WO2021243813A1 - 一种检测新型冠状病毒的试剂盒和检测装置及其制备方法 - Google Patents

一种检测新型冠状病毒的试剂盒和检测装置及其制备方法 Download PDF

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WO2021243813A1
WO2021243813A1 PCT/CN2020/102212 CN2020102212W WO2021243813A1 WO 2021243813 A1 WO2021243813 A1 WO 2021243813A1 CN 2020102212 W CN2020102212 W CN 2020102212W WO 2021243813 A1 WO2021243813 A1 WO 2021243813A1
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protein
coronavirus
gold
sample
antibody
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PCT/CN2020/102212
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French (fr)
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马哲
赵文祥
田子琦
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湖南珐驷特生物科技有限公司
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Priority to CN202080002802.XA priority Critical patent/CN112384805A/zh
Publication of WO2021243813A1 publication Critical patent/WO2021243813A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

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  • the invention relates to the detection of antibodies, in particular to a colloidal gold chromatography kit for detecting SARS-CoV-2 coronavirus Spike protein antibodies, a detection device and a preparation method thereof.
  • coronavirus pneumonia (COVID-19) has become a public health emergency of international concern. As of now, there have been a total of 7 million confirmed cases worldwide and more than 400,000 deaths. For the new crown virus epidemic, speeding up the testing and confirmation of suspects and close contacts is an extremely effective prevention and control measure.
  • the new type of coronavirus is a ⁇ -coronavirus with a diameter of about 60-140nm and an envelope.
  • SARS-CoV-2 encodes 15 non-structural proteins (nsp1 ⁇ nsp10, nsp12 ⁇ nsp16), 4 structural proteins (S, E, M and N) and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14).
  • S-RBD receptor binding domain of the spike protein binds to its receptor angiotensin converting enzyme 2 (ACE2). This binding helps the virus attach to the surface of the target cell and infect the target cell.
  • the main methods include molecular detection, cell culture detection and antibody detection.
  • PCR method detects whether the SARS-CoV-2 specific RNA sequence is contained in the patient's sample for early pathogenic diagnosis of COVID-19.
  • this detection method has certain false negatives, 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 detect; in addition, the reliability and sensitivity of diagnostic reagents are limited.
  • metagenomic sequencing is to extract the new coronavirus RNA from the patient's lower respiratory tract secretions, construct a viral cDNA library, and then perform high-throughput sequencing , Through database comparison analysis, to identify whether the genome sequence is highly homologous to SARS-CoV-2.
  • mNGS for SARS-CoV-2 detection are high specificity and sensitivity, but also obvious disadvantages, high instrument configuration requirements and long detection cycle; cell culture method is a kind of aseptic operation technique, which requires aseptic operation room and ultra-clean work Tables, cell incubators, centrifuges, microscopes, etc., the experimental technology is complex, and requires experienced professional and technical personnel to operate, and the experimental period is very long, not suitable for rapid detection of diseases; antibody detection is mainly 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 more complicated, and repeated washing is required.
  • ELISA enzyme-linked immunosorbent reaction
  • the washing times are not enough or too many, it will easily cause false positives and false negatives, and will easily cause harm to the operator. And environmental pollution, the experiment time is long, it takes more than two hours to get the test results.
  • This method must be equipped with a microplate reader and a plate washer, which is difficult to achieve in primary laboratories and small outpatient clinics.
  • the reagents for detecting virus 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, so as to prevent the spread of the epidemic early; blood testing solves the inaccurate sampling of throat swabs The problem.
  • the accuracy of the new coronavirus detected at present needs to be further improved.
  • the mutation of the new coronavirus quickly makes detection more difficult. Therefore, based on the current severe epidemic prevention situation, an efficient and accurate The detection method is very necessary.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art, and provide a kit or test strip for accurate detection of novel coronavirus and its preparation method and application, which can be used in hospitals, airports, customs, homes and other places, and can Judge the result within a few minutes, so as to prevent the spread of the epidemic early.
