-human IgG antibody is an equine Fc fragment.

In one embodiment, the concentration of the anti-human IgG antibody in the luminescent component is 1-400. mu.g/mL.

In one embodiment, the kit further comprises a sample dilution component, wherein the sample dilution component comprises a blocking agent and dithiothreitol.

In one embodiment, the sample dilution composition further comprises one or more of PBS, BSA, IgG of isotype origin to the Fab fragment of the anti-human IgG antibody, cholesterol, trehalose, mannitol, glycine, arginine, glutathione, casein, and surfactants.

In one embodiment, the concentration of each substance in the sample dilution composition is: PBS 10 mM-100 mM, BSA 0.1 wt% -5 wt%, IgG 10 mu g/mL-200 mu g/mL from the same species as the Fab fragment of the anti-human IgG antibody, blocker 40 mu g/mL-60 mu g/mL, dithiothreitol 0.1 mM-10 mM, cholesterol 1 mM-10 mM, trehalose 1 mM-10 mM, mannitol 1 mM-200 mM, glycine 1 mM-50 mM, arginine 1 mM-50 mM, glutathione 1 mM-10 mM, casein 1 mM-50 mM, and surfactant 0.1 wt% -2 wt%.

In one embodiment, the blocking agent comprises one or more of MAK33-IgG1/IgG1 Poly, MAK33-IgG1/Fab1 Poly, Poly MAK IgG2b/Fab2a, and IEP-Framework.

In one embodiment, the preparation method of the novel coronavirus protein-magnetic bead complex comprises the following steps: the novel coronavirus protein is treated with a3 mM-7 mM sodium dodecyl sulfate solution to obtain a denatured antigen, and then the denatured antigen is used to coat magnetic beads.

In one embodiment, the luminescent label comprises one or more of acridinium ester, acridinium ester sulfonamide, acridinium ester toluene sulfonamide, acridinium ester p-methyl sulfonamide, acridinium ester trifluoromethyl sulfonamide.

The novel coronavirus detection kit disclosed by the invention is based on a two-step indirect immunity method principle of direct chemiluminescence, and can be used for quickly judging the concentration of the novel coronavirus IgG antibody in a sample to be detected by using a corresponding chemiluminescence determinator. In the detection process, RNA extraction operation is not required to be carried out on the sample, the detection steps are fewer, the operation time is shortened, and the false negative result caused by low RNA abundance can be avoided. And an automatic chemiluminescence determinator can be used in a matched manner, so that the contact between personnel and samples possibly carrying pathogens is reduced, and the infection probability is reduced to the greatest extent. Meanwhile, the chemiluminescence technology has sensitive detection and high detection speed, and is far superior to nucleic acid detection.

In the antigen components of the novel coronavirus detection kit, all novel coronavirus antigens which can possibly generate antibodies are coated on the surfaces of the magnetic beads, so that the missing detection of individual patients caused by different antibodies generated in vivo is avoided to the greatest extent.

In the luminescent component of the novel coronavirus detection kit, the Fc segment of the antibody is replaced by the recombinant mouse anti-human IgG antibody of the horse Fc segment, so that possible interference of heterophagy antibody HAMA can be avoided on the premise of ensuring the affinity of the antibody, and the detection accuracy is further improved.

The novel coronavirus detection kit disclosed by the invention dilutes a sample to be detected by using a sample dilution component containing substances such as a blocking agent, dithiothreitol and the like, so that possible interference of a heterophagy antibody HAMA and interference of rheumatoid factors RF can be reduced to the greatest extent, and the detection accuracy is further improved.

In the preparation process of the protein-magnetic bead compound, the novel coronavirus related antigen is treated by SDS with certain concentration to release epitope, so that the possibility of combining with novel coronavirus IgG antibodies in different samples is increased, the detection sensitivity is improved, and the possibility of false negative or missing detection is further reduced.

Drawings

FIG. 1 is a map of plasmid pAc-kappa-CH 3 andpAAc-lambda-CH 3 (Progen).

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

According to research results, after a patient is infected with the novel coronavirus, the patient can generate IgM antibodies in about 3-7 days, the IgG antibodies can rise after 1 week, the IgM antibodies are the antibodies which appear earliest in humoral immune reaction, the detection of the early IgM antibodies can shorten the window period of detection after the virus infects the human body, the detection of the IgM antibodies and the IgG antibodies simultaneously in clinic can assist diagnosis and monitoring of the disease condition, and the method has important significance for judgment after recovery. Therefore, when detecting novel coronavirus IgG antibodies, how to comprehensively detect the content and the change of all novel coronavirus IgG antibodies in blood of infected persons is the key point of IgG antibody detection. Therefore, the selection and proportioning of the antigen is very important when developing the serological detection of the novel coronavirus pneumonia and quantifying the content of the novel coronavirus antibody in the blood of a suspected patient or a confirmed patient.

The chemiluminescence technology is mainly based on the principle that the concentration of an object to be detected in a detection system and the chemiluminescence intensity of the system form quantitative linear inertia under a certain condition, and a special instrument is used for detecting the chemiluminescence intensity, so that the content of the object to be detected is determined. According to the reaction principle, the chemiluminescence technology can be divided into an indirect method, a capture method, a competition method and a sandwich method, wherein the indirect method is to coat an antigen on the surface of a solid phase, the antibody to be detected can be rapidly enriched according to the immunological principle of antigen-antibody combination, and the content of the antibody to be detected can be detected through a proper secondary antibody.

The novel coronavirus gene encodes various structural proteins, mainly including spike glycoprotein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N), which can be used as antigens of novel human immune coronavirus, the commercially available antigens are mostly S protein and N protein, and the content of antibodies excited by S protein and (or) N protein can not be comprehensively reflected by the generation and titer change of specific IgG antibodies in an infected person. Meanwhile, since viral infection causes a strong immune response, a large amount of Rheumatoid Factor (RF) may be produced, which may affect the accurate measurement of the content of specific IgG and IgM antibodies in a human body. Therefore, how to reduce or eliminate RF interference is also a key issue to overcome to accurately measure IgG and IgM antibodies specific for the new coronavirus pneumonia.

The novel coronavirus detection kit provided by the embodiment of the invention comprises an antigen component and a luminescent component. The antigen component contains a novel coronavirus protein-magnetic bead complex, and the novel coronavirus protein-magnetic bead complex comprises an S1 protein-magnetic bead complex, an S2 protein-magnetic bead complex, an E protein-magnetic bead complex, an M protein-magnetic bead complex and an N protein-magnetic bead complex. The luminescent component contains anti-human IgG antibody, and the anti-human IgG antibody is marked with a luminescent marker. Wherein, the S1 protein is Spike protein Spike-S1 subunit, the S2 protein is Spike protein Spike-S2 subunit, the N protein is nucleocapsid protein, the E protein is capsid protein, and the M protein is membrane protein.

