CN112384805A

CN112384805A - Kit and detection device for detecting novel coronavirus and preparation method thereof

Info

Publication number: CN112384805A
Application number: CN202080002802.XA
Authority: CN
Inventors: 马哲; 赵文祥; 田子琦
Original assignee: Hunan Fasiter Biotechnology Co ltd
Current assignee: Hunan Fasiter Biotechnology Co ltd
Priority date: 2020-05-31
Filing date: 2020-07-15
Publication date: 2021-02-19

Abstract

The invention relates to a kit and a detection device for detecting novel coronavirus and a preparation method thereof, wherein whether the serum of a sample to be detected contains an antibody against the novel coronavirus is indirectly detected by utilizing SARS-CoV-2 Spike protein Spike, an S-a1 protein fragment and an S-a2 protein fragment. The detection reagent has the advantages of accurate detection, high detection efficiency and stable and accurate detection result, and can be identified by naked eyes, and experiments show that the detection device can achieve the sensitivity of detecting 0.016 mu g S protein IgG antibodies and 0.031 mu g S protein IgM antibodies by naked eye observation. The invention is suitable for places such as hospitals, airports, customs, families and the like, can judge results within a few minutes, thereby preventing epidemic spread as soon as possible and having very important popularization and application values.

Description

Kit and detection device for detecting novel coronavirus and preparation method thereof

Technical Field

The invention relates to the detection of antibody, in particular to a colloidal gold chromatography kit for detecting SARS-CoV-2 coronavirus Spike protein antibody, a detection device and a preparation method thereof.

Background

New coronavirus pneumonia (COVID-19) has become a public health emergent event of international concern, and 700 thousands of cases are diagnosed globally until now, and the number of dead people exceeds 40 thousands. For the new coronavirus epidemic situation, the method accelerates the detection, screening and confirmation of suspected and closely contacted people and the like, and is an extremely effective prevention and control measure.

The novel coronavirus (SARS-CoV-2) is a beta genus coronavirus having a diameter of about 60 to 140nm and an envelope. SARS-CoV-2 encodes 15 non-structural proteins (nsp 1-nsp 10, nsp 12-nsp 16), 4 structural proteins (S, E, M and N), and 8 auxiliary proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf 14). The spike protein of the novel coronavirus facilitates viral entry into target cells. The receptor binding domain (S-RBD) of the spike protein binds to its receptor angiotensin converting enzyme 2(ACE2), which facilitates attachment of the virus to the surface of target cells, thereby infecting them.

Researchers in various countries around the world are researching rapid and accurate novel coronavirus detection methods, and the main methods at present are molecular detection, cell culture detection and antibody detection.

One is a molecular assay (PCR method) which detects whether a patient sample contains an RNA sequence specific for SARS-CoV-2 and thereby diagnoses the early etiology of COVID-19. However, the detection method has certain false negative, and the positive rate is only 30-50%. The reason is that the different parts and methods of clinical sample collection cause the too small loading of SARS-CoV-2 and the failure of detection; in addition, diagnostic reagents have limited reliability and sensitivity. Meanwhile, the method needs professional personnel to operate and requires high experimental working conditions; metagenomic sequencing (mNGS) is to extract RNA of a novel coronavirus from lower respiratory secretion of a patient, construct a virus cDNA library, perform high-throughput sequencing, and identify whether a genome sequence is highly homologous with SARS-CoV-2 by database alignment analysis. The advantages of the mNGS for detecting SARS-CoV-2 are that the specificity and the sensitivity are high, the defects are obvious, the requirement of the instrument configuration is high, and the detection period is long; the cell culture method is an aseptic technique, needs an aseptic operation room, an ultra-clean workbench, a cell culture box, a centrifuge, a microscope and the like, has complex experimental technique, needs operation of experienced professional technicians, has long experimental period and is not suitable for rapid detection of diseases; antibody detection the main enzyme-linked immunosorbent assay (ELISA): the ELISA method requires high antigen purity and good specificity, otherwise nonspecific reaction can occur, the operation procedure is complex, repeated washing is needed, false positive and false negative can be easily caused if the washing times are insufficient or excessive, the operator is easily damaged, the environment is easily polluted, the experiment time is long, and the detection result can be obtained in more than two hours. The method must be provided with a microplate reader and a plate washer, which are difficult to access in basic laboratories and small clinics. The reagent for detecting the virus antibody by the colloidal gold chromatography can be applied to places such as hospitals, airports, customs, families and the like, and can judge results within a few minutes, so that epidemic spread can be prevented as soon as possible; blood detection solves the problem that sampling of throat swabs is not accurate to the greatest extent.

However, the accuracy of the existing detection of the novel coronavirus needs to be further improved, and particularly, the rapid variation of the novel coronavirus brings higher difficulty to the detection, so that it is necessary to develop an efficient and accurate detection method as soon as possible based on the current severe epidemic situation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a novel coronavirus detection kit or reagent strip, a preparation method and application thereof, which can be applied to places such as hospitals, airports, customs, families and the like, and can judge results within a few minutes so as to prevent epidemic spread as soon as possible.

In order to achieve the above objects, the assay of the present invention utilizes novel coronavirus S proteins and specific fragments thereof to detect IgM and IgG antibodies of the novel coronavirus Spike protein Spike. Among them, the S protein is a membrane protein, which constitutes an envelope particle of a virus, and is a main component of a virus-infected host cell. The novel coronavirus is intended to infect cells by first introducing its genetic material RNA into the host cell, a process which is mainly carried out by the S protein. The protuberance of the S protein is a key site for its interaction with receptors and membrane fusion. However, the entire length of the S protein is prone to mutation, and if the S protein is simply detected, the mutated virus is likely to be unable to be detected. In contrast, through intensive research, the inventors find out specific fragments S-a1 and S-a2 of the S protein, so that after the S protein is mutated, a target analyte can be detected by detecting the specific fragments S-a1 and S-a2, the application range of the kit is improved, and the result accuracy of the kit is ensured.