  • the detection of the present invention uses the new coronavirus S protein and its specific fragments to detect the IgM and IgG antibodies of the new coronavirus spike protein Spike.
  • S protein is a membrane protein, which constitutes the envelope particles of the virus and is the main component of the virus infecting host cells.
  • the new coronavirus To infect cells, the new coronavirus must first introduce its genetic material RNA into the host cell. This process is mainly done through the S protein.
  • the protrusion of S protein is the key part of its interaction with 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.
  • the inventors conducted in-depth research to find the specific fragments S-a1 and S-a2 of the S protein, so that after the S protein is mutated, it can still be detected by detecting the specific fragments S-a1 and S-a2.
  • the target analyte not only increases the scope of use of the kit, but also ensures the accuracy of the results of the kit.
  • the present invention provides a kit for detecting a novel coronavirus, which includes the 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 as follows: The list is shown in SEQ ID NO: 3, and the amino acid sequence of the S-a2 protein fragment is shown in SEQ ID NO: 5 in the sequence list.
  • the kit also includes mouse IgG antibodies, and further includes mouse anti-human IgG antibodies and/or mouse anti-human IgM antibodies, and goats
  • the anti-mouse IgG antibody further includes an S protein IgG antibody as a standard and/or an S protein IgM antibody, and further includes a sample processing solution.
  • the novel coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.
  • the kit is used for indirect detection of whether the serum of the sample to be tested contains antibodies against the novel coronavirus, preferably including the kit and related reagents for indirect detection.
  • the kit is used for colloidal gold chromatography for detection, so the kit includes a detection device and a sample processing solution, preferably the sample diluent contains 0.2% (volume) Tween 20 and 0.6% (Volume) 0.1M PBS solution of Casein, the test device reagent strip or test card.
  • the detection device is prepared by the following method:
  • Another aspect of the present invention provides a device for detecting a new type of coronavirus, wherein the device includes a sample pad, a gold label binding pad, a reaction pad, and an absorbent pad that are sequentially connected; wherein the gold label binding pad is coated with a colloidal gold label
  • the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of the sample to be tested, and further on the reaction pad
  • a mouse anti-human IgG antibody or a mouse anti-human IgM antibody, and a goat anti-mouse IgG antibody are sequentially arranged along the flow direction of the sample to be tested, respectively denoted as the T line (that is, the G line or the M line) and the C line, preferably the reaction A mouse anti-human IgG antibody, a mouse anti-human IgM antibody, and a goat anti-mouse Ig
  • the novel coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.
  • the antibody when the sample to be tested contains human anti-new coronavirus antibodies, the antibody first binds to the gold-labeled antigen. Due to the chromatographic effect, the reaction complex moves forward along the coating membrane. When it encounters the coated anti-antibody, it forms The antibody-gold-labeled antigen-anti-antibody complex is enriched on the coated line to form a red precipitation line, which is a positive result, so as to quickly diagnose whether the sample contains IgM/IgG of the new coronavirus (SARS-CoV-2) antibody.
  • SARS-CoV-2 new coronavirus
  • the present invention further provides a preparation method for preparing the aforementioned device for detecting novel coronavirus, which includes the following steps:
  • the absorbent paper, the NC film prepared in step (1), the gold label bonding pad prepared in step (2) and the sample pad are overlapped and pasted on the bottom plate, and then cut into strips and assembled.
  • the reagent strip may be further prepared into a test card with sample holes.
  • the novel coronavirus is a beta coronavirus, especially SARS-CoV-2 coronavirus, SARS virus, MERS virus, and more preferably SARS-CoV-2 coronavirus.
  • 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, and mark them 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 proportions.
  • the sample pad is a glass fiber membrane or non-woven fabric or filter paper impregnated with a sample pad treatment solution, and further preferably the sample pad treatment solution includes the following components: Tween 20, Triton x-405, Casein, BSA, PEG-20000 and NaCl.