The novel coronavirus detection kit disclosed by the invention is based on a two-step indirect immunity method principle of direct chemiluminescence, and can be used for quickly judging the concentration of the novel coronavirus IgG antibody in a sample to be detected by using a corresponding chemiluminescence determinator. In the detection process, RNA extraction operation is not required to be carried out on the sample, the detection steps are fewer, the operation time is shortened, and the false negative result caused by low RNA abundance can be avoided. And an automatic chemiluminescence determinator can be used in a matched manner, so that the contact between personnel and samples possibly carrying pathogens is reduced, and the infection probability is reduced to the greatest extent. Meanwhile, the chemiluminescence technology has sensitive detection and high detection speed, and is far superior to nucleic acid detection. In the antigen components of the novel coronavirus detection kit, all novel coronavirus antigens which can possibly generate antibodies are coated on the surfaces of paramagnetic magnetic beads, so that the detection omission of individual patients due to different antibodies generated in vivo is avoided to the greatest extent.

In one embodiment, the molar ratio of the S1 protein, the S2 protein, the E protein, the M protein and the N protein in the antigen component is (2-5): 1-5): 2-5): 0.1-3, preferably (3-4): 1.5-2.5): 3-4): 0.3-0.7. The ratio is an optimal ratio obtained by researching the change of time and abundance of antibodies aiming at different antigens generated by different individuals, and the detection rate and accuracy are optimal.

In a specific example, the antigen component has a total concentration of the novel coronavirus protein-magnetic bead complexes (including different bead sizes and surface groups) of 0.1mg/mL to 0.5mg/mL, preferably 0.3 mg/mL. Optionally, the antigen component further comprises a magnetic bead diluent, wherein the magnetic bead diluent comprises 10 mM-100 mM PBS, 0.1 wt% -5 wt% BSA, preservative and the like, and the pH is 5.5-8.5.

In a specific example, the anti-human IgG antibody is a murine anti-human IgG antibody. Preferably, the Fc fragment of the above-mentioned anti-human IgG antibody is an Fc fragment derived from a species other than mouse. Preferably, the species other than murine is selected from any of the following genera: horses, pigs, rabbits, dogs and sheep. More preferably, the source of the Fc fragment of the anti-human IgG antibody is an equine Fc fragment. By using the recombinant mouse anti-human IgG antibody with the replaced Fc segment of the antibody, possible interference of heterophagy antibody HAMA can be avoided on the premise of ensuring the affinity of the antibody, and the detection accuracy is further improved.

In a specific example, the concentration of the anti-human IgG antibody in the luminescent component is 1. mu.g/mL-400. mu.g/mL, preferably 200. mu.g/mL. Optionally, the luminescent component further comprises a marker diluent, wherein the marker diluent comprises 10 mM-100 mM PBS, 0.1 wt% -5 wt% BSA, Mouse IgG, a preservative and the like, and the pH is 5.5-8.5.

In one particular example, the luminescent label includes one or more of acridinium ester, acridinium ester sulfonamide, acridinium ester toluene sulfonamide, acridinium ester p-methyl sulfonamide, acridinium ester trifluoromethyl sulfonamide, but is not limited thereto.

In one embodiment, the novel coronavirus detection kit further comprises a sample dilution component, wherein the sample dilution component comprises a blocking agent and dithiothreitol. Preferably, the sample dilution composition further comprises one or more of PBS, BSA, IgG of source homologous to Fab fragments of the anti-human IgG antibody, cholesterol, trehalose, mannitol, glycine, arginine, glutathione, casein, surfactants and preservatives. By using the sample dilution component containing the blocking agent, dithiothreitol and other substances to dilute the sample to be detected, possible interference of heterophagy antibody HAMA and interference of rheumatoid factor RF can be reduced to the greatest extent, and the detection accuracy is further improved.

In one specific example, the concentrations of each substance in the sample dilution composition are: BS 10 mM-100 mM, BSA 0.1 wt% -5 wt%, IgG 10 mug/mL-200 mug/mL from the same species as Fab fragment of anti-human IgG antibody, blocker 40 mug/mL-60 mug/mL, dithiothreitol 0.1 mM-10 mM, cholesterol 1 mM-10 mM, trehalose 1 mM-10 mM, mannitol 1 mM-200 mM, glycine 1 mM-50 mM, arginine 1 mM-50 mM, glutathione 1 mM-10 mM, casein 1 mM-50 mM, surfactant 0.1 wt% -2 wt%, and preservative 0.1 wt% -0.5 wt%. Within the concentration range, the kit has better detection sensitivity and anti-interference capability.

In one particular example, the blocking agent includes one or more of, but is not limited to, MAK33-IgG1/IgG1 Poly, MAK33-IgG1/Fab1 Poly, Poly MAK IgG2b/Fab2a, and IEP-Framework. Alternatively, surfactants include, but are not limited to, Tween-20, Tween-80, and the like, and preservatives include, but are not limited to, NaN₃ProClin-300, and the like.

In one specific example, the preparation method of the novel coronavirus protein-magnetic bead complex comprises the following steps: the novel coronavirus protein is treated with a3 mM-7 mM sodium dodecyl sulfate solution to obtain a denatured antigen, and then the denatured antigen is used to coat the magnetic beads. Preferably, the concentration of SDS is 4mM to 6 mM. Because the types of the antigens are more and the conformation is complex, in the preparation process of the protein-magnetic bead compound, the recombinant novel coronavirus related antigen is treated by SDS with certain concentration to release the epitope, so that the possibility of combining with novel coronavirus IgG antibodies in different samples is increased, the detection sensitivity is improved, and the possibility of false negative or missed detection is further reduced.

The invention develops the novel coronavirus detection kit aiming at the defects of a nucleic acid detection method and possible omission of a chemical reflection detection technology according to the principle of a two-step indirect immunity method of a direct chemiluminescence technology. The kit is matched with a corresponding chemiluminescence determinator, and can realize automation (automatic sampling, automatic detection and automatic reporting), high flux (200 persons/hour) and rapid detection (25min reports) of the novel coronavirus pneumonia IgG antibody.

The present invention is described in further detail below with reference to specific examples.

Example 1

First, preparation of antigen

According to the gene sequence of the novel coronavirus and the composition of the shell of the novel coronavirus, a 293 expression system is applied to express and purify each protein (Spike protein Spike-S1 subunit, Spike-S2 subunit, nucleocapsid protein, capsid protein and membrane protein) which forms the novel coronavirus structural protein respectively as a coating antigen.

For example, expression purification of S1 protein:

selecting an RBD (receptor binding domain) protein of a novel coronavirus spike protein, wherein the amino acid sequence is as follows:

NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(SEQ ID NO:1)；

a signal peptide having the amino acid sequence: MFVFLVLLPLVSSQCV (SEQ ID NO: 2);

a transmembrane domain having the amino acid sequence: ELWLVLVAVGAGLLLLGLIILLL (SEQ ID NO: 3);

the tag peptide segment has the amino acid sequence as follows: WGQGGTHGQWNKPSKP (SEQ ID NO: 4).

