A kit and a detection device for detecting a novel coronavirus and a preparation method thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to antibody detection, and specifically to a colloidal gold chromatography kit for detecting SARS-CoV-2 coronavirus Spike protein antibodies, a detection device and a preparation method thereof. Background Art

[0002] The new coronavirus (SARS-CoV-2) is an enveloped beta coronavirus with a diameter of approximately 60 to 140 nm. SARS-CoV-2 encodes 15 nonstructural proteins (nsp1 to nsp10, nsp12 to nsp16), 4 structural proteins (S, E, M and N) and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14). The spike protein of the new coronavirus helps the virus enter target cells. The receptor binding domain (S-RBD) of the spike protein binds to its receptor angiotensin-converting enzyme 2 (ACE2), which helps the virus attach to the surface of target cells and then infect them.

[0003] Researchers around the world are working on rapid and accurate methods for detecting the novel coronavirus. The main methods currently used include molecular testing, cell culture testing, and antibody testing.

[0004] One is a molecular test (PCR method), which detects whether the patient sample contains SARS-CoV-2-specific RNA sequences to make an early etiological diagnosis of COVID-19. However, this detection method has a certain false negative rate, and the positive rate is only 30%-50%. The reason is that the location and method of clinical sample collection are different, resulting in too little SARS-CoV-2 load to be detected; in addition, the reliability and sensitivity of diagnostic reagents are limited. At the same time, this method requires professional personnel to operate and requires very high experimental working conditions; metagenomic sequencing (mNGS) is to extract the RNA of the new coronavirus from the patient's lower respiratory tract secretions, construct a viral cDNA library, and then perform high-throughput sequencing. Through database comparison analysis, it is identified whether the genome sequence is highly homologous to SARS-CoV-2. The advantages of mNGS detection of SARS-CoV-2 are high specificity and sensitivity, but its disadvantages are also obvious. The instrument configuration requirements are high and the detection cycle is long. The cell culture method is a sterile operation technology that requires a sterile operation room, a clean bench, a cell culture box, a centrifuge, a microscope, etc. The experimental technology is complex and requires experienced professional technicians to operate. The experimental cycle is very long and is not suitable for rapid detection of diseases. Antibody detection mainly uses enzyme-linked immunosorbent reaction (ELISA): The ELISA method requires high antigen purity and good specificity, otherwise non-specific reactions will occur. The operation procedure is relatively complicated and requires repeated washing. If the number of washing times is insufficient or too many, it is easy to cause false positives and false negatives, and it is easy to cause harm to the operator and environmental pollution. The experiment time is long and it takes more than two hours to get the test results. This method must be equipped with an enzyme labeler and a plate washer, which is difficult to achieve in grassroots laboratories and small clinics. Among them, the reagents for detecting viral antibodies by colloidal gold chromatography can be used in hospitals, airports, customs, homes and other places. The results can be judged within a few minutes, thereby preventing the spread of the epidemic as early as possible; blood testing solves the problem of inaccurate sampling of throat swabs. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a kit or reagent strip for accurately detecting the new coronavirus and a preparation method and application thereof, which can be used in hospitals, airports, customs, homes and other places, and can judge the results within a few minutes, thereby preventing the spread of the epidemic as early as possible.

[0006] In order to achieve the above-mentioned purpose, the detection of the present invention utilizes the novel coronavirus S protein and its specific fragments to detect IgM and IgG antibodies of the novel coronavirus spike protein Spike. Among them, the S protein is a membrane protein, which constitutes the viral envelope particles and is the main component of the virus infecting host cells. In order for the novel coronavirus to infect cells, it must first introduce its genetic material RNA into the host cell, and this process is mainly completed by the S protein. The protrusions of the S protein are the key parts of its interaction with the receptor and membrane fusion. However, the full length of the S protein is prone to mutation. If the S protein is simply detected, it is likely that the mutated virus cannot be detected. In this regard, the inventors have conducted in-depth research and found the specific fragments S-a1 and S-a2 of the S protein, so that after the S protein mutates, the target analyte can still be detected by detecting the specific fragments S-a1 and S-a2, which not only improves the scope of use of this kit, but also ensures the accuracy of the results of this kit.

[0007] Therefore, the present invention provides a kit for detecting the new coronavirus, which comprises the S protein, S-a1 protein fragment and S-a2 protein fragment of the SARS-CoV-2 coronavirus, wherein the amino acid sequence of the S-a1 protein fragment is shown in the sequence listing SEQ ID NO:3, and the amino acid sequence of the S-a2 protein fragment is shown in the sequence listing SEQ ID NO:5.

[0008] Furthermore, in order to utilize the antibody detection method, that is, to detect antibodies against the S protein of the new coronavirus, the kit also includes mouse IgG antibodies, further includes mouse anti-human IgG antibodies and / or mouse anti-human IgM antibodies, and goat anti-mouse IgG antibodies, further includes S protein IgG antibodies and / or S protein IgM antibodies as standards, and further includes sample processing fluid.

[0009] Among them, the new coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.

[0010] In a specific embodiment, the kit is used for indirect detection of whether the serum of the sample to be tested contains antibodies against the new coronavirus. Preferably, the kit also includes related reagents for indirect detection.