Therefore, the invention provides a kit for detecting novel coronavirus, which comprises S protein, S-a1 protein fragment and S-a2 protein fragment of SARS-CoV-2 coronavirus, wherein the amino acid sequence of the S-a1 protein fragment is shown as SEQ ID NO. 3 of a sequence table, and the amino acid sequence of the S-a2 protein fragment is shown as SEQ ID NO. 5 of the sequence table.

Further, in order to utilize an antibody detection method, namely to detect a novel coronavirus to S protein antibody, the kit further comprises a mouse IgG antibody, a mouse anti-human IgG antibody and/or a mouse anti-human IgM antibody, a goat anti-mouse IgG antibody, an S protein IgG antibody as a standard and/or an S protein IgM antibody, and a sample treatment solution.

Wherein the novel coronavirus is a beta coronavirus, particularly SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.

In a specific embodiment, the kit is used for indirect detection to determine whether the serum of a sample to be detected contains the antibody against the novel coronavirus, and preferably comprises the kit and related reagents for indirect detection.

Preferably, the kit is for detection by colloidal gold chromatography, whereby the kit comprises a detection device and a sample processing solution, preferably the sample dilution is a 0.1MPBS solution containing 0.2% (v) Tween 20 and 0.6% (v) Casein the detection device reagent strip or card. In one embodiment, the detection device is prepared by the following method:

(1) respectively spraying a mouse anti-human IgG antibody, and/or a mouse anti-human IgM antibody, and/or a goat anti-mouse IgG antibody onto the NC membrane;

(2) adding colloidal gold into mixed protein of the S protein, the S-a1 protein fragment and the S-a2 protein fragment, adding a mouse IgG antibody, washing and precipitating by using a labeling washing solution, dissolving by using a gold-labeled antibody preserving solution, uniformly paving on a glass fiber membrane (pre-treating by using a gold-labeled binding pad sealing solution), and freeze-drying to obtain a gold-labeled binding pad;

(3) and (3) overlapping and sticking the absorbent paper, the NC film obtained in the step (1), the gold-labeled bonding pad and the sample pad on a rubber plate, and cutting to obtain the reagent strip.

In another aspect, the present invention provides a device for detecting a novel coronavirus, wherein the device comprises a sample pad, a gold-labeled conjugate pad, a reaction pad and a water-absorbent pad, which are connected in sequence; wherein the gold-labeled conjugate pad is coated with colloidal gold labeled S protein, mixed protein of S-a1 protein fragment and S-a2 protein fragment and mouse IgG antibody; the reaction pad is sequentially provided with a detection line and a quality control line along the flowing direction of a sample to be detected, further the reaction pad is sequentially provided with a mouse anti-human IgG antibody or a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody along the flowing direction of the sample to be detected and respectively marked as a T line (namely a G line or an M line) and a C line, and preferably the reaction pad is sequentially provided with a mouse anti-human IgG antibody, a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody along the flowing direction of the sample to be detected and respectively marked as a G line, an M line and a C line; the device is a test reagent strip, and is more preferably a test card with a sample hole.

In particular, the novel coronavirus is a beta coronavirus, in particular a SARS-CoV-2 coronavirus, a SARS virus, a MERS virus, more preferably a SARS-CoV-2 coronavirus.

When the sample to be detected contains the human anti-novel coronavirus antibody, the antibody is firstly combined with the gold-labeled antigen, the reaction compound moves forwards along the envelope film due to the chromatography effect, and when the envelope antibody meets, the antibody-gold-labeled antigen-anti-antibody compound is formed and is enriched on the envelope line to form a red precipitation line which is a positive result, so that whether the sample contains the IgM/IgG antibody of the novel coronavirus (SARS-CoV-2) or not is quickly diagnosed.

The invention further provides a preparation method of the device for detecting the novel coronavirus, which comprises the following steps:

(1) respectively spraying a mouse anti-human IgG antibody and/or a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody onto an NC membrane;

(2) adding colloidal gold into mixed protein of the S protein, the S-a1 protein fragment and the S-a2 protein fragment, adding a mouse IgG antibody, washing and precipitating by using a labeling washing solution, dissolving by using a gold-labeled antibody preserving solution, uniformly paving on a fiber membrane pretreated by using a gold-labeled binding pad sealing solution in advance, and freeze-drying to obtain a gold-labeled binding pad;

(3) and (3) overlapping and sticking the absorbent paper, the NC film prepared in the step (1), the gold-labeled bonding pad prepared in the step (2) and the sample pad on a bottom plate, further cutting into strips, and assembling to obtain the reagent strip or further preparing the detection card with the sample holes.

In a preferred embodiment, the step (1) is that a mouse anti-human IgG antibody, a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody are respectively sprayed on an NC membrane and respectively marked as a G line, an M line and a C line; the mixed protein in the step (2) is formed by mixing S protein, S-a1 protein fragments and S-a2 protein fragments in equal proportion.

In one embodiment, the specific steps are as follows:

(1) diluting the antibody with 0.01mol/L PBS, spraying the antibody with the concentration of the mouse anti-human IgG being 1.0mg/mL on an NC membrane, and marking as a G line; or spraying the mouse anti-human IgM antibody with the concentration of 1.0mg/mL on an NC membrane, and marking as an M line; or respectively spraying a mouse anti-human IgG antibody and a mouse anti-human IgM antibody on an NC membrane, and respectively marking as a G line and an M line; the goat anti-mouse IgG antibody concentration is 1.0mg/mL, and the goat anti-mouse IgG antibody is sprayed on an NC membrane, marked as C line and is ready for use after completion.