  • the specific components of the gold-labeled washing solution used are: 1% BSA, 0.01 mol/L PBS, pH 9; the gold-labeled antibody preservation solution: 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN 3 0.01mol/L PBS, pH 9; the gold-labeled binding pad blocking solution: 0.01M PBS, 1% BSA, 1% Tween-20, pH 7.5.
  • the present invention also provides the application of the mixed protein of S protein, S-a1 protein fragment and S-a2 protein fragment of SARS-CoV-2 coronavirus in preparing reagents for detecting novel coronavirus, wherein the amino acid sequence of S-a1 protein fragment is as
  • the sequence table SEQ ID NO: 3 is shown in the sequence table SEQ ID NO: 5 amino acid sequence of the S-a2 protein fragment.
  • the application is to detect whether there are antibodies against the new coronavirus in the sample by colloidal gold chromatography.
  • the new coronavirus is ⁇ -coronavirus, especially SARS-CoV-2 coronavirus.
  • the sample to be tested is selected from samples of convalescent people and samples of healthy people.
  • the samples are preferably nasopharyngeal swabs, sputum, alveolar lavage fluid, blood, and urine. At least one of them.
  • the detection device of the present application can detect both IgM and IgG antibodies at the same time.
  • 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 eliminating the virus, and can be used to evaluate the immune response of patients or the immune effect of vaccines.
  • This kit is used to detect virus infection from the perspective of immune response, which is exactly complementary to the current universal virus nucleic acid detection kit, which can effectively avoid the generation of false negative results.
  • the reagent of the present invention adopts colloidal gold immunochromatography technology, selects the purified new coronavirus (SARS-CoV-2) spike protein Spike antigen and its two specific fragments as the solid phase, and uses indirect method to detect whether the serum contains Human anti-new coronavirus antibodies.
  • the test substance detected by the detection reagent strip of the present invention is S protein, which avoids the problem of high false positives in N protein detection, and has reliable detection results and high stability; more particularly, the present invention
  • the invented reagent strip can detect the full length of S protein, as well as S-a1 protein fragments and S-a2 protein fragments.
  • 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 scope of application of the detection kit is expanded.
  • the full length of S protein can be measured, and specific fragments can also be identified, which greatly improves the identification specificity and accuracy of the kit.
  • experiments show that the reagent strip of the present invention can achieve the sensitivity of detecting 0.016 ⁇ g S protein IgG antibody and 0.031 ⁇ g S protein IgM antibody by naked eye observation, and the detection result is stable and accurate and can be recognized by the naked eye. It is suitable for hospitals, airports, customs, homes and other places. The ability to judge the result within a few minutes, so as to prevent the spread of the epidemic early, has very important promotion and application value.
  • Figure 1 shows the alignment results of S-a1 and S-a2 with the S protein sequence of different coronaviruses
  • Figure 2 is a PAGE identification diagram of S protein
  • FIG. 3 shows the results of binding activity of S-a1, S-a2 and S protein to IgG antibodies in the serum of convalescent patients
  • Example 4 is a schematic diagram of the detection result of the sensitivity of the reagent strip prepared in Example 4.
  • Example 5 is a schematic diagram of the specific detection result of the positive reference product of the reagent strip prepared in Example 4;
  • FIG. 6 is a schematic diagram of the specific detection results of the reagent strip prepared in Example 4 on 13 blood samples that were positive by PCR;
  • Figure 7 is a schematic diagram of the specific detection results of the reagent strip prepared in Example 4 on normal human blood samples
  • Figure 8 is a schematic diagram of the specific detection results treated with DTT
  • FIG. 9 is a schematic diagram of the stability test result of the reagent strip prepared in Example 4.
  • FIG. 10 is a schematic diagram of the stability results of the sample test results of the reagent strip prepared in Example 4.
  • Figure 11 shows the sample stabilization results of different sampling sources.