[0011] Preferably, the kit is used for detection by colloidal gold chromatography, and thus the kit comprises a detection device and a sample treatment solution, the sample treatment solution is a 0.1M PBS solution containing 0.2% (volume) Tween 20 and 0.6% (volume) Casein, and the detection device is a reagent strip or a detection card. In a specific embodiment, the detection device is prepared by the following method:

[0012] (1) Spraying mouse anti-human IgG antibody, and / or mouse anti-human IgM antibody, and / or goat anti-mouse IgG antibody onto the NC membrane respectively;

[0013] (2) adding colloidal gold to a mixed protein of S protein, S-a1 protein fragment and S-a2 protein fragment, adding mouse IgG antibody, washing the precipitate with labeling washing solution, dissolving it with gold-labeled antibody storage solution, and then evenly spreading it on a glass fiber membrane (pre-treated with a gold-labeled binding pad blocking solution), and freeze-drying it to obtain a gold-labeled binding pad;

[0014] (3) Overlap the absorbent paper, the NC membrane obtained in step (1), the gold label conjugate pad, and the sample pad and stick them on a plastic plate, and cut them into strips to obtain a reagent strip.

[0015] On the other hand, the present invention provides a device for detecting the novel coronavirus, wherein the device comprises a sample pad, a gold-labeled binding pad, a reaction pad and a water-absorbing pad connected in sequence; wherein the gold-labeled binding pad is coated with colloidal gold-labeled S protein, a mixed protein of S-a1 protein fragment and S-a2 protein fragment, and mouse IgG antibody; wherein the amino acid sequence of the S-a1 protein fragment is shown in the sequence list SEQ ID NO:3, and the amino acid sequence of the S-a2 protein fragment is shown in the sequence list SEQ ID NO:5; a detection line and a quality control line are sequentially arranged on the reaction pad along the flow direction of the sample to be tested; further, mouse anti-human IgG antibody or mouse anti-human IgM antibody, and goat anti-mouse IgG antibody are sequentially arranged on the reaction pad along the flow direction of the sample to be tested, respectively recorded as T line (i.e., G line or M line), and C line; preferably, mouse anti-human IgG antibody, mouse anti-human IgM antibody, and goat anti-mouse IgG antibody are sequentially arranged on the reaction pad along the flow direction of the sample to be tested, respectively recorded as G line, M line, and C line; the device is a detection reagent strip, more preferably a detection card with a sample hole.

[0016] Specifically, the novel coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.

[0017] Among them, when the sample to be tested contains human anti-new coronavirus antibodies, the antibodies first bind to the gold-labeled antigen. Due to the chromatography effect, the reaction complex moves forward along the coating membrane. When it encounters the coated anti-antibody, an antibody-gold-labeled antigen-anti-antibody complex is formed and enriched on the coating line to form a red precipitation line, which is a positive result, thereby quickly diagnosing whether the sample contains IgM / IgG antibodies to the new coronavirus (SARS-CoV-2).

[0018] The present invention further provides a method for preparing the above-mentioned device for detecting the novel coronavirus, which comprises the following steps:

[0019] (1) Spraying mouse anti-human IgG antibody, mouse anti-human IgM antibody, and goat anti-mouse IgG antibody onto the NC membrane respectively;

[0020] (2) adding colloidal gold to a mixed protein of S protein, S-a1 protein fragment and S-a2 protein fragment, adding mouse IgG antibody, washing the precipitate with a labeling washing solution, dissolving it with a gold-labeled antibody storage solution, and then evenly spreading it on a fiber membrane pre-treated with a gold-labeled binding pad blocking solution, and freeze-drying it to obtain a gold-labeled binding pad;

[0021] (3) The absorbent paper, the NC membrane prepared in step (1), the gold label conjugate pad prepared in step (2), and the sample pad are overlapped and pasted on a base plate, and then cut into strips and assembled to obtain a reagent strip or further prepared into a test card with a sample hole.

[0022] Specifically, the novel coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.

[0023] In a preferred embodiment, the step (1) is to spray mouse anti-human IgG antibody, mouse anti-human IgM antibody, and goat anti-mouse IgG antibody onto the NC membrane, respectively, which are recorded as G line, M line, and C line, respectively; the mixed protein in the step (2) is a mixture of S protein, S-a1 protein fragment, and S-a2 protein fragment in equal proportions.

[0024] In a specific implementation, the specific steps are as follows:

[0025] (1) Dilute the antibody with 0.01 mol / L PBS, spray the mouse anti-human IgG antibody at a concentration of 1.0 mg / mL onto the NC membrane, which is recorded as the G line; or spray the mouse anti-human IgM antibody at a concentration of 1.0 mg / mL onto the NC membrane, which is recorded as the M line; or spray the mouse anti-human IgG antibody and mouse anti-human IgM antibody onto the NC membrane, which are recorded as the G line and the M line respectively; spray the goat anti-mouse IgG antibody at a concentration of 1.0 mg / mL onto the NC membrane, which is recorded as the C line, and set aside for use after completion;

[0026] (2) adjusting the pH value of colloidal gold to 9 with 0.1 M K2CO3 solution, adding the aforementioned S protein, S-a1 protein fragment and S-a2 protein fragment mixed in equal proportions at 12 μg / mL, and adding 12 μg / mL mouse IgG antibody at the same time, mixing thoroughly, standing and centrifuging to discard the supernatant, washing the precipitate with gold label washing solution, discarding the supernatant, and dissolving the precipitate with gold label antibody storage solution with one tenth of the initial colloidal gold volume; spreading the labeled colloidal gold evenly on the glass fiber membrane, and freeze-drying to obtain a gold label binding pad;

[0027] (3) Overlapping the absorbent paper, the NC membrane prepared in step (1), the gold-labeled conjugate pad prepared in step (2), and the sample pad in sequence on a polyvinyl chloride base plate or a plastic plate, cutting them into strips, and assembling them to obtain a reagent strip; or preparing a test card with a sample hole.