Sequentially connecting the signal peptide, the RBD protein, the transmembrane domain and the tag peptide segment, and fusing into the RBD recombinant antigen, wherein the DNA sequence of the complete fusion protein is as follows:

ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATATTACAAACTTGTGCCCTTT TGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACT GTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCT ACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCA GACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAG GCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAG ATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAG CACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACT AATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACT GTTTGTGGACCTAAAAAGTCTACTGAGCTGTGGCTGGTGCTGGTGGCCGTGGGCGCTGGACTGCTGC TGCTGGGACTGATCATCCTGCTGCTGTGGGGTCAAGGCGGTACACATGGTCAATGGAACAAGCCTTC GAAGCCGTGA(SEQ ID NO:5)。

the DNA of the RBD recombinant protein was obtained by gene synthesis (general biological System (Anhui) Co., Ltd.). The DNA upstream of the RBD recombinant antigen is provided with Hind III site and Kozak sequence, and the downstream is provided with EcoR I site. The DNA is cut by corresponding restriction enzyme and is connected to a shuttle expression vector pcDNA3.1(+) cut by Hind III and EcoR I to obtain a recombinant plasmid pcDNA3.1(+) -RBD.

The recombinant plasmid was transferred to E.coli clone Top 10, and a single colony was selected and inoculated into 100mL of LB medium containing 100. mu.g/mL ampicillin, and cultured overnight at 37 ℃ and 200rpm for extraction of the recombinant plasmid. The recombinant plasmid was extracted using an endotoxin-free plasmid extraction kit (Tiangen Biochemical technology (Beijing) Ltd., DP 120). The 293T cell is plated and cultured until the confluency is about 85 percent, and then the recombinant plasmid pcDNA3.1(+) -RBD is transfected.

One dish of cells was transfected with 10. mu.g of plasmid DNA, the mass ratio of plasmid DNA to PEI was 1: 5. And uniformly mixing the plasmid DNA and the PEI with 250 mu L of PBS respectively, then vortex and uniformly mixing the two mixtures for 1min, standing for 15min at room temperature, uniformly dripping the mixture into a cell culture dish, uniformly mixing the mixture and then placing the mixture in an incubator for culturing for 48 h. After scraping the transfected cells, PBS resuspended and washed the cells twice, and then 1mL 10mM Na was applied to one dish of cells₂HPO₄50mM NaCl/1% Triton X-100, pH7.4 buffer (buffer A) for heavy suspension, and placing in 4 deg.C environment for extraction for 30 min; 16000g, centrifuging for 15min to obtain supernatant as the extracted recombinant RBD antigen, and storing at-20 deg.C.

Expression and purification of N protein:

selecting a nucleocapsid protein (N protein) having the amino acid sequence:

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRG QGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGAL NTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARM AGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRR GPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFK DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADS TQA(SEQ ID NO:6)；

and the sequence of the oligo-lysine is RRRRR, and the amino acid sequence of the connecting peptide is GSGS. The oligo-lysine, the N protein and the oligo-lysine are all connected by connecting peptide and fused into N protein recombinant protein, and the DNA sequence of the N protein recombinant protein is

ATGCGTCGCAGACGTCGAGGATCTGGCTCGATGAGTGATAACGGCCCGCAGAATCAGCGCAATGCCC CGCGCATTACCTTTGGTGGCCCGAGTGATAGTACCGGTAGTAATCAGAATGGTGAACGCAGCGGCGCC CGCAGTAAACAGCGTCGTCCGCAGGGTCTGCCGAATAATACCGCAAGCTGGTTTACCGCCCTGACCC AGCATGGCAAAGAAGATCTGAAATTTCCGCGCGGTCAGGGTGTTCCGATTAATACCAATAGCAGTCCG GATGATCAGATTGGTTATTATCGTCGTGCCACCCGTCGTATTCGCGGCGGCGATGGTAAAATGAAAGAT CTGAGTCCGCGTTGGTATTTTTATTATCTGGGCACCGGTCCGGAAGCAGGCCTGCCTTATGGTGCCAAT AAGGATGGCATTATTTGGGTGGCAACCGAAGGCGCCCTGAATACCCCGAAAGATCATATTGGTACCCG TAATCCGGCCAATAATGCAGCAATTGTGCTGCAGCTGCCGCAGGGCACCACCCTGCCTAAAGGCTTTT ATGCAGAAGGTAGTCGCGGTGGCAGCCAGGCAAGCAGCCGTAGTAGTAGCCGCAGTCGTAATAGCAG TCGCAATAGCACCCCGGGTAGCAGTCGTGGTACCAGCCCGGCACGCATGGCAGGCAATGGTGGTGAC GCCGCACTGGCACTGCTGCTGCTGGATCGTCTGAATCAGCTGGAAAGCAAAATGAGTGGCAAAGGTC AGCAGCAGCAGGGCCAGACCGTGACCAAAAAATCTGCCGCAGAAGCCAGCAAAAAACCGCGTCAG AAACGTACCGCCACCAAAGCCTATAATGTGACCCAGGCATTTGGCCGTCGCGGCCCGGAACAGACCC AGGGTAATTTTGGTGACCAGGAACTGATTCGCCAGGGCACCGATTATAAACATTGGCCGCAGATTGCA CAGTTTGCACCGAGTGCCAGTGCCTTTTTCGGCATGAGCCGCATTGGCATGGAAGTTACCCCGAGTGG TACCTGGCTGACCTATACCGGTGCCATTAAGCTGGATGATAAAGATCCGAATTTTAAAGATCAGGTTAT TCTGCTGAACAAACATATTGATGCCTATAAAACCTTCCCGCCGACCGAACCGAAAAAAGATAAAAAG AAAAAGGCAGATGAGACCCAGGCACTGCCGCAGCGCCAGAAAAAACAGCAGACCGTGACACTGCT GCCGGCAGCAGATCTGGATGATTTTAGTAAACAGCTGCAGCAGAGCATGAGTAGCGCCGATAGTACCC AGGCCGGATCTGGCTCGCGTCGCAGACGTCGATAA(SEQ ID NO:7)。

The DNA of the N protein recombinant protein was obtained by gene synthesis (general biological System (Anhui) Co., Ltd.). The DNA of the N protein recombinant protein has an Nco I site at the upstream and a Hind III site at the downstream. The DNA was digested with the corresponding restriction enzymes, ligated to expression vector pET-28a (+) digested with Nco I and Hind III, and yielded recombinant plasmid pET-28a (+) -N.

The recombinant plasmid was transferred into Escherichia coli expression strain BL21(DE3), and a single colony was picked up and inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, and cultured overnight at 37 ℃ and 200 rpm. The following day, 1% of the overnight culture was aspirated into 1L of fresh LB medium containing 50. mu.g/mL kanamycin, cultured at 37 ℃ at 200rpm to OD₆₀₀The expression was induced with IPTG (isopropylthiogalactoside) at a final concentration of 1mM for 20h at 18 ℃. Centrifuging at 4 deg.C and 12000g for 3min to collect thallus, and precooling thallus per liter bacterial liquid with 40mL 50mM Na precooled on ice₂HPO₄Suspending in buffer solution (buffer solution A) of pH7.4, adding small amount of RNase, crushing with a high pressure homogenizer, centrifuging at 12000g at 4 deg.C for 30min, filtering the supernatant with 0.22 μm filter membrane, and passing through cation exchange column.