(2) With 0.1M K₂CO₃Adjusting the pH value of the colloidal gold to 9, adding proteins mixed in equal proportion of the S protein, the S-a1 protein fragment and the S-a2 protein fragment according to 12 mu g/mL, simultaneously adding a mouse IgG antibody of 12 mu g/mL, fully and uniformly mixing, standing, centrifuging, discarding supernatant, washing precipitate with a gold-labeled washing solution, discarding supernatant, and dissolving precipitate with a gold-labeled antibody storage solution of one tenth of the initial volume of the colloidal gold; uniformly spreading the marked colloidal gold on a glass fiber membrane, and freeze-drying to obtain a gold-labeled bonding pad;

(3) sequentially overlapping and sticking the absorbent paper, the NC film prepared in the step (1), the gold-labeled bonding pad prepared in the step (2) and the sample pad on a polyvinyl chloride bottom plate or a rubber plate, slitting and assembling to obtain a reagent strip; or prepared into a detection card with sample holes.

Wherein, preferably, the sample pad is a glass fiber membrane or a non-woven fabric or a filter paper which is impregnated with a sample pad treatment solution, and further preferably, the sample pad treatment solution comprises the following components: tween 20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.

The gold-labeled washing liquid specifically adopted in the steps comprises the following components: 1% BSA, 0.01mol/L PBS, pH 9; the gold-labeled antibody preservation solution comprises: 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN₃0.01mol/L PBS, pH 9; the gold label bonding pad sealing liquid: 0.01M PBS, 1% BSA, 1% Tween-20, pH7.5.

The invention also provides application of the S protein, the S-a1 protein fragment and the S-a2 protein fragment mixed protein of SARS-CoV-2 coronavirus in preparing a reagent for detecting novel coronavirus, wherein the amino acid sequence of the S-a1 protein fragment is shown as SEQ ID NO. 3 in a sequence table, and the amino acid sequence of the S-a2 protein fragment is shown as SEQ ID NO. 5 in the sequence table.

In a preferred embodiment, said use is the detection of the presence or absence of antibodies against a novel coronavirus, preferably said novel coronavirus is a β -coronavirus, especially a SARS-CoV-2 coronavirus, a SARS virus, a MERS virus, more preferably a SARS-CoV-2 coronavirus, in a sample by colloidal gold chromatography

In a more specific application, the sample to be detected is selected from samples of convalescent persons and samples of healthy persons, wherein the samples are preferably at least one of nasopharyngeal swabs, sputum, alveolar lavage, blood and urine.

The detection device, such as a reagent strip or a detection card, can detect IgM and IgG antibodies simultaneously. Wherein IgM is produced early in viral infection and can be used to evaluate early infection of the virus; IgG is gradually increased in the later stage of infection, mainly plays a role in thoroughly eliminating viruses, and can be used for evaluating the in vivo immune response condition of a patient or the immune effect of a vaccine. The kit detects the infection of the virus from the angle of immune response, is just complementary with the current universal virus nucleic acid detection kit, and can effectively avoid the generation of false negative results. The reagent of the present invention adopts colloidal gold immunochromatography technology, selects purified novel coronavirus (SARS-CoV-2) Spike protein Spike antigen and two specific fragments thereof as solid phase substances, and detects whether serum contains human anti-novel coronavirus antibodies by an indirect method. Particularly, compared with the prior art, the to-be-detected object detected by the detection reagent strip is S protein, so that the problem of high false positive of N protein detection is solved, and the detection result is reliable and high in stability; more particularly, the reagent strip can detect the full length of the S protein, and can also detect the S-a1 protein fragment and the S-a2 protein fragment, so that the highly conserved protein fragments S-a1 and S-a2 can be identified when the S protein is mutated, the detection specificity of the kit is improved, and the application range of the detection kit is expanded. Meanwhile, the full length of the S protein can be determined, and specific fragments can be identified, so that the identification specificity and accuracy of the kit are greatly improved. Meanwhile, experiments show that the reagent strip can achieve the sensitivity of detecting 0.016 mu g S protein IgG antibody and 0.031 mu g S protein IgM antibody by visual observation, the detection result is stable and accurate, can be visually identified, and is suitable for places such as hospitals, airports, customs, families and the like, the result can be judged within a few minutes, so that the epidemic spread can be prevented as soon as possible, and the reagent strip has very important popularization and application values.

Drawings

FIG. 1 shows the alignment of S-a1 and S-a2 with different coronavirus S protein sequences;

FIG. 2 is a PAGE identification of the S protein;

FIG. 3 shows the results of the binding activity of S-a1, S-a2 and S protein to IgG antibodies in the serum of convalescent patients;

FIG. 4 is a graph showing the results of sensitivity measurements of the reagent strip prepared in example 4;

FIG. 5 is a diagram showing the specific test result of the test strip prepared in example 4 for the positive reference substance;

FIG. 6 is a schematic diagram showing the results of specific tests on 13 blood samples positive by PCR using the reagent strip prepared in example 4;

FIG. 7 is a schematic diagram showing the results of specific measurement of a blood sample of a normal person by the reagent strip prepared in example 4;

FIG. 8 is a diagram showing the results of specific detection by DTT treatment;

FIG. 9 is a graph showing the stability test results of the test strip prepared in example 4;

FIG. 10 is a graph showing the stability of the test results of the sample using the test strip prepared in example 4;

FIG. 11 shows the sample stabilization results of different sampling sources.

Detailed Description

In order to more clearly describe the technical contents of the present invention, the following further description is given in conjunction with specific embodiments.

In the following examples, only the parts of the present disclosure related to the improvement will be described in detail, and other steps or raw material sources not mentioned in the present disclosure are the same as those in the prior art, and will not be described herein.

Example 1: construction of S protein (spike protein) encoding gene fragment and strong antigenic S protein fragment

Synthesis of S protein-encoding Gene

The full-length base sequence of the Spike protein is shown as SEQ ID NO. 1, and the full-length nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The full-length coding gene of Spike protein was synthesized by a conventional method.