[0028] Preferably, the sample pad is a glass fiber membrane, non-woven fabric or filter paper that has been impregnated with a sample pad treatment solution. More preferably, the sample pad treatment solution comprises the following components: Tween 20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.

[0029] In the above steps, the gold-labeled washing solution specifically used is: 1% BSA, 0.01 mol / L PBS, pH 9; the gold-labeled antibody storage solution: 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN3 in 0.01 mol / L PBS, pH 9; the gold-labeled binding pad blocking solution: 0.01M PBS, 1% BSA, 1% Tween-20, pH 7.5.

[0030] The present invention also provides the use of a mixed protein comprising the S protein, S-a1 protein fragment and S-a2 protein fragment of the SARS-CoV-2 coronavirus in the preparation of a reagent for detecting the new coronavirus, wherein the amino acid sequence of the S-a1 protein fragment is shown in the sequence listing SEQ ID NO:3, and the amino acid sequence of the S-a2 protein fragment is shown in the sequence listing SEQ ID NO:5.

[0031] In a preferred embodiment, the application is to detect whether there are antibodies against the new coronavirus in the sample by colloidal gold chromatography. Preferably, the new coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.

[0032] In a more specific application, the samples for detection are selected from samples of people in recovery and healthy people, and the samples are preferably at least one of nasopharyngeal swabs, sputum, alveolar lavage fluid, blood, and urine.

[0033] The detection device of the present application, such as a reagent strip or a test card, can detect both IgM and IgG antibodies at the same time. Among them, IgM is produced in the early stage of viral infection and can be used to evaluate the early infection of the virus; IgG gradually increases in the later stage of infection, mainly playing a role in completely clearing the virus, and can be used to evaluate the immune response in the patient or the immune effect of the vaccine. This kit detects viral infection from the perspective of immune response, which complements the currently common viral 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 new coronavirus (SARS-CoV-2) spike protein Spike antigen and its two specific fragments as solid phases, and uses an indirect method to detect whether the serum contains human anti-new coronavirus antibodies. Among them, especially compared with the prior art, the test strip of the present invention detects the S protein, which avoids the problem of high false positives in N protein detection, and the test results are reliable and stable; more especially, the test strip of the present invention 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 when the S protein mutates, its highly conservative protein fragments S-a1 and S-a2 can be identified, which improves the detection specificity of the test kit and expands the scope of application of the test kit. At the same time, the full length of the S protein can be determined, and specific fragments can also be identified, which greatly improves the recognition specificity and accuracy of the test kit. At the same time, experiments show that the test strip of the present invention can achieve the sensitivity of detecting 0.016μg S protein IgG antibodies and 0.031μg S protein IgM antibodies by naked eye observation, and the test results are stable and accurate and can be identified by the naked eye. It is suitable for hospitals, airports, customs, homes and other places, and can judge the results within a few minutes, thereby preventing the spread of the epidemic as early as possible, and has very important promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the comparison between S-a1 and S-a2 and the S protein sequences of different coronaviruses;

[0035] Figure 2 This is the PAGE identification diagram of S protein;

[0036] Figure 3 The results are for the binding activity of S-a1, S-a2 and S protein with IgG antibodies in the serum of convalescent patients;

[0037] Figure 4 A schematic diagram showing the test results of the sensitivity of the reagent strip prepared in Example 4;

[0038] Figure 5 A schematic diagram of the specific detection results of the reagent strip prepared in Example 4 for a positive reference;

[0039] Figure 6A schematic diagram of the specific detection results of the reagent strip prepared in Example 4 on 13 blood samples that were positive by PCR identification;

[0040] Figure 7 This is a schematic diagram of the specific detection results of the reagent strip prepared in Example 4 on normal human blood samples;

[0041] Figure 8 It is a schematic diagram of the specific detection results after DTT treatment;

[0042] Fig. 9 A schematic diagram of the stability test results of the reagent strip prepared in Example 4;

[0043] Fig.10 This is a schematic diagram of the stability results of the sample test results of the reagent strip prepared by Example 4;

[0044] Fig.11 Stable results for samples from different sampling sources. DETAILED DESCRIPTION

[0045] In order to more clearly describe the technical content of the present invention, further description is given below in conjunction with specific embodiments.

[0046] In the following examples, only the improved parts of the present invention are described in detail, and the other steps or sources of raw materials not mentioned are the same as those in the prior art and are not described in detail here.

[0047] Example 1: Construction of S protein (spike protein) encoding gene fragment and strong antigenic S protein fragment

[0048] 1. Synthesis of S protein encoding gene

[0049] The full-length amino acid sequence of the Spike protein is shown in SEQ ID NO: 1, and the full-length nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2. The full-length encoding gene of the Spike protein was synthesized by conventional methods.