The cation exchange column was equilibrated with 10 column volumes of buffer a, the supernatant was added, and unbound protein was washed off with at least 10 column volumes of buffer a. 50mM Na was prepared₂HPO₄500mM NaCl, pH7.4 buffer (buffer B), the protein of interest is eluted with a linear gradient of 20 column volumes of buffer containing 0mM to 500mM NaCl. Fractions of the target protein with a purity of 90% were selected, pooled and extensively dialyzed against 10mM Tris 7.5 buffer (buffer C) at 4 ℃. To further remove impurities, the dialyzed N protein was applied to a Buffer C pre-equilibrated anion exchange column, and the permeate was collected and thoroughly dialyzed into PBS Buffer. After the N protein is ultrafiltered and concentrated, the protein is stored at the temperature of minus 20 ℃ for standby.

Preparation of antigen-magnetic bead complexes

Taking 0.2mL of the envelope antigen, adding 0.02mL of phosphate solution containing 5mM of Sodium Dodecyl Sulfate (SDS), mixing uniformly, and incubating for 30min at 37 ℃ to open the secondary structure of the antigen and release an antibody binding epitope, thereby finally obtaining the recombinant envelope antigen denatured by SDS. 10mg of the suspension of nanobeads modified with carboxyl groups at the ends was washed several times with 10 mM-20 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer, pH6.0, and resuspended in 0.3 mL. 0.1mL of a 20mg/mL aqueous solution of N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) was added thereto, and the mixture was mixed at 25 ℃ for 30 minutes. After magnetic separation, the magnetic beads were resuspended to 1.0mL with 20mM phosphate buffer, 0.2mg of the coated antigen was added to the mixture, and the mixture was reacted at 25 ℃ for 2 hours. And (3) carrying out magnetic separation, removing unbound coating antigen, and resuspending the unbound coating antigen to 10mg/mL by using a Tris buffer solution containing 1% BSA and having the pH of 7.5 to obtain the carboxylated magnetic particles coated by the recombinant antigen, and storing the carboxylated magnetic particles at the temperature of 2-8 ℃ for later use.

Preparation of recombinant mouse anti-human IgG antibody

(1) The Xho1 enzyme cutting site is added at the upstream of the antibody heavy chain variable region gene, the Nhe1 enzyme cutting site is added at the downstream of the gene, the gene is cloned on a plasmid pAc-kappa-CH 3and Ac-lambda-CH 3(Progen), the map is shown in figure 1, and the gene sequence of the antibody heavy chain variable region is shown in Table 1.

(2) The equine antibody heavy chain constant region gene (CH1-CH3) (FC fragment) is shown in Table 1, with Nhe1 cleavage site added at the upstream of the equine antibody heavy chain constant region gene (CH1-CH3) (FC fragment), and BamHI cleavage site added at the downstream of the gene, and cloned into plasmid pAc-kappa-CH 3and Ac-lambda-CH 3 (Progen).

(3) Adding Sac1 enzyme cutting site at the upstream of the antibody light chain variable region gene and adding Hind111 enzyme cutting site at the downstream of the gene, cloning to plasmid pAc-kappa-CH 3 andpAAc-lambda-CH 3(Progen), wherein the sequence of the antibody light chain variable region gene is shown in Table 1; the complete construction plasmid pAc-. kappa. -CH3 andpAAc-. lamda. -CH3(Progen) was finally obtained.

(4) Cell transfection

HEK293 cells were prepared in logarithmic growth phase with a viability rate higher than 90%. On the day of transfection, sampling and counting the cell density and the survival rate, wherein the cell density should be (3-5) multiplied by 10⁶cells/mL, the activity rate is higher than 90%. Adjusting cell density to 3X 10⁶cells/mL, cell fluid volume per bottle 20 mL. Diluting 20 μ g DNA (10 μ g clone construction plasmid 1# +10 μ g clone construction plasmid 2#) with 150mM NaCl to total volume of 0.5mL, and mixing gently; diluting 100 mu LSinofection transfection reagent by 150mM NaCl until the total volume is 0.5mL, and mildly and uniformly mixing; the diluted DNA and transfection reagent are simultaneously and independently kept still for about 5 minutes and then are gently mixed, the total volume is 1mL, and then the chamber is filled with the DNA and the transfection reagentThe mixture was allowed to warm for 10 minutes. And dropwise adding the transfection solution into the cell culture solution, slightly shaking the culture flask while dropwise adding, uniformly shaking, putting the culture flask back to the shaking table for continuous culture, and collecting cell supernatant after 48 hours of transfection.

(5) Cell supernatant antibody purification

proteinG affinity chromatography was used. Firstly, preparing a proteinG affinity chromatographic column, balancing the column by PBS, taking ascites to pass through the column, then washing the column by PBS, eluting by 50nmol/L glycine hydrochloride solution, collecting eluent, measuring the OD value of each collecting pipe, reserving the eluent in a peak area, dialyzing the eluent and collecting the eluent.

TABLE 1 antibody Gene sequences

Preparation of antigen component

The antigen-magnetic bead compound prepared by the method and the magnetic bead diluent. Wherein the molar ratio of the S1 protein, the S2 protein, the N protein, the E protein and the M protein is 3.5:2:3.5:0.5: 0.5. The total concentration of the antigen-magnetic bead complex was 0.3mg/mL, and the magnetic bead diluent contained 10mM PBS, 0.5 wt% BSA, 0.2% Proclin 300, etc., and had a pH of 7.4.

Preparation of luminescent component

The recombinant mouse anti-human IgG antibody labeled with the chemiluminescent marker and a marker diluent. Wherein the concentration of the Mouse anti-human IgG antibody is 200. mu.g/mL, the marker diluent contains 10mM PBS, 0.5 wt% BSA, 0.5mg/mL Mouse IgG, 0.2% Proclin 300 and the like, and the pH is 7.4.

Sixthly, preparation of sample dilution component

The concentrations of the substances are respectively as follows: PBS 10mM, BSA 0.5 wt%, Mouse IgG 50. mu.g/mL, blocker 50. mu.g/mL, dithiothreitol 1mM, cholesterol 1mM, trehalose 5mM, mannitol 50mM, glycine 10mM, arginine 5mM, glutathione 1mM, casein 2mM, surfactant 2 wt%, 0.2% Proclin 300.

Example 2

This example is substantially the same as example 1 except that different concentrations of SDS (2mM, 7mM, 10mM) were used in the preparation process of the antigen-magnetic bead complexes.

The resulting kits were tested for 10 positive samples using different concentrations of SDS. As can be seen from the above table, when 2mM SDS was used, individual samples were tested negative because of insufficient exposure of antigen upon treatment. The treatment effect was best when 5mM SDS was used, and 10 positive samples were detected. In contrast, in the case of 7mM and 10mM, the protein is completely denatured due to the use of a larger concentration, so that the antigen site is destroyed, and the result of a partial positive test is negative.

Example 3

This example is essentially the same as example 1, except that in the antigen component, different molar ratios of S1 protein, S2 protein, E protein, M protein and N protein were used, respectively.