Designing the following upstream primer and downstream primer according to the full-length sequence, wherein the primer sequences are as follows:

primer and method for producing the same	Sequence Listing numbering	Sequence (5 '-3')
			Forward direction S-F	SEQ ID NO:7	ggggggGCGGCCGCATGTTTGTTTTTCTTGTTTT
Reverse S-R	SEQ ID NO:8	ggggggCCATGGTTATGTGTAATGTAA

And (3) taking the S protein coding sequence synthesized in the front as a template, and carrying out PCR amplification to obtain more Spike protein coding sequences. The PCR system comprises the following components:

	volume (uL)
		S-F(2μM)	5
S-R(2μM)	5
		Template plasmid (100ng/uL)	1
Primer mixture (SEQ ID NO:7 and SEQ ID NO:8)	5
		Water (W)	34
Total volume	50

The reaction solution was added to the PCR tube as above and mixed well. Placing the PCR reaction tube in a PCR instrument, and setting reaction conditions as follows: denaturation at 95 deg.C for 5 min; 30S at 95 ℃, 30S at 58 ℃ and 4min at 72 ℃; 10min at 72 ℃; the product can be stored for a short time at 4 ℃ and used for subsequent experiments.

Synthesis of the coding sequences for the S-a1 and S-a2 fragments

Through the analysis and alignment of Protein knowledgebase, the specific sequence with high immunogenicity in Spike is screened. The alignment of the two sequences with different coronavirus Spike protein sequences is shown in FIG. 1. Through analysis, the two sequences S-a1 and S-a2 positioned in different regions of the RBD are determined to obtain peptide fragments a1 and a2 through database analysis, and meanwhile, the effect is not lower than that of the S protein through related experiment verification, so that the method plays an important role in obtaining the finally obtained detection reagent card with good effect.

Wherein the first conserved region is a S-a1 fragment, the amino acid sequence of which is shown as SEQ ID NO. 3, and the coded nucleotide sequence of which is shown as SEQ ID NO. 4; the amino acid sequence of the other conserved region S-a2 fragment is shown in SEQ ID NO. 5, and the coded nucleotide sequence is shown in SEQ ID NO. 6. The coding sequences for the S-a1 and S-a2 fragments were also synthesized using conventional methods.

Wherein:

the full-length base sequence of Spike protein is shown below (SEQ ID NO:1)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

The full-length sequence of the Spike protein coding gene is shown as follows: (SEQ ID NO:2)

ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAA

The amino acid sequence of S-a1 is shown below: (SEQ ID NO:3)

VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF

The base sequence of S-a1 is shown as follows: (SEQ ID NO:4)

GTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTT

The amino acid sequence of S-a2 is shown below: (SEQ ID NO:5)

TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

The base sequence of S-a2 is shown as follows: (SEQ ID NO:6)

ACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC

Designing an upstream primer and a downstream primer aiming at the coding sequences of the S-a1 and S-a2 fragments respectively according to the sequences, wherein the primer sequences are as follows:

primer and method for producing the same	Sequence Listing numbering	Sequence (5 '-3')
			Positive direction S-a1-F	SEQ ID NO:9	ggggggGCGGCCGCGTCCAACCAACAGAATCTAT
Reverse S-a1-R	SEQ ID NO:10	ggggggCCATGGAAAATCATCTGGTAATTT
			Positive direction S-a2-F	SEQ ID NO:11	ggggggGCGGCCGCACAGGCTGCGTTATAGCTTGGA
Reverse S-a1-R	SEQ ID NO:12	ggggggCCATGGGAAATTGACACATTTTT

The nucleotide sequence of the S-a1 was amplified by PCR using the nucleotide sequence of the S-a1 fragment synthesized above as a template. Wherein, the PCR system is as follows:

	volume (uL)
		S-a1-F(2μM)	5
S-a1-R(2μM)	5
		Template plasmid (100ng/uL)	1
Primer mixture (SEQ ID NO:9 and SEQ ID NO:10)	5
		Water (W)	34
Total volume	50

The base sequence of the S-a2 fragment synthesized above was used as a template, and the base sequence of S-a2 was amplified by a PCR method. Wherein, the PCR system is as follows:

	volume (uL)
		S-a2-F(2μM)	5
S-a2-R(2μM)	5
		Template plasmid (100ng/uL)	1
Primer mixture (SEQ ID NO:11 and SEQ ID NO:12)	5
		Water (W)	34
Total volume	50

Construction of S protein, S-a1 and S-a2 expression plasmids

Pretreatment of pCDNA vectors

The enzyme digestion system is as follows:

pCDNA	50μg
		NotI-HF	5μL
NcoI-HF	5μL
		CutSmart
10×	20μL
		ddH2O	up 200μL
total of	200μL

Carrying out water bath digestion at 37 ℃ for 5h, and carrying out DNA electrophoresis after the digestion is finished. First, the electrophoresis tank was cleaned with pure water and replaced with a new 1 × TAE buffer. Adding 25 μ L10 × loading buffer into the enzyme-digested system, mixing uniformly, applying sample 50 μ L per well, and performing 150V electrophoresis for 30 min. Cutting off the target fragment of about 4000bp on a gel cutting instrument, transferring the target fragment into a pre-weighed 5mL centrifuge tube, weighing to obtain the weight of a gel block, and recovering the target fragment by using a Tiangen universal DNA purification recovery kit. The resulting product was assayed for DNA concentration using Nano-300 and diluted to 50 ng/. mu.L.

3.2. Connection of

The linking system is as follows:

	volume (μ L)
		The above-mentioned pretreated pCDNA (NcoI/NotI)	0.5
Full-length S protein gene or S-a1 or S-a2 fragment	8
		T4 Ligase	0.5
T4 Ligase Buffer	1
		Total volume	10

The full-length S protein gene or the fragment S-a1 or S-a2 is ligated to pCDNA vector according to the above ligation system, ligated at 16 ℃ for 30min, transformed into plates using TOP10 competence, and then the bacteria are picked the next day for detection. And comparing the sequencing results to obtain three expression plasmids with correct sequences, namely pCDNA-S, pCDNA-S-a1 and pCDNA-S-a2, which are used for subsequent experiments.