[0050] The following upstream primers and downstream primers were designed based on the above full-length sequence, and the primer sequences are as follows:

[0051]

[0052] Using the previously synthesized S protein coding sequence as a template, PCR amplification was performed to obtain more Spike protein coding sequences. The PCR system is as follows:

[0053]

[0054] Add the reaction solution to the PCR tube according to the table above and mix well. Place the PCR reaction tube in the PCR instrument and set the reaction conditions: 95℃ denaturation for 5min; 95℃ 30S, 58℃ 30S, 72℃ 4min; 72℃ 10min; the product can be stored at 4℃ for a short time for subsequent experiments.

[0055] 2. Synthesis of the coding sequences of S-a1 and S-a2 fragments

[0056] After protein knowledgebase analysis and comparison, the specific sequences with high immunogenicity in Spike were screened. The results of the comparison between the two sequences and the Spike protein sequences of different coronaviruses are shown in Figure 1 . After analysis, it was determined that two sequences S-a1 and S-a2 located in different regions of RBD were obtained through database analysis of peptides a1 and a2. At the same time, relevant experiments also verified that the effect was not lower than that of S protein, which played an important role in the good effect of the final detection reagent card.

[0057] The first conserved region is the S-a1 fragment, whose amino acid sequence is shown in SEQ ID NO: 3 and whose encoded nucleotide sequence is shown in SEQ ID NO: 4; the other conserved region is the S-a2 fragment, whose amino acid sequence is shown in SEQ ID NO: 5 and whose encoded nucleotide sequence is shown in SEQ ID NO: 6. The coding sequences of the S-a1 fragment and the S-a2 fragment were synthesized by conventional methods.

[0058] in:

[0059] The full-length amino acid sequence of the Spike protein is shown below (SEQ ID NO: 1):

[0060]

[0061] The full-length sequence of the Spike protein encoding gene is as follows: (SEQ ID NO: 2)

[0062]

[0063] The amino acid sequence of S-a1 is as follows: (SEQ ID NO: 3)

[0064] VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF

[0065] The base sequence of S-a1 is as follows: (SEQ ID NO: 4)

[0066] GTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTT TCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTT

[0067] The amino acid sequence of S-a2 is as follows: (SEQ ID NO: 5)

[0068] TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0069] The base sequence of S-a2 is as follows: (SEQ ID NO: 6)

[0070] ACAGGCTCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACAACCTTGTAATGGTGTTGAAGGT TTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC

[0071] According to the above sequences, upstream primers and downstream primers were designed for the coding sequences of S-a1 and S-a2 fragments, respectively. The primer sequences are as follows:

[0072]

[0073] The base sequence of the previously synthesized S-a1 fragment was used as a template and the S-a1 base sequence was amplified by PCR. The PCR system was as follows:

[0074]

[0075] Add the reaction solution to the PCR tube according to the table above and mix well. Place the PCR reaction tube in the PCR instrument and set the reaction conditions: 95℃ denaturation for 5min; 95℃ 30S, 58℃ 30S, 72℃ 4min; 72℃ 10min; the product can be stored at 4℃ for a short time for subsequent experiments.

[0076] The base sequence of the previously synthesized S-a2 fragment was used as a template to amplify the S-a2 base sequence by PCR. The PCR system is as follows:

[0077]

[0078] Add the reaction solution to the PCR tube according to the table above and mix well. Place the PCR reaction tube in the PCR instrument and set the reaction conditions: 95℃ denaturation for 5min; 95℃ 30S, 58℃ 30S, 72℃ 4min; 72℃ 10min; the product can be stored at 4℃ for a short time for subsequent experiments.

[0079] 3. Construction of S protein, S-a1 and S-a2 expression plasmids

[0080] 3.1. Pretreatment of pCDNA vector

[0081] The enzyme digestion system is as follows:

[0082]

[0083] Digest in a 37℃ water bath for 5h, and perform DNA electrophoresis after digestion. First, use pure water to clean the electrophoresis tank and replace with a new 1×TAE buffer. Add 25 µL of 10× loading buffer to the digested system, mix well and apply 50 µL to each well, and perform electrophoresis at 150 V for 30 min. Cut the target fragment of about 4000 bp on the gel cutter, transfer it to a pre-weighed 5mL centrifuge tube, weigh it, and get the weight of the gel block. Use the Tiangen Universal DNA Purification and Recovery Kit to recover the target fragment. The DNA concentration of the obtained product was determined using Nano-300 and diluted to 50 ng / µL.

[0084] 3.2. Connection

[0085] The connection system is as follows:

[0086]

[0087] According to the above connection system, the full-length S protein gene or S-a1 or S-a2 fragment was connected to the pCDNA vector for 30 minutes at 16°C, and the TOP10 competent cells were used for transformation and plating. The next day, the bacteria were picked and sent for testing. After comparing the sequencing results, three expression plasmids with correct sequences were obtained, namely pCDNA-S, pCDNA-S-a1, and pCDNA-S-a2, for subsequent experiments.