As can be seen from the above table, 10 positive samples (positive samples Nos. 1 to 10) and 10 negative samples (negative samples Nos. 10 to 20) were tested. The proportion of the E protein and the M protein compound is increased, so that the conditions that the positive sample has missed detection and the negative sample has false positive are probably caused by detecting antibodies generated by other coronavirus stimulating organisms; the contents of S1 and S2 proteins and the contents of M protein and N protein are increased at the same time, although a positive sample can be detected, a negative sample also generates false positive possibly due to the detection of antibodies generated by other coronavirus-stimulated organisms; the content of the N protein compound is increased independently, positive results can be detected, but false positive appears in negative samples; the selected difference of the novel coronavirus and the antigen sites of other coronaviruses is 3.5:2:3.5:0.5:0.5, and other structural proteins are considered, so that the specificity is ensured, and the detection rate is improved.

Example 4

This example is essentially the same as example 1 except that in the luminescent component, the anti-human IgG antibody is a murine anti-human IgG antibody substituted with another Fc fragment.

As can be seen from the above table, 10 positive samples (positive samples Nos. 1 to 10) and 10 negative samples (negative samples Nos. 10 to 20) were tested. The Fc end of the mouse anti-human IgG interferes with a negative sample due to the possible existence of HAMA antibody in the sample, so that the detection result is false positive; the problem of false positive can be improved by replacing a mouse anti-human Fc end with a sheep anti-human Fc segment, but a false positive result still exists; the replacement with the horse Fc fragment can improve false positive, and positive results can be detected.

The specific steps of detection using the novel coronavirus detection kit are as follows. The kit can be matched with an iFlash-3000 series chemiluminescence determinator produced by the company for use, wherein the steps of cleaning solution, pre-excitation solution, excitation solution and corresponding cleaning and luminescence reading are default settings of the determinator, and other steps are finished by manual setting programs and processing by the determinator.

1. Pre-diluting a sample: setting a program to suck 10 mu l of sample into a reaction cup, adding a reagent sample dilution component, preferably in a dilution ratio of 1:10(1: 1-1: 20), uniformly mixing, and incubating for 5min at 37 ℃ to eliminate or reduce false positive or false negative caused by interference of an autophagy antibody HAMA and a rheumatoid factor RF.

2. Adding magnetic beads: mu.l of the above antigen component reagent was added to the cuvette and incubated at 37 ℃ for 5min to allow the novel coronavirus IgG antibody to sufficiently react with the antigen immobilized on the surface of the magnetic beads, followed by performing a washing procedure to wash away the unbound and adsorbed sample.

3. Adding acridine: add 100 μ l of luminescent component into the reaction cup, and incubate at 37 ℃ for 5min to make the antibody with luminescent marker fully combine with the analyte, then execute washing procedure to wash away the unbound antibody.

4. And (3) determining the concentration of the substance to be detected: the chemiluminescent labels bound to the magnetic beads via a series of reactions are excited according to a luminescence program preset by the instrument, and the luminescence values are read by a chemiluminescent analyzer.

5. And (3) generating a report: and reporting the content of the novel coronavirus IgG antibody in the patient blood sample according to the calibration curve and the reference interval so as to assist clinical judgment of the disease condition.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shenzhen Shenhuilong Biotech stock Co., Ltd