Example 2

This example differs from example 1 only in that, unlike the primers for amplifying the gene encoding S protein in this example, the following upstream and downstream primers were synthesized based on the S protein sequence:

primer and method for producing the same	Sequence Listing numbering	Sequence (5 '-3')
			Forward direction S-F	SEQ ID NO:13	cgtccggCAGTGCGTGAACCTGACCACCCGCACCCAGCTGCCGCCGGCGTATgc
Reverse S-R	SEQ ID NO:14	ggccgcGGTATAATGCAGTTTCACGCCTTTCAGCACccg

Example 3: expression purification of S protein, S-a1 and S-a2 protein fragments

1. Cell transfection:

i. 293F cells were cultured to a density of approximately 1X10⁶/mL；

ii.1mL of fresh medium resuspends 50 μ L PEI, 1mL of fresh medium resuspends 10 μ g of three plasmids, namely pCDNA-S, pCDNA-S-a1 and pCDNA-S-a2, respectively, mixing uniformly, and standing at room temperature for 5 min;

mixing the PEI premix with the plasmid premix uniformly, and standing for 20min at room temperature;

the PEI-plasmid complex was added dropwise to 20ml 293F cells, incubated for 5min on a CO2 shaker, and the supernatant was collected after three days of incubation.

2. And (3) purifying and identifying the protein:

i. collecting the supernatant into a 50mL centrifuge tube, centrifuging at 10000rpm for 10min, and taking the supernatant into a clean centrifuge tube;

washing the medium with a column pre-packed with 2mL of ProteinA packing with 10 bed volumes of deionized water;

iii.30mL PBS to equilibrate the media;

transferring the supernatant to a column and adjusting the flow rate to allow the liquid to flow out at a rate of 2s per drop;

v. wash sample with 30mL PBS;

eluting with vi.10mL of elution buffer solution at the speed of 10s per drop, collecting 1.5mL of eluent per EP tube, simultaneously dropwise adding 20 mu L of neutralization buffer solution, and continuously collecting 7 eluent;

using Nano-300 to determine the antibody concentration of each tube, combining the collected liquid with the concentration of more than 0.1mg/mL, putting the combined liquid into a dialysis bag, and adding PBS for dialysis;

filtering the antibody solution by using a 0.22 mu m filter membrane, collecting the antibody solution into a sterile centrifuge tube, and storing for later use;

mu.g of the Sample was diluted to 20. mu.L with 1 XPBS, 6. mu.L of 4 XPDS Sample Buffer and 4. mu.L of DTT were added, boiled in boiling water for 10min, at 12000rpm, and centrifuged for 1 min. The gel pieces were then visualized and photographed after being placed in water and destained for about 30min (the results are shown in FIG. 2 (the emphasis is on the full length S protein, and since S-a1 and S-a2 have only about 110 amino acids, the conventional PAGE gel in the laboratory shows no bands with a molecular weight of 10kD or less, and thus no indication is provided).

Example 4: detection of the binding Activity of S-a1, S-a2 and S proteins to IgG antibodies in the serum of convalescent patients

Binding of S-a1, S-a2 and S protein to IgG antibodies in the serum of convalescent patients was detected by ELISA. The method comprises the following specific steps:

1. diluting S-a1, S-a2 and S protein to the concentration of 1 mu g/ml, adding 100 mu L/hole of the diluted protein into a 96-well plate, sealing, and coating overnight at 4 ℃;

2. the supernatant was discarded and washed 3 times with 200. mu.L of PBST (TWEEN-200.1%) per well;

3. blocking 200 mu L of 2% BSA blocking solution in each hole for 1 hour at room temperature, removing the liquid from the enzyme label plate after blocking, and washing 3 times with 200 mu L of PBST (TWEEN-200.1%) in each hole;

4. adding 1 mu g/mL anti-human IgG-HRP antibody 100 mu L into each hole, and incubating for 2 hours at room temperature;

5. discarding the supernatant, drying the supernatant by using absorbent paper, and washing for 5 times by using 100 mu L of Wash Buffer;

6. adding 100 μ L of 1X TMB into each well, shaking in dark at room temperature, and developing for 10 min;

7. adding 100 mu L of stop solution into each hole;

8. reading a light absorption value of 450nm by using a microplate reader;

the results of the experiments are shown in FIG. 3, where it can be found that S-a1, S-a2 have slightly higher binding activity than Spike protein.

Example 5: preparation and use of detection device

Using the mixture of Spike, S-a1 and S-a2 of SARS-CoV-2 prepared as described above as a solid phase, the presence or absence of an antibody against the novel coronavirus in serum was examined by the principle of indirect method. The method comprises the following specific steps:

1. preparation of coating film: diluting the antibody with 0.01mol/L PBS, spraying the diluted antibody with the concentration of 1.0mg/mL of the mouse anti-human IgG antibody on an NC membrane, and marking as a G line; spraying the mouse anti-human IgM antibody with the concentration of 1.0mg/mL on an NC membrane, and marking as an M line; the goat anti-mouse IgG antibody concentration is 1.0mg/mL, and the goat anti-mouse IgG antibody is sprayed on an NC membrane, marked as C line and is ready for use after completion.

2. Preparation of gold labeled Spike, S-a1 and S-a2 mixtures: 0.1M of K₂CO₃Adjusting the pH value of the colloidal gold to 9 according to the solution 12 μ g/mL of protein mixed by equal weight proportions of Spike, S-a1 and S-a2 prepared in the previous examples (i.e., the final concentrations of Spike, S-a1 and S-a2 are 12 μ g/mL respectively) was added, while 12 μ g/mL of mouse IgG antibody was added, mixed well, left to stand for 30 minutes, centrifuged at 13000rpm for 45 minutes, the supernatant was discarded, the precipitate was washed twice with a gold-labeled washing solution, the supernatant was discarded last, and the precipitate was dissolved with a gold-labeled antibody preservation solution in one tenth of the initial colloidal gold volume. Uniformly spreading the marked colloidal gold on a glass fiber membrane to serve as a gold-labeled bonding pad (which is sealed by using a gold-labeled bonding pad sealing solution for 30 minutes in advance), drying at room temperature, spreading 3 square centimeters of the solution per milliliter, freeze-drying to obtain the gold-labeled bonding pad, sealing a bag, and storing at 4 ℃ for later use.