[0088] Example 2

[0089] The difference between this embodiment and embodiment 1 is that the primers for amplifying the S protein encoding gene in this embodiment are different, and the following upstream primers and downstream primers are synthesized according to the S protein sequence:

[0090]

[0091] Example 3: Expression and purification of S protein, S-a1 and S-a2 protein fragments

[0092] 1. Cell transfection:

[0093] i. Grow 293F cells to a density of about 1x10 6 / mL;

[0094] ii. Resuspend 50 μL PEI in 1 mL fresh culture medium, and resuspend 10 μg of three plasmids, pCDNA-S, pCDNA-S-a1, and pCDNA-S-a2, in 1 mL fresh culture medium, mix well, and let stand at room temperature for 5 minutes;

[0095] iii. Mix the PEI premix and plasmid premix and let stand at room temperature for 20 min;

[0096] iv. Add the PEI-plasmid complex dropwise into 20 ml of 293F cells, let it rest for 5 min, then culture it in a CO2 shaker. Collect the supernatant after three days of culture.

[0097] 2. Protein purification and identification:

[0098] i. Collect the supernatant into a 50 mL centrifuge tube, centrifuge at 10,000 rpm for 10 min, and transfer the supernatant to a clean centrifuge tube;

[0099] ii. Wash the column pre-loaded with 2 mL of ProteinA filler with 10 times the column bed volume of deionized water;

[0100] iii.30 mL PBS to balance the medium;

[0101] iv. Transfer the supernatant to the column and adjust the flow rate so that the liquid flows out at a rate of 2 drops per second;

[0102] v. Wash the sample with 30 mL PBS;

[0103] vi. 10 mL of elution buffer was added at a rate of 10 s per drop, and 1.5 mL of the eluate was collected in each EP tube. At the same time, 20 μL of neutralization buffer was added dropwise, and 7 tubes were collected continuously;

[0104] vii. Determine the antibody concentration of each tube using Nano-300, combine the collected solutions with a concentration > 0.1 mg / mL into a dialysis bag, and dialyze with PBS;

[0105] viii. Filter the antibody solution through a 0.22 μm filter membrane into a sterile centrifuge tube and store for later use;

[0106] ix. Take 5 μg of sample and dilute it to 20 μL with 1×PBS, add 6 μL 4× LDS Sample Buffer and 4 μL DTT, boil in water for 10 min, centrifuge at 12000 rpm for 1 min. Load 20 μL, run the PAGE gel at 150 V for 60 min and use InstantBlue protein electrophoresis to stain for 30 min, place the gel in water, decolorize for about 30 min, observe and take pictures, the results are shown in the figure. Figure 2(The focus is on the results of the full-length S protein. Since S-a1 and S-a2 only have about 110 amino acids, the PAGE gel currently available in the laboratory is difficult to display bands with a molecular weight of 10KD or less, so no results are provided).

[0107] Example 4: Detection of the binding activity of S-a1, S-a2 and S protein with IgG antibodies in the serum of convalescent patients

[0108] ELISA was used to detect the binding of S-a1, S-a2 and S protein to IgG antibodies in the serum of convalescent patients. The specific steps are as follows:

[0109] 1. Dilute S-a1, S-a2 and S protein to a concentration of 1 μg / ml, add 100 μL / well of the diluted protein to a 96-well plate, seal, and coat overnight at 4°C;

[0110] 2. Discard the supernatant and wash each well three times with 200 μL PBST (TWEEN-20 0.1%);

[0111] 3. Add 200 μL 2% BSA blocking solution to each well, block at room temperature for 1 hour, discard the blocked ELISA plate, and wash 3 times with 200 μL PBST (TWEEN-20 0.1%) per well;

[0112] 4. Add 100 μL of 1 μg / mL anti-human IgG-HRP antibody to each well and incubate at room temperature for 2 hours;

[0113] 5. Discard the supernatant, dry it with absorbent paper, and wash 5 times with 100μL Wash Buffer;

[0114] 6. Add 100 µL 1X TMB to each well and shake at room temperature in the dark for 10 minutes;

[0115] 7. Add 100 µL stop solution to each well;

[0116] 8. Use a microplate reader to read the absorbance at 450 nm;

[0117] The experimental results are shown in Figure 3 As shown, it can be found that the binding activity of S-a1 and S-a2 is slightly higher than that of Spike protein.

[0118] Example 5: Preparation and use of detection device

[0119] The above-prepared mixture of SARS-CoV-2 Spike, S-a1 and S-a2 was used as a solid phase to detect whether the serum contained antibodies against the new coronavirus using the indirect method principle. The specific steps are as follows:

[0120] 1. Preparation of coating membrane: dilute the antibody with 0.01 mol / L PBS, spray the mouse anti-human IgG antibody at a concentration of 1.0 mg / mL onto the NC membrane, recorded as the G line; spray the mouse anti-human IgM antibody at a concentration of 1.0 mg / mL onto the NC membrane, recorded as the M line; spray the goat anti-mouse IgG antibody at a concentration of 1.0 mg / mL onto the NC membrane, recorded as the C line, and set aside after completion.

[0121] 2. Preparation of the mixture of gold-labeled Spike, S-a1 and S-a2: 0.1M K2CO3 solution was used to adjust the pH value of colloidal gold to 9, and the Spike, S-a1 and S-a2 prepared in the above example were added at 12μg / mL in equal proportions by weight (i.e., the final concentrations of Spike, S-a1 and S-a2 were 12μg / mL, respectively). At the same time, 12μg / mL mouse IgG antibody was added, and the mixture was fully mixed, allowed to stand for 30 minutes, centrifuged at 13000rpm for 45 minutes, and the supernatant was discarded. The precipitate was washed twice with gold-labeled washing solution, and the supernatant was discarded for the last time. The precipitate was dissolved with gold-labeled antibody preservation solution of one-tenth of the initial colloidal gold volume. The labeled colloidal gold was evenly spread on the glass fiber membrane as a gold-labeled binding pad (pre-sealed with gold-labeled binding pad blocking solution for 30 minutes), and dried at room temperature, 3 square centimeters per milliliter of solution, freeze-dried to form a gold-labeled binding pad, sealed in a bag, and stored at 4°C for later use.