<120> novel coronavirus detection kit

<140> 2020101267123

<141> 2020-02-28

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 201

<212> PRT

<213> Artificial Sequence

<400> 1

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

1 5 10 15

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

20 25 30

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

35 40 45

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

50 55 60

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

65 70 75 80

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

85 90 95

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

100 105 110

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

115 120 125

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

130 135 140

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

145 150 155 160

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

165 170 175

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

180 185 190

Thr Val Cys Gly Pro Lys Lys Ser Thr

195 200

<210> 2

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 2

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

<210> 3

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 3

Glu Leu Trp Leu Val Leu Val Ala Val Gly Ala Gly Leu Leu Leu Leu

1 5 10 15

Gly Leu Ile Ile Leu Leu Leu

20

<210> 4

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 4

Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys Pro Ser Lys Pro

1 5 10 15

<210> 5

<211> 771

<212> PRT

<213> Artificial Sequence

<400> 5

Ala Thr Gly Thr Thr Thr Gly Thr Thr Thr Thr Thr Cys Thr Thr Gly

1 5 10 15

Thr Thr Thr Thr Ala Thr Thr Gly Cys Cys Ala Cys Thr Ala Gly Thr

20 25 30

Cys Thr Cys Thr Ala Gly Thr Cys Ala Gly Thr Gly Thr Gly Thr Thr

35 40 45

Ala Ala Thr Ala Thr Thr Ala Cys Ala Ala Ala Cys Thr Thr Gly Thr

50 55 60

Gly Cys Cys Cys Thr Thr Thr Thr Gly Gly Thr Gly Ala Ala Gly Thr

65 70 75 80

Thr Thr Thr Thr Ala Ala Cys Gly Cys Cys Ala Cys Cys Ala Gly Ala

85 90 95

Thr Thr Thr Gly Cys Ala Thr Cys Thr Gly Thr Thr Thr Ala Thr Gly

100 105 110

Cys Thr Thr Gly Gly Ala Ala Cys Ala Gly Gly Ala Ala Gly Ala Gly

115 120 125

Ala Ala Thr Cys Ala Gly Cys Ala Ala Cys Thr Gly Thr Gly Thr Thr

130 135 140

Gly Cys Thr Gly Ala Thr Thr Ala Thr Thr Cys Thr Gly Thr Cys Cys

145 150 155 160

Thr Ala Thr Ala Thr Ala Ala Thr Thr Cys Cys Gly Cys Ala Thr Cys

165 170 175

Ala Thr Thr Thr Thr Cys Cys Ala Cys Thr Thr Thr Thr Ala Ala Gly

180 185 190

Thr Gly Thr Thr Ala Thr Gly Gly Ala Gly Thr Gly Thr Cys Thr Cys

195 200 205

Cys Thr Ala Cys Thr Ala Ala Ala Thr Thr Ala Ala Ala Thr Gly Ala

210 215 220

Thr Cys Thr Cys Thr Gly Cys Thr Thr Thr Ala Cys Thr Ala Ala Thr

225 230 235 240

Gly Thr Cys Thr Ala Thr Gly Cys Ala Gly Ala Thr Thr Cys Ala Thr

245 250 255

Thr Thr Gly Thr Ala Ala Thr Thr Ala Gly Ala Gly Gly Thr Gly Ala

260 265 270

Thr Gly Ala Ala Gly Thr Cys Ala Gly Ala Cys Ala Ala Ala Thr Cys

275 280 285

Gly Cys Thr Cys Cys Ala Gly Gly Gly Cys Ala Ala Ala Cys Thr Gly

290 295 300

Gly Ala Ala Ala Gly Ala Thr Thr Gly Cys Thr Gly Ala Thr Thr Ala

305 310 315 320

Thr Ala Ala Thr Thr Ala Thr Ala Ala Ala Thr Thr Ala Cys Cys Ala

325 330 335

Gly Ala Thr Gly Ala Thr Thr Thr Thr Ala Cys Ala Gly Gly Cys Thr

340 345 350

Gly Cys Gly Thr Thr Ala Thr Ala Gly Cys Thr Thr Gly Gly Ala Ala

355 360 365

Thr Thr Cys Thr Ala Ala Cys Ala Ala Thr Cys Thr Thr Gly Ala Thr

370 375 380

Thr Cys Thr Ala Ala Gly Gly Thr Thr Gly Gly Thr Gly Gly Thr Ala

385 390 395 400

Ala Thr Thr Ala Thr Ala Ala Thr Thr Ala Cys Cys Thr Gly Thr Ala

405 410 415

Thr Ala Gly Ala Thr Thr Gly Thr Thr Thr Ala Gly Gly Ala Ala Gly

420 425 430

Thr Cys Thr Ala Ala Thr Cys Thr Cys Ala Ala Ala Cys Cys Thr Thr

435 440 445

Thr Thr Gly Ala Gly Ala Gly Ala Gly Ala Thr Ala Thr Thr Thr Cys

450 455 460

Ala Ala Cys Thr Gly Ala Ala Ala Thr Cys Thr Ala Thr Cys Ala Gly

465 470 475 480

Gly Cys Cys Gly Gly Thr Ala Gly Cys Ala Cys Ala Cys Cys Thr Thr

485 490 495

Gly Thr Ala Ala Thr Gly Gly Thr Gly Thr Thr Gly Ala Ala Gly Gly

500 505 510

Thr Thr Thr Thr Ala Ala Thr Thr Gly Thr Thr Ala Cys Thr Thr Thr

515 520 525

Cys Cys Thr Thr Thr Ala Cys Ala Ala Thr Cys Ala Thr Ala Thr Gly

530 535 540

Gly Thr Thr Thr Cys Cys Ala Ala Cys Cys Cys Ala Cys Thr Ala Ala

545 550 555 560

Thr Gly Gly Thr Gly Thr Thr Gly Gly Thr Thr Ala Cys Cys Ala Ala

565 570 575

Cys Cys Ala Thr Ala Cys Ala Gly Ala Gly Thr Ala Gly Thr Ala Gly

580 585 590

Thr Ala Cys Thr Thr Thr Cys Thr Thr Thr Thr Gly Ala Ala Cys Thr

595 600 605

Thr Cys Thr Ala Cys Ala Thr Gly Cys Ala Cys Cys Ala Gly Cys Ala

610 615 620

Ala Cys Thr Gly Thr Thr Thr Gly Thr Gly Gly Ala Cys Cys Thr Ala

625 630 635 640

Ala Ala Ala Ala Gly Thr Cys Thr Ala Cys Thr Gly Ala Gly Cys Thr

645 650 655

Gly Thr Gly Gly Cys Thr Gly Gly Thr Gly Cys Thr Gly Gly Thr Gly

660 665 670

Gly Cys Cys Gly Thr Gly Gly Gly Cys Gly Cys Thr Gly Gly Ala Cys

675 680 685

Thr Gly Cys Thr Gly Cys Thr Gly Cys Thr Gly Gly Gly Ala Cys Thr

690 695 700

Gly Ala Thr Cys Ala Thr Cys Cys Thr Gly Cys Thr Gly Cys Thr Gly

705 710 715 720

Thr Gly Gly Gly Gly Thr Cys Ala Ala Gly Gly Cys Gly Gly Thr Ala

725 730 735

Cys Ala Cys Ala Thr Gly Gly Thr Cys Ala Ala Thr Gly Gly Ala Ala

740 745 750

Cys Ala Ala Gly Cys Cys Thr Thr Cys Gly Ala Ala Gly Cys Cys Gly

755 760 765

Thr Gly Ala

770

<210> 6

<211> 419

<212> PRT

<213> Artificial Sequence

<400> 6

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 5 10 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170 175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

180 185 190

Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

210 215 220

Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Ala Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