3. Assembling: and (3) overlapping and sticking the absorbent paper, the NC film prepared in the step (1), the gold-labeled combining pad prepared in the step (2) and the sample pad on a rubber plate in sequence, cutting the rubber plate into strips and assembling the strips to generate a finished product detection reagent strip, or preparing the finished product detection reagent strip into a detection card with sample holes.

Wherein, the gold-labeled washing solution: 1% BSA, 0.01mol/L PBS, pH 9; gold-labeled antibody preservation solution: 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN3 in 0.01mol/L PBS, pH 9; gold label bonding pad sealing liquid: 0.01M PBS, 1% BSA, 1% Tween-20, pH7.5.

The following describes the method of using the test strip of the present invention with a blood sample as an example:

1. test card preparation test cards were removed from the test kit and allowed to equilibrate to room temperature. The foil bag is torn open and the test card is placed horizontally.

2. Blood collection: 1) disinfecting the blood sampling finger by using an alcohol disinfection sheet, and after the alcohol is completely volatilized about half a minute, sampling blood by using a disposable blood sampling device; 2) the head of the disposable capillary is slightly squeezed and slowly released, fingertip blood is collected into the capillary under the action of negative pressure, and bubbles are prevented from being generated during sampling.

3. Blood sample detection: 1 drop (about 30. mu.L) of blood sample was dropped vertically into the sample well in the form of a test card, and then 3 drops (about 120. mu.L) of diluent was added into the sample well. (in the case of a general reagent strip, the sample pad is immersed in the sample to be tested and taken out to wait for the subsequent reading)

4. Reading a result: results were read within half an hour after ten minutes after the sample was added. Window C is the control line, G is the IgG line, M is the IgM line.

Interpretation of the results:

IgG positive results: the control line and IgG line are visible on the test strip. The detection result is positive for IgG antibody. This indicates that the patient is in the convalescent phase of infection.

Positive IgM result: control line C and IgM lines were visible on the test strips. Positive IgM antibody detection indicates that the patient is in the acute phase of infection.

IgM/IgG double positive results: control lines, IgM lines, IgG lines are clearly visible on the test strip. Positive detection of IgM and IgG antibodies. Indicating that the patient is in the acute advanced stage of infection.

4. Negative results: the control line is the only line visible in the test strip. No IgG or IgM antibodies were detected. The results do not exclude infection. If symptoms persist, a new sample should be taken from the patient within 3-5 days and then retested.

5. Invalid result: if the control line is not present in the test strip, the test result is invalid regardless of whether there are other visible lines in the test strip. The test card needs to be replaced and retested.

Example 6: sensitivity test

The sensitivity of the detection card prepared in example 5 was determined by using the S protein IgG antibody as a standard in amounts of 0.5. mu.g, 0.25. mu.g, 0.125. mu.g, 0.0625. mu.g, 0.031. mu.g, 0.016. mu.g, 0.0078. mu.g, 0.0039. mu.g, 0.0019. mu.g and 0.001. mu.g, and experiments showed that the detection card prepared in example 4 could achieve the sensitivity for detecting the 0.016. mu. g S protein IgG antibody by visual observation (see FIG. 2 in particular).

The detection sensitivity of the detection card is detected by using the S protein IgM antibody as a standard substance in the detection amounts of 0.5. mu.g, 0.25. mu.g, 0.125. mu.g, 0.0625. mu.g, 0.031. mu.g, 0.016. mu.g, 0.0078. mu.g, 0.0039. mu.g, 0.0019. mu.g and 0.001. mu.g respectively, and experiments show that the detection card prepared in example 4 can achieve the sensitivity of detecting the 0.031. mu. g S protein IgM antibody by visual observation, and the specific results are shown in FIG. 4 (wherein the detection card is visible but weak at 0.031. mu.g, and the detection card is not obvious in the picture due to the difference between visual observation and the picture, but is obvious in the picture at 0.0625. mu.g).

Example 7: experiment of specificity

The test card prepared in example 5 is used to test ten positive reference products of SARS-CoV-2IgG and IgM, and the test results show that the test quantity of IgG is 0.125 mug, the test quantity of IgM is 0.25 mug, the consistency of the test results is good, and the results are shown in FIG. 5.

13 samples with positive recovery period identified by PCR were tested by the test card prepared in example 4, and the results are shown in FIG. 6. The blood samples from 10 healthy persons were negative by the test card prepared in example 4, and the results are shown in FIG. 7.

And taking 5 positive reference substances and 5 recovery period samples identified to be positive by PCR, carrying out water bath treatment for 30min at 37 ℃ by using 0.01M DTT, and then carrying out detection. The IgM standard was tested in duplicate using 5 test cards prepared in example 4, each measuring 0.5. mu.g, and after treating the samples, the IgM specificity of the test cards was measured again at 0.5. mu.g. After DTT treatment, the IgM detection results were reduced and IgG detection was not affected, as shown in FIG. 8.

Example 8: stability test

1. Reagent stability

The test cards prepared in example 5 were divided into 3 groups of 10 cards each, placed at 37 ℃ for 2 days, 5 days and 7 days, and then used to test five IgG/IgM standard samples, and the test cards stored at room temperature were added each time as a control, that is, the test cards were treated at 37 ℃ for 0 day. The results showed that the samples stored at 37 ℃ for 2 days, 5 days and 7 days did not affect the test results compared to the samples stored normally, as shown in FIG. 9.

2. Stability of sample detection result

The 3 samples with recovery period are divided into four groups, and are respectively stored for 0 hour, 1 hour, 2 hours and 5 hours at 37 ℃, each group of samples are parallelly detected twice by the detection card prepared in the embodiment 4, compared with the 0 hour samples, the detection of the samples is not influenced when the samples are stored for 5 hours at 37 ℃, and the result is shown in figure 10.

3. Sample stability for different sampling sources

3 samples of recovery-period whole blood, serum and plasma positive by PCR identification are tested by using the detection card prepared in example 4, 3 samples of parallel positive reference substances and negative control are arranged, the detection results of the three types of samples are similar, and the results are shown in FIG. 11.