[0122] 3. Assembly: overlap the absorbent paper, the NC membrane prepared in step (1), the gold label conjugate pad prepared in step (2), and the sample pad on the plastic plate in sequence, cut them into strips and assemble them to produce a finished test reagent strip, or prepare a test card with a sample hole.

[0123] Among them, gold label washing solution: 1% BSA, 0.01 mol / L PBS, pH9; gold label antibody storage solution: 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN3 in 0.01 mol / L PBS, pH9; gold label binding pad blocking solution: 0.01M PBS, 1% BSA, 1% Tween-20, pH7.5.

[0124] The following is an example of a blood sample to describe the method of using the test strip of the present invention:

[0125] 1. Test Card Preparation: Remove the test card from the test kit and equilibrate to room temperature. Tear open the foil pouch and place the test card horizontally.

[0126] 2. Blood collection: 1) Disinfect the finger for blood collection with an alcohol disinfectant. After about half a minute, wait for the alcohol to completely evaporate, and then use a disposable blood collection device to collect blood; 2) Gently squeeze the head of the disposable capillary tube, and then slowly release it. Under the action of negative pressure, the fingertip blood is collected into the capillary tube. Avoid bubbles when sampling.

[0127] 3. Blood sample test: vertically drip 1 drop (about 30μL) of blood sample into the sample well of the test card, and then add 3 drops of diluent (about 120μL) into the sample well. (If it is a general reagent strip, immerse the sample pad in the sample to be tested, take it out and wait for the subsequent reading of the results)

[0128] 4. Read the results: Read the results within half an hour after adding the sample ten minutes later. Window C is the control line, G is the IgG line, and M is the IgM line.

[0129] Result interpretation:

[0130] 1. IgG positive result: The control line and IgG line are visible on the test strip. The test result is IgG antibody positive. This means that the patient is in the recovery stage of infection.

[0131] 2. IgM positive result: The control line C line and the IgM line are visible on the test strip. A positive IgM antibody test indicates that the patient is in the acute stage of infection.

[0132] 3. IgM / IgG double positive result: The control line, IgM line, and IgG line are clearly visible on the test strip. IgM and IgG antibodies are positive. Indicates that the patient is in the acute late stage of infection.

[0133] 4. Negative result: The control line is the only visible line in the test strip. No IgG or IgM antibodies are detected. The result does not rule out infection. If symptoms persist, a new sample should be drawn from the patient within 3-5 days and the test repeated.

[0134] 5. Invalid result: If the control line does not appear in the test strip, the test result is invalid regardless of whether there are other visible lines in the test strip. Replace the test card and retest.

[0135] Example 6: Sensitivity experiment

[0136] Using S protein IgG antibody as a standard, the detection amounts were 0.5 μg, 0.25 μg, 0.125 μg, 0.0625 μg, 0.031 μg, 0.016 μg, 0.0078 μg, 0.0039 μg, 0.0019 μg, and 0.001 μg, respectively, to test the sensitivity of the test card prepared in Example 5. Experiments have shown that the test card prepared in Example 4 can achieve the sensitivity of detecting 0.016 μg of S protein IgG antibody by naked eye observation (see for details). Figure 2 ).

[0137] Using S protein IgM antibody as a standard, the detection amounts were 0.5 μg, 0.25 μg, 0.125 μg, 0.0625 μg, 0.031 μg, 0.016 μg, 0.0078 μg, 0.0039 μg, 0.0019 μg, and 0.001 μg, respectively, to test the sensitivity of the test card. Experiments have shown that the test card prepared in Example 4 can achieve the sensitivity of detecting 0.031 μg of S protein IgM antibody by naked eye observation. The specific results are shown in Figure 4 (It is visible to the naked eye but weak at 0.031μg. Due to the difference between naked eye observation and photograph, it is not very obvious in the photograph, but it is also quite obvious in the photograph at 0.0625μg).

[0138] Example 7: Specificity Experiment

[0139] The test card prepared in Example 5 was used to detect ten SARS-CoV-2 IgG and IgM positive reference samples. The experimental results showed that the detection amount of IgG was 0.125 μg, and the detection amount of IgM was 0.25 μg. The experimental results were consistent. Figure 5 .

[0140] Thirteen samples of the convalescent period that were positive by PCR were tested by the test card prepared in Example 4, and the results were all positive. Figure 6 10 blood samples of healthy people were tested by the test card prepared in Example 4, and the results were all negative. Figure 7 .

[0141] Five positive reference samples and five convalescent samples that were positive by PCR were treated with 0.01M DTT in a 37°C water bath for 30 minutes and then tested. The IgM standard was tested repeatedly using five test cards prepared in Example 4, each with a test amount of 0.5 μg. After the samples were treated, they were tested again with a test amount of 0.5 μg to test the IgM specificity of the test card. After the test card prepared in Example 4 was tested, the IgM test results were weakened after DTT treatment, while the IgG test was not affected. Figure 8 shown.