385 390 395 400

Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

405 410 415

Thr Gln Ala

<210> 7

<211> 1317

<212> PRT

<213> Artificial Sequence

<400> 7

Ala Thr Gly Cys Gly Thr Cys Gly Cys Ala Gly Ala Cys Gly Thr Cys

1 5 10 15

Gly Ala Gly Gly Ala Thr Cys Thr Gly Gly Cys Thr Cys Gly Ala Thr

20 25 30

Gly Ala Gly Thr Gly Ala Thr Ala Ala Cys Gly Gly Cys Cys Cys Gly

35 40 45

Cys Ala Gly Ala Ala Thr Cys Ala Gly Cys Gly Cys Ala Ala Thr Gly

50 55 60

Cys Cys Cys Cys Gly Cys Gly Cys Ala Thr Thr Ala Cys Cys Thr Thr

65 70 75 80

Thr Gly Gly Thr Gly Gly Cys Cys Cys Gly Ala Gly Thr Gly Ala Thr

85 90 95

Ala Gly Thr Ala Cys Cys Gly Gly Thr Ala Gly Thr Ala Ala Thr Cys

100 105 110

Ala Gly Ala Ala Thr Gly Gly Thr Gly Ala Ala Cys Gly Cys Ala Gly

115 120 125

Cys Gly Gly Cys Gly Cys Cys Cys Gly Cys Ala Gly Thr Ala Ala Ala

130 135 140

Cys Ala Gly Cys Gly Thr Cys Gly Thr Cys Cys Gly Cys Ala Gly Gly

145 150 155 160

Gly Thr Cys Thr Gly Cys Cys Gly Ala Ala Thr Ala Ala Thr Ala Cys

165 170 175

Cys Gly Cys Ala Ala Gly Cys Thr Gly Gly Thr Thr Thr Ala Cys Cys

180 185 190

Gly Cys Cys Cys Thr Gly Ala Cys Cys Cys Ala Gly Cys Ala Thr Gly

195 200 205

Gly Cys Ala Ala Ala Gly Ala Ala Gly Ala Thr Cys Thr Gly Ala Ala

210 215 220

Ala Thr Thr Thr Cys Cys Gly Cys Gly Cys Gly Gly Thr Cys Ala Gly

225 230 235 240

Gly Gly Thr Gly Thr Thr Cys Cys Gly Ala Thr Thr Ala Ala Thr Ala

245 250 255

Cys Cys Ala Ala Thr Ala Gly Cys Ala Gly Thr Cys Cys Gly Gly Ala

260 265 270

Thr Gly Ala Thr Cys Ala Gly Ala Thr Thr Gly Gly Thr Thr Ala Thr

275 280 285

Thr Ala Thr Cys Gly Thr Cys Gly Thr Gly Cys Cys Ala Cys Cys Cys

290 295 300

Gly Thr Cys Gly Thr Ala Thr Thr Cys Gly Cys Gly Gly Cys Gly Gly

305 310 315 320

Cys Gly Ala Thr Gly Gly Thr Ala Ala Ala Ala Thr Gly Ala Ala Ala

325 330 335

Gly Ala Thr Cys Thr Gly Ala Gly Thr Cys Cys Gly Cys Gly Thr Thr

340 345 350

Gly Gly Thr Ala Thr Thr Thr Thr Thr Ala Thr Thr Ala Thr Cys Thr

355 360 365

Gly Gly Gly Cys Ala Cys Cys Gly Gly Thr Cys Cys Gly Gly Ala Ala

370 375 380

Gly Cys Ala Gly Gly Cys Cys Thr Gly Cys Cys Thr Thr Ala Thr Gly

385 390 395 400

Gly Thr Gly Cys Cys Ala Ala Thr Ala Ala Gly Gly Ala Thr Gly Gly

405 410 415

Cys Ala Thr Thr Ala Thr Thr Thr Gly Gly Gly Thr Gly Gly Cys Ala

420 425 430

Ala Cys Cys Gly Ala Ala Gly Gly Cys Gly Cys Cys Cys Thr Gly Ala

435 440 445

Ala Thr Ala Cys Cys Cys Cys Gly Ala Ala Ala Gly Ala Thr Cys Ala

450 455 460

Thr Ala Thr Thr Gly Gly Thr Ala Cys Cys Cys Gly Thr Ala Ala Thr

465 470 475 480

Cys Cys Gly Gly Cys Cys Ala Ala Thr Ala Ala Thr Gly Cys Ala Gly

485 490 495

Cys Ala Ala Thr Thr Gly Thr Gly Cys Thr Gly Cys Ala Gly Cys Thr

500 505 510

Gly Cys Cys Gly Cys Ala Gly Gly Gly Cys Ala Cys Cys Ala Cys Cys

515 520 525

Cys Thr Gly Cys Cys Thr Ala Ala Ala Gly Gly Cys Thr Thr Thr Thr

530 535 540

Ala Thr Gly Cys Ala Gly Ala Ala Gly Gly Thr Ala Gly Thr Cys Gly

545 550 555 560

Cys Gly Gly Thr Gly Gly Cys Ala Gly Cys Cys Ala Gly Gly Cys Ala

565 570 575

Ala Gly Cys Ala Gly Cys Cys Gly Thr Ala Gly Thr Ala Gly Thr Ala

580 585 590

Gly Cys Cys Gly Cys Ala Gly Thr Cys Gly Thr Ala Ala Thr Ala Gly

595 600 605

Cys Ala Gly Thr Cys Gly Cys Ala Ala Thr Ala Gly Cys Ala Cys Cys

610 615 620

Cys Cys Gly Gly Gly Thr Ala Gly Cys Ala Gly Thr Cys Gly Thr Gly

625 630 635 640

Gly Thr Ala Cys Cys Ala Gly Cys Cys Cys Gly Gly Cys Ala Cys Gly

645 650 655

Cys Ala Thr Gly Gly Cys Ala Gly Gly Cys Ala Ala Thr Gly Gly Thr

660 665 670

Gly Gly Thr Gly Ala Cys Gly Cys Cys Gly Cys Ala Cys Thr Gly Gly

675 680 685

Cys Ala Cys Thr Gly Cys Thr Gly Cys Thr Gly Cys Thr Gly Gly Ala

690 695 700

Thr Cys Gly Thr Cys Thr Gly Ala Ala Thr Cys Ala Gly Cys Thr Gly

705 710 715 720

Gly Ala Ala Ala Gly Cys Ala Ala Ala Ala Thr Gly Ala Gly Thr Gly

725 730 735

Gly Cys Ala Ala Ala Gly Gly Thr Cys Ala Gly Cys Ala Gly Cys Ala

740 745 750

Gly Cys Ala Gly Gly Gly Cys Cys Ala Gly Ala Cys Cys Gly Thr Gly

755 760 765

Ala Cys Cys Ala Ala Ala Ala Ala Ala Thr Cys Thr Gly Cys Cys Gly

770 775 780

Cys Ala Gly Ala Ala Gly Cys Cys Ala Gly Cys Ala Ala Ala Ala Ala

785 790 795 800

Ala Cys Cys Gly Cys Gly Thr Cys Ala Gly Ala Ala Ala Cys Gly Thr

805 810 815

Ala Cys Cys Gly Cys Cys Ala Cys Cys Ala Ala Ala Gly Cys Cys Thr

820 825 830

Ala Thr Ala Ala Thr Gly Thr Gly Ala Cys Cys Cys Ala Gly Gly Cys

835 840 845

Ala Thr Thr Thr Gly Gly Cys Cys Gly Thr Cys Gly Cys Gly Gly Cys

850 855 860

Cys Cys Gly Gly Ala Ala Cys Ala Gly Ala Cys Cys Cys Ala Gly Gly

865 870 875 880

Gly Thr Ala Ala Thr Thr Thr Thr Gly Gly Thr Gly Ala Cys Cys Ala

885 890 895

Gly Gly Ala Ala Cys Thr Gly Ala Thr Thr Cys Gly Cys Cys Ala Gly

900 905 910

Gly Gly Cys Ala Cys Cys Gly Ala Thr Thr Ala Thr Ala Ala Ala Cys

915 920 925

Ala Thr Thr Gly Gly Cys Cys Gly Cys Ala Gly Ala Thr Thr Gly Cys

930 935 940

Ala Cys Ala Gly Thr Thr Thr Gly Cys Ala Cys Cys Gly Ala Gly Thr

945 950 955 960

Gly Cys Cys Ala Gly Thr Gly Cys Cys Thr Thr Thr Thr Thr Cys Gly

965 970 975

Gly Cys Ala Thr Gly Ala Gly Cys Cys Gly Cys Ala Thr Thr Gly Gly

980 985 990

Cys Ala Thr Gly Gly Ala Ala Gly Thr Thr Ala Cys Cys Cys Cys Gly

995 1000 1005

Ala Gly Thr Gly Gly Thr Ala Cys Cys Thr Gly Gly Cys Thr Gly Ala

1010 1015 1020

Cys Cys Thr Ala Thr Ala Cys Cys Gly Gly Thr Gly Cys Cys Ala Thr

1025 1030 1035 1040

Thr Ala Ala Gly Cys Thr Gly Gly Ala Thr Gly Ala Thr Ala Ala Ala

1045 1050 1055

Gly Ala Thr Cys Cys Gly Ala Ala Thr Thr Thr Thr Ala Ala Ala Gly

1060 1065 1070

Ala Thr Cys Ala Gly Gly Thr Thr Ala Thr Thr Cys Thr Gly Cys Thr

1075 1080 1085

Gly Ala Ala Cys Ala Ala Ala Cys Ala Thr Ala Thr Thr Gly Ala Thr

1090 1095 1100

Gly Cys Cys Thr Ala Thr Ala Ala Ala Ala Cys Cys Thr Thr Cys Cys

1105 1110 1115 1120

Cys Gly Cys Cys Gly Ala Cys Cys Gly Ala Ala Cys Cys Gly Ala Ala

1125 1130 1135