In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The description is thus to be regarded as illustrative instead of limiting.

Sequence listing

<110> Hunan enamel horse TeBiotech Co., Ltd

<120> kit and detection device for detecting novel coronavirus and preparation method thereof

<150> 2020104813444

<151> 2020-05-31

<150> 2020104941705

<151> 2020-06-03

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1273

<212> PRT

<213> Unknown (Unknown)

<220>

<221> UNSURE

<222> (1)..(1273)

<400> 1

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

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

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His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

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Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

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Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

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

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Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

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Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

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Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

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Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

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Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

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Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

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

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Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

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

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

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Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

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Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

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Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

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Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

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Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

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Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

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Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

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Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

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His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

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Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

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

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Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

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Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

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Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

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Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

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Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

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Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

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Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

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Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

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Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

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Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

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Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

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Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

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Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

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Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

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Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

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Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

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Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

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Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

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Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

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Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

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Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

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Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

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Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

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Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

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Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

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Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

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Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

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Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

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

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Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

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Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val

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Leu Lys Gly Val Lys Leu His Tyr Thr

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<210> 2

<211> 3822

<212> DNA

<213> Unknown (Unknown)

<220>

<221> unsure

<222> (1)..(3822)

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atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860

cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920

aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980

gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040

cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100

gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160

agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220

tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280

acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340

gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400

aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460

ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520

cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580

ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640

acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700

caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760

aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820

acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880

acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940

ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000

cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060

tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120

gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180

gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240

atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300

cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360

tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420

ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480

tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540

aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600

caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660

atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720

tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780

tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

<210> 3

<211> 110

<212> PRT

<213> Unknown (Unknown)

<220>

<221> UNSURE

<222> (1)..(110)

<400> 3

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

1 5 10 15

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

20 25 30

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

35 40 45

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

50 55 60

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

65 70 75 80

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

85 90 95

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

100 105 110

<210> 4

<211> 330

<212> DNA

<213> Unknown (Unknown)

<220>

<221> unsure

<222> (1)..(330)

<400> 4

gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg cccttttggt 60

gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa gagaatcagc 120

aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac ttttaagtgt 180

tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta tgcagattca 240

tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg aaagattgct 300

gattataatt ataaattacc agatgatttt 330

<210> 5

<211> 112

<212> PRT

<213> Unknown (Unknown)

<220>

<221> UNSURE

<222> (1)..(112)

<400> 5

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

1 5 10 15

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

20 25 30

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

35 40 45

Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln

50 55 60

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

65 70 75 80

Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys

85 90 95

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

100 105 110

<210> 6

<211> 336

<212> DNA

<213> Unknown (Unknown)

<220>

<221> unsure

<222> (1)..(336)

<400> 6

acaggctgcg ttatagcttg gaattctaac aatcttgatt ctaaggttgg tggtaattat 60

aattacctgt atagattgtt taggaagtct aatctcaaac cttttgagag agatatttca 120

actgaaatct atcaggccgg tagcacacct tgtaatggtg ttgaaggttt taattgttac 180

tttcctttac aatcatatgg tttccaaccc actaatggtg ttggttacca accatacaga 240

gtagtagtac tttcttttga acttctacat gcaccagcaa ctgtttgtgg acctaaaaag 300

tctactaatt tggttaaaaa caaatgtgtc aatttc 336

<210> 7

<211> 34

<212> DNA

<213> Unknown (Unknown)

<400> 7

gggggggcgg ccgcatgttt gtttttcttg tttt 34

<210> 8

<211> 27

<212> DNA

<213> Unknown (Unknown)

<400> 8

ggggggccat ggttatgtgt aatgtaa 27

<210> 9

<211> 34

<212> DNA

<213> Unknown (Unknown)

<400> 9

gggggggcgg ccgcgtccaa ccaacagaat ctat 34

<210> 10

<211> 30

<212> DNA

<213> Unknown (Unknown)

<400> 10

ggggggccat ggaaaatcat ctggtaattt 30

<210> 11

<211> 36

<212> DNA

<213> Unknown (Unknown)

<400> 11

gggggggcgg ccgcacaggc tgcgttatag cttgga 36

<210> 12

<211> 29

<212> DNA

<213> Unknown (Unknown)

<400> 12

ggggggccat gggaaattga cacattttt 29

<210> 13

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> unsure

<222> (1)..(54)

<400> 13

cgtccggcag tgcgtgaacc tgaccacccg cacccagctg ccgccggcgt atgc 54

<210> 14

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> unsure

<222> (1)..(39)

<400> 14

ggccgcggta taatgcagtt tcacgccttt cagcacccg 39

Claims

1. A kit for detecting novel coronavirus is characterized by comprising an S protein, an S-a1 protein fragment and an S-a2 protein fragment of SARS-CoV-2 coronavirus and further comprising a mouse IgG antibody, wherein the amino acid sequence of the S-a1 protein fragment is shown as a sequence table SEQ ID NO:3, and the amino acid sequence of the S-a2 protein fragment is shown as a sequence table SEQ ID NO: 5.

2. The kit of claim 1, further comprising a murine anti-human IgG antibody, and/or a murine anti-human IgM antibody, and a goat anti-mouse IgG antibody, further comprising an S protein IgG antibody as a standard and/or an S protein IgM antibody, and further comprising a sample treatment solution.

3. The kit of claim 1, wherein the novel coronavirus is a β -coronavirus, in particular a SARS-CoV-2 coronavirus, a SARS virus, a MERS virus, more preferably a SARS-CoV-2 coronavirus.

4. The kit according to claim 3, wherein the kit is used for indirect detection of antibodies against the novel coronavirus in the serum of a sample to be detected, preferably wherein the kit further comprises related reagents for indirect detection.

5. The kit of claims 1 to 4, wherein the kit is for detection by colloidal gold chromatography, the kit comprising a detection device and a sample processing solution, preferably the sample diluent is a 0.1MPBS solution containing 0.2% (v/v) Tween 20 and 0.6% (v/v) Casein, the detection device reagent strip or card.