[0142] Example 8: Stability test

[0143] 1. Reagent stability

[0144] The test cards prepared in Example 5 were divided into 3 groups, each with 10 cards. After being placed at 37°C for 2 days, 5 days and 7 days, they were used to test 5 IgG / IgM standards respectively. Each time, a test card stored at room temperature was added as a control, i.e., 0 days at 37°C. The results showed that the test results were not affected by the storage at 37°C for 2 days, 5 days and 7 days compared with the samples stored normally. Fig. 9 .

[0145] 2. Stability of sample test results

[0146] The three recovery period samples were divided into four groups and stored at 37°C for 0 hours, 1 hour, 2 hours and 5 hours respectively. Each group of samples was tested twice in parallel using the test card prepared in Example 4. Compared with the 0 hour sample, the 5 hours at 37°C did not affect the detection of the sample. The results are shown in Table 1. Fig.10 .

[0147] 3. Sample stability from different sampling sources

[0148] Three samples of whole blood, serum and plasma from the convalescent period that were positive by PCR were tested using the test card prepared in Example 4. Three parallel positive reference samples and three negative controls were set up at the same time. The test results of the three types of samples were similar. Fig.11 shown.

[0149] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description should be regarded as illustrative rather than restrictive. Sequence Listing <110> Hunan Fasi Biotechnology Co., Ltd. <120> A kit and a detection device for detecting a novel coronavirus and a 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 <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 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 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 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 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 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 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 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu 1010 1015 1020 Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val 1025 1030 1035 1040 Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala 1045 1050 1055 Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu 1060 1065 1070 Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His 1075 1080 1085 Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val 1090 1095 1100 Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr 1105 1110 1115 1120 Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr 1125 1130 1135 Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu 1140 1145 1150 Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp 1155 1160 1165 Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp 1170 1175 1180 Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu 1185 1190 1195 1200 Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile 1205 1210 1215 Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile 1220 1225 1230 Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val 1250 1255 1260 Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 2 <211> 3822 <212> DNA <213> Unknown <220> <221> unsure <222> (1)..(3822) <400> 2 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 ataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420 ttgggtgttt attaccacaaaacaaaa agttggatgg aaagtgagtt cagagtttat 480 tctagtgcga atattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540 ggaaaacagg gtaatttca aaatcttagg gatttgtgt ttaagaat tgatggttat 600 tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660 tcggctttag aaccattggt agatttgcca atagtatta acatcactag gtttcaact 720 ttacttgctt tacatagaag tatttgact cctggtgat cttctcagg ttggacagct 780 ggtgctgcag cttattatgt gggttatctt caactagga ctttctatt aaaatataat 840 gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctc agaaacaaag 900 tgtacgttga atccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960 caccacag aatctattgt tagattcct atattacaa acttgtgccc ttttggtgaa 1020 gttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagg atcagcac 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 tctttttggtg 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 <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 <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 <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 <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 <400> 7 gggggggcgg ccgcatgttt gtttttcttg tttt 34 <210> 8 <211> 27 <212> DNA <213> Unknown <400> 8 ggggggccat ggttatgtgt aatgtaa 27 <210> 9 <211> 34 <212> DNA <213> Unknown <400> 9 gggggggcgg ccgcgtccaa ccaacagaat ctat 34 <210> 10 <211> 30 <212> DNA <213> Unknown <400> 10 ggggggccat ggaaaatcat ctggtaattt 30 <210> 11 <211> 36 <212> DNA <213> Unknown <400> 11 gggggggcgg ccgcacaggc tgcgttatag cttgga 36 <210> 12 <211> 29 <212> DNA <213> Unknown <400> 12 ggggggccat gggaaattga cacattttt 29 <210> 13 <211> 54 <212> DNA <213> 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 <220> <221> unsure <222> (1)..(39) <400> 14 ggccgcggta taatgcagtt tcacgccttt cagcacccg 39

Claims

1. A kit for detecting a novel coronavirus, characterized in that: It includes the S protein, S-a1 protein fragment and S-a2 protein fragment of the SARS-CoV-2 coronavirus, and also includes mouse IgG, wherein the amino acid sequence of the S-a1 protein fragment is shown in the sequence listing SEQ ID NO:3, and the amino acid sequence of the S-a2 protein fragment is shown in the sequence listing SEQ ID NO:

5.

2. The kit according to claim 1, characterized in that It also includes mouse anti-human IgG antibody, and / or mouse anti-human IgM antibody, and goat anti-mouse IgG antibody, and also includes S protein IgG antibody and / or S protein IgM antibody as standard, and includes sample processing liquid.

3. The kit according to claim 1, characterized in that The kit is used for indirect detection of whether the serum of the sample to be tested contains antibodies against the new coronavirus, and the kit also includes related reagents for indirect detection.

4. The kit according to any one of claims 1 to 3, characterized in that The kit is used for detection by colloidal gold chromatography, and comprises a detection device and a sample processing solution, which is a 0.1M PBS solution containing 0.2% (volume) Tween 20 and 0.6% (volume) Casein, and the detection device is a reagent strip or a detection card.