Ala Ala Ala Ala Gly Ala Thr Ala Ala Ala Ala Ala Gly Ala Ala Ala

1140 1145 1150

Ala Ala Gly Gly Cys Ala Gly Ala Thr Gly Ala Gly Ala Cys Cys Cys

1155 1160 1165

Ala Gly Gly Cys Ala Cys Thr Gly Cys Cys Gly Cys Ala Gly Cys Gly

1170 1175 1180

Cys Cys Ala Gly Ala Ala Ala Ala Ala Ala Cys Ala Gly Cys Ala Gly

1185 1190 1195 1200

Ala Cys Cys Gly Thr Gly Ala Cys Ala Cys Thr Gly Cys Thr Gly Cys

1205 1210 1215

Cys Gly Gly Cys Ala Gly Cys Ala Gly Ala Thr Cys Thr Gly Gly Ala

1220 1225 1230

Thr Gly Ala Thr Thr Thr Thr Ala Gly Thr Ala Ala Ala Cys Ala Gly

1235 1240 1245

Cys Thr Gly Cys Ala Gly Cys Ala Gly Ala Gly Cys Ala Thr Gly Ala

1250 1255 1260

Gly Thr Ala Gly Cys Gly Cys Cys Gly Ala Thr Ala Gly Thr Ala Cys

1265 1270 1275 1280

Cys Cys Ala Gly Gly Cys Cys Gly Gly Ala Thr Cys Thr Gly Gly Cys

1285 1290 1295

Thr Cys Gly Cys Gly Thr Cys Gly Cys Ala Gly Ala Cys Gly Thr Cys

1300 1305 1310

Gly Ala Thr Ala Ala

1315

<210> 8

<211> 420

<212> DNA

<213> Artificial Sequence

<400> 8

atggctcctg cccaattttt aggtttgctt ctcctatgtt tccagggcac tcgttgcgaa 60

gttcaactgg tcgagtctgg agggggtgta gtgcagcccg gccgctcctt acgattgtca 120

tgtgcagcgt cgggatttac cttcagtgat tatccaatga attgggttcg gcaagctccg 180

gggaaaggtc ttgaatgggt cagctctatt tccggctcag gagggtcgac atactatgcc 240

gacagtgtaa agggtagatt tacgatcagc agggataact ctaaaaatac tctctaccta 300

cagatgaact ccctgcgtgc agaggacacc gcggtgtatt actgcgctaa gggcttattc 360

atggttacaa cgtatgcctt tgattactgg ggacaaggga ctaccgtcac agtatcatcg 420

<210> 9

<211> 1065

<212> DNA

<213> Artificial Sequence

<400> 9

gcctccacca ccgccccgaa ggtcttccct ctggctccca gctgtgggac cacatctgac 60

tccacggtgg ccctgggctg cctggtctcc agctacttcc cagagccagt gacagtgtcc 120

tggaactcgg gcacgctgac cagcggtgtg cgcaccttcc cgtccgtcct gcagtcctcg 180

gggctctact ccctcagcag catggtgact gtgcctgcca gcagcttgga gagcaagacc 240

tacatctgca acgtagccca cccggccagc agcaccaagg tggacaagag aatcgagccc 300

gtcctcccaa agcctacgac acctgcacct acagtgccgc taacaaccac agttccagtt 360

gagacgacta caccaccctg tccctgcgag tgtcccaaat gcccagctcc tgagctgcta 420

ggagggcctt ccgtgttcat cttcccccca aaaccgaagg acgtcctcat gatcacccga 480

acgcctgagg tcacctgcct ggttgtggac gtgagccatg acagctccga tgtcctgttc 540

acctggtatg tggacggcac agaggtgaag actgccaaga caatgccgaa cgaggaacag 600

aacaacagca cttaccgcgt ggtcagcgtc ctccgcatcc agcaccagga ctggctgaac 660

ggaaagaagt tcaagtgtaa ggtcaacaac caagccctcc cagcccctgt agagaggacc 720

atctccaagg ccacagggca aacccgggtg ccgcaggtgt atgtcctggc cccgcaccca 780

gatgagctgt ccaagaacaa ggtcagcgtg acctgcctgg tcaaggactt cttaccaacc 840

gacatcaccg tcgagtggca gagcaatgag catccagagc cagagggcaa gtacagaacc 900

actgaagccc agaaggacag cgacgggtcc tacttcctgt acagcaagct cactgtggag 960

acggacaggt ggcagcaggg aacgacattc acgtgtgtgg tgatgcatga ggctctccac 1020

aatcacgtca tgcagaagaa cgtctcccac tctccgggta aatga 1065

<210> 10

<211> 339

<212> DNA

<213> Artificial Sequence

<400> 10

atggctcctg cccaattttt aggtttgctt ctcctatgtt tccagggcac tcgttgcgaa 60

ctgacccaat ctccctcctc attatcggca agtgttggag atcgcgtcac aattacgtgt 120

cgagcgagcc agtctatctc ctcatatttg aattggtacc aacagaaacc agggaaggct 180

ccgaaacttc tcatatatgc cgcatcgagt ctacaaagcg gtgtaccttc tcggttttcc 240

ggctcaggat cggggactga cttcaccctg acaattagta gcttacagcc cgaggatttt 300

gcgacgtact attgccaaca gtcttactcc actccaccg 339

Claims

1. A novel coronavirus detection kit is characterized by comprising an antigen component and a luminescent component; the antigen component contains novel coronavirus protein-magnetic bead complexes, wherein the novel coronavirus protein-magnetic bead complexes comprise S1 protein-magnetic bead complexes, S2 protein-magnetic bead complexes, E protein-magnetic bead complexes, M protein-magnetic bead complexes and N protein-magnetic bead complexes; the luminescent component contains an anti-human IgG antibody, and the anti-human IgG antibody is marked with a luminescent marker.

2. The novel coronavirus detection kit of claim 1, wherein the antigen component comprises S1 protein, S2 protein, E protein, M protein and N protein in a molar ratio of (2-5): 1-5): 2-5: (0.1-3): 0.1-3.

3. The novel coronavirus detection kit of claim 2, wherein the antigen component is characterized in that the total concentration of the novel coronavirus protein-magnetic bead complexes is 0.1mg/mL to 0.5 mg/mL.

4. The novel coronavirus detection kit according to claim 1, wherein the anti-human IgG antibody is a murine anti-human IgG antibody.

5. The novel coronavirus detection kit according to claim 4, wherein the Fc fragment of the anti-human IgG antibody is an Fc fragment derived from a species other than mouse.

6. The novel coronavirus detection kit of claim 5, wherein the Fc fragment of the anti-human IgG antibody is derived from an equine Fc fragment.

7. The novel coronavirus detection kit of claim 1, further comprising a sample dilution component, wherein the sample dilution component comprises a blocking agent and dithiothreitol.

8. The novel coronavirus detection kit of claim 7, wherein the blocking agent comprises one or more of MAK33-IgG1/IgG1 Poly, MAK33-IgG1/Fab1 Poly, Poly MAK IgG2b/Fab2a, and IEP-Framework.

9. The novel coronavirus detection kit according to claim 1, wherein the novel coronavirus protein-magnetic bead complex is prepared by a method comprising the following steps: the novel coronavirus protein is treated with a3 mM-7 mM sodium dodecyl sulfate solution to obtain a denatured antigen, and then the denatured antigen is used to coat magnetic beads.

10. The novel coronavirus detection kit according to any one of claims 1 to 9, wherein the luminescent marker comprises one or more of acridinium ester, acridinium ester sulfonamide, acridinium ester toluene sulfonamide, acridinium ester p-methyl sulfonamide, and acridinium ester trifluoromethyl sulfonamide.