6. The kit of claim 5, wherein the test strip is prepared by the method comprising:

(2) adding colloidal gold into mixed protein of the S protein, the S-a1 protein fragment and the S-a2 protein fragment, adding a mouse IgG antibody, washing and precipitating by using a labeling washing solution, dissolving by using a gold-labeled antibody preserving solution, uniformly paving on a glass fiber membrane, and freeze-drying to obtain a gold-labeled binding pad;

7. A device for detecting novel coronavirus is characterized by comprising a sample pad, a gold-labeled combination pad, a reaction pad and a water absorption pad which are sequentially connected; wherein the gold-labeled conjugate pad is coated with colloidal gold labeled S protein, mixed protein of S-a1 protein fragment and S-a2 protein fragment, and mouse IgG antibody; the reaction pad is sequentially provided with a detection line and a quality control line along the flowing direction of a sample to be detected, further the reaction pad is sequentially provided with a mouse anti-human IgG antibody or a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody along the flowing direction of the sample to be detected and respectively marked as a G line, an M line and a C line, and preferably the reaction pad is sequentially provided with a mouse anti-human IgG antibody or a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody along the flowing direction of the sample to be detected and respectively marked as a G line, an M line and a C line; the device is a test reagent strip, and is more preferably a test card with a sample hole.

8. The device according to claim 7, wherein the novel coronavirus is a β -coronavirus, in particular a SARS-CoV-2 coronavirus, a SARS virus, a MERS virus, more preferably a SARS-CoV-2 coronavirus.

9. The method for preparing the device for detecting a novel coronavirus according to claim 7 or 8, which comprises the steps of:

(1) respectively spraying a mouse anti-human IgG antibody, and/or a mouse anti-human IgM antibody and a goat anti-mouse IgG antibody onto the NC membrane;

(2) adding colloidal gold into mixed protein of the S protein, the S-a1 protein fragment and the S-a2 protein fragment, adding a mouse IgG antibody, washing and precipitating by using a labeling washing solution, dissolving by using a gold-labeled antibody preserving solution, uniformly paving on a fibrous membrane, and freeze-drying to obtain a gold-labeled binding pad; wherein the fiber membrane is pretreated by gold-labeled bonding pad sealing liquid in advance;

(3) and (3) overlapping and sticking the water absorption paper, the NC film prepared in the step (1), the gold-labeled bonding pad prepared in the step (2) and the sample pad on the bottom plate.

10. The method of claim 9, wherein the novel coronavirus is a beta coronavirus, particularly a SARS-CoV-2 coronavirus, a SARS virus, a MERS virus, and more preferably a SARS-CoV-2 coronavirus.

11. The method according to claim 10, wherein in the step (1), the mouse anti-human IgG antibody, the mouse anti-human IgM antibody, and the goat anti-mouse IgG antibody are sprayed onto the NC membrane, and the antibodies are marked as G line, M line, and C line; the mixed protein in the step (2) is formed by mixing S protein, S-a1 protein fragments and S-a2 protein fragments in equal proportion.

12. The preparation method according to claim 11, comprising the following specific steps:

(1) diluting the antibody with 0.01mol/L PBS, spraying the mouse anti-human IgG antibody with the concentration of 1.0mg/mL onto the NC membrane, and marking as a G line, or spraying the mouse anti-human IgM antibody with the concentration of 1.0mg/mL onto the NC membrane, and marking as an M line; or respectively spraying a mouse anti-human IgG antibody and a mouse anti-human IgM antibody on an NC membrane, and respectively marking as a G line and an M line; spraying the goat anti-mouse IgG antibody with the concentration of 1.0mg/mL onto an NC membrane, marking as a C line, and reserving after completion;

(2) with 0.1M K₂CO₃Adjusting the pH value of the colloidal gold to 9, adding proteins mixed in equal proportion of the S protein, the S-a1 protein fragment and the S-a2 protein fragment according to 12 mu g/mL, simultaneously adding a mouse IgG antibody of 12 mu g/mL, fully and uniformly mixing, standing, centrifuging, discarding supernatant, washing precipitate with a gold-labeled washing solution, discarding supernatant, and dissolving precipitate with a gold-labeled antibody storage solution of one tenth of the initial volume of the colloidal gold; uniformly spreading the marked colloidal gold on a glass fiber membrane pretreated by a gold-labeled bonding pad sealing solution in advance, and freeze-drying to obtain a gold-labeled bonding pad;

(3) sequentially overlapping and sticking the absorbent paper, the NC film prepared in the step (1), the gold-labeled bonding pad prepared in the step (2) and the sample pad on a polyvinyl chloride bottom plate or a rubber plate to obtain the device, further cutting the device into strips, and assembling to obtain reagent strips or further preparing a detection card with sample holes;

13. The method according to claim 12, wherein the gold-labeled washing solution comprises: 1% BSA, 0.01mol/L PBS, pH 9; the gold-labeled antibody preservation solution comprises: 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN₃0.01mol/L PBS, pH 9; the gold label bonding pad sealing liquid: 0.01M PBS, 1% BSA, 1% Tween-20, pH7.5.

14. Comprises the application of the S protein, the S-a1 protein fragment and the S-a2 protein fragment mixed protein of SARS-CoV-2 coronavirus in the preparation of reagents for detecting novel coronavirus, wherein the amino acid sequence of the S-a1 protein fragment is shown as the sequence table SEQ ID NO. 3, and the amino acid sequence of the S-a2 protein fragment is shown as the sequence table SEQ ID NO. 5.

15. Use according to claim 14, characterized in that it consists in detecting in the sample, by colloidal gold chromatography, the presence or absence of antibodies against a novel coronavirus, preferably said novel coronavirus is a β coronavirus, in particular a SARS-CoV-2 coronavirus, a SARS virus, a MERS virus, more preferably a SARS-CoV-2 coronavirus.

16. The use according to claim 15, wherein the sample to be tested is selected from the group consisting of samples of convalescent persons and samples of healthy persons, preferably at least one of nasopharyngeal swabs, sputum, alveolar lavage, blood, urine.