5. The kit according to claim 4, characterized in that The reagent strip is prepared by the following method: (1) Spraying mouse anti-human IgG antibody and / or mouse anti-human IgM antibody, and goat anti-mouse IgG antibody onto the NC membrane respectively; (2) adding colloidal gold to a mixed protein of S protein, S-a1 protein fragment and S-a2 protein fragment, adding mouse IgG, washing the precipitate with a labeling washing solution, dissolving it with a gold-labeled antibody preservation solution, and then evenly spreading it on a glass fiber membrane, and freeze-drying it to form a gold-labeled binding pad; (3) Overlap the absorbent paper, the NC membrane obtained in step (1), the gold label conjugate pad, and the sample pad and stick them on a plastic plate, and cut them into strips to obtain a reagent strip.

6. A device for detecting a novel coronavirus, characterized in that: The device comprises a sample pad, a gold-labeled binding pad, a reaction pad and a water-absorbing pad connected in sequence; wherein the gold-labeled binding pad is coated with a mixed protein of S protein, S-a1 protein fragment and S-a2 protein fragment labeled with colloidal gold, wherein the amino acid sequence of the S-a1 protein fragment is shown in SEQ ID NO:3 of the sequence list, and the amino acid sequence of the S-a2 protein fragment is shown in SEQ ID NO:5 of the sequence list, and mouse IgG; a detection line and a quality control line are sequentially arranged on the reaction pad along the flow direction of the sample to be tested, and mouse anti-human IgG antibody or mouse anti-human IgM antibody and goat anti-mouse IgG antibody are sequentially arranged on the reaction pad along the flow direction of the sample to be tested, and are respectively recorded as G line or M line and C line, or mouse anti-human IgG antibody, mouse anti-human IgM antibody and goat anti-mouse IgG antibody are sequentially arranged on the reaction pad along the flow direction of the sample to be tested, and are respectively recorded as G line, M line and C line; the device is a detection card with a sample hole.

7. A method for preparing a device for detecting a novel coronavirus as claimed in claim 6, comprising the following steps: (1) Spraying mouse anti-human IgG antibody, mouse anti-human IgM antibody, and goat anti-mouse IgG antibody onto the NC membrane respectively; (2) adding colloidal gold to a mixed protein of S protein, S-a1 protein fragment and S-a2 protein fragment, adding mouse IgG, washing the precipitate with a labeling washing solution, dissolving it with a gold-labeled antibody preservation solution, and then evenly spreading it on a glass fiber membrane, and freeze-drying it to form a gold-labeled binding pad; wherein the glass fiber membrane is pre-treated with a gold-labeled binding pad blocking solution; (3) Overlap the absorbent paper, the NC membrane prepared in step (1), the gold label binding pad prepared in step (2), and the sample pad and stick them on the base plate.

8. The preparation method according to claim 7, characterized in that: The step (1) is to spray mouse anti-human IgG antibody, mouse anti-human IgM antibody, and goat anti-mouse IgG antibody onto the NC membrane, respectively, which are recorded as G line, M line, and C line, respectively; the mixed protein in the step (2) is a mixture of S protein, S-a1 protein fragment, and S-a2 protein fragment in equal proportions.

9. The preparation method according to claim 8, characterized in that: The specific steps are as follows: (1) Dilute the antibody with 0.01 mol / L PBS, spray 1.0 mg / mL mouse anti-human IgG antibody onto the NC membrane, marked as G line, spray 1.0 mg / mL mouse anti-human IgM antibody onto the NC membrane, marked as M line; spray 1.0 mg / mL goat anti-mouse IgG antibody onto the NC membrane, marked as C line, and set aside after completion; (2) adjusting the pH value of colloidal gold to 9 with 0.1 M K2CO3 solution, adding the aforementioned S protein, S-a1 protein fragment and S-a2 protein fragment mixed in equal proportions at 12 μg / mL, and adding 12 μg / mL mouse IgG at the same time, mixing thoroughly, standing and centrifuging to discard the supernatant, washing the precipitate with gold label washing solution, discarding the supernatant, and dissolving the precipitate with gold label antibody storage solution with a volume of one-tenth of the initial colloidal gold; evenly spreading the labeled colloidal gold on a glass fiber membrane pre-treated with a gold label binding pad blocking solution, and freeze-drying to obtain a gold label binding pad; (3) overlapping and pasting the absorbent paper, the NC membrane prepared in step (1), the gold-labeled binding pad prepared in step (2), and the sample pad on a polyvinyl chloride base plate or a plastic plate, cutting them into strips, and assembling them into a reagent strip or preparing a test card with a sample hole; The sample pad is a glass fiber membrane, non-woven fabric or filter paper that has been impregnated with a sample pad treatment solution, and the sample pad treatment solution includes the following components: Tween 20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.

10. The preparation method according to claim 9, characterized in that: in, The gold-labeled washing solution comprises 1% BSA, 0.01 mol / L PBS, pH 9; the gold-labeled antibody storage solution comprises 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN3 in 0.01 mol / L PBS, pH 9; the gold-labeled binding pad blocking solution comprises 0.01M PBS, 1% BSA, 1% Tween-20, pH 7.

5.

11. The use of a mixed protein comprising the S protein, S-a1 protein fragment and S-a2 protein fragment of the SARS-CoV-2 coronavirus in the preparation of a reagent for detecting the new coronavirus, wherein the amino acid sequence of the S-a1 protein fragment is shown in the sequence listing SEQ ID NO: 3, and the amino acid sequence of the S-a2 protein fragment is shown in the sequence listing SEQ ID NO: 5.

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