  • Example 1 Construction of S protein (spike protein) coding gene fragment and strong antigenic S protein fragment
  • the full-length base sequence of Spike protein is shown in SEQ ID NO:1, and the full-length nucleotide sequence of its coding gene is shown in SEQ ID NO:2. Synthesize the full-length gene encoding Spike protein by conventional methods.
  • upstream primers and downstream primers are designed, and the primer sequences are as follows:
  • PCR amplification obtains more Spike protein coding sequences.
  • the PCR system is as follows:
  • reaction solution to the PCR tube according to the above table, and mix well. Place the PCR reaction tube in a PCR machine and set the reaction conditions: denaturation at 95°C for 5 minutes; 95°C for 30S, 58°C for 30S, 72°C for 4 minutes; 72°C for 10 minutes; the product can be stored briefly at 4°C for subsequent experiments.
  • the first conserved region is the S-a1 fragment, its amino acid sequence is shown in SEQ ID NO: 3, and the encoded nucleotide sequence is shown in SEQ ID NO: 4; the other conserved region S-a2 fragment, its amino acid sequence The sequence is shown in SEQ ID NO: 5, and the encoded nucleotide sequence is shown in SEQ ID NO: 6.
  • the coding sequence of S-a1 fragment and S-a2 fragment was synthesized by the same conventional method.
  • the full-length sequence of Spike protein coding gene is as follows: (SEQ ID NO: 2)
  • amino acid sequence of S-a1 is as follows: (SEQ ID NO: 3)
  • the base sequence of S-a1 is as follows: (SEQ ID NO: 4)
  • amino acid sequence of S-a2 is as follows: (SEQ ID NO: 5)
  • the base sequence of S-a2 is as follows: (SEQ ID NO: 6)
  • the upstream and downstream primers are designed for the coding sequence of the S-a1 and S-a2 fragments.
  • the primer sequences are as follows:
  • the S-a1 base sequence was amplified by the PCR method.
  • the PCR system is as follows:
  • reaction solution to the PCR tube according to the above table, and mix well. Place the PCR reaction tube in the PCR machine and set the reaction conditions: denaturation at 95°C for 5 minutes; 95°C for 30S, 58°C for 30S, 72°C for 4 minutes; 72°C for 10 minutes; the product can be stored briefly at 4°C for subsequent experiments.
  • the S-a2 base sequence was amplified by the PCR method.
  • the PCR system is as follows:
  • reaction solution to the PCR tube according to the above table, and mix well. Place the PCR reaction tube in the PCR machine and set the reaction conditions: denaturation at 95°C for 5 minutes; 95°C for 30S, 58°C for 30S, 72°C for 4 minutes; 72°C for 10 minutes; the product can be stored at 4°C for a short period of time for subsequent experiments.
  • the digestion system is as follows:
  • the DNA concentration of the obtained product was measured with Nano-300 and diluted to 50ng/ ⁇ L.
  • connection system is as follows:
  • Example 4 Detection of binding activity of S-a1, S-a2 and S protein to IgG antibody in the serum of convalescent patients
  • ELISA was used to detect the binding of S-a1, S-a2 and S protein to the IgG antibody in the serum of convalescent patients. Specific steps are as follows:
  • Preparation of coating film 0.01mol/L PBS diluted antibody, the mouse anti-human IgG antibody concentration is 1.0mg/mL sprayed on the NC membrane, marked as G line; the mouse anti-human IgM antibody concentration is 1.0mg/mL sprayed On the NC membrane, mark it as the M line; the goat anti-mouse IgG antibody concentration is 1.0mg/mL, spray it on the NC membrane, mark it as the C line, and use it for later use.
  • gold label washing solution 1% BSA, 0.01mol/L PBS, pH 9
  • gold label antibody preservation solution 1% BSA, 0.5% PEG20000, 2% sucrose, 0.05% NaN3 0.01mol/L PBS, pH 9
  • gold Standard binding pad blocking solution 0.01M PBS, 1% BSA, 1% Tween-20, pH 7.5.
  • Test card preparation Take out the test card from the test kit and equilibrate to room temperature. Tear off the foil bag and place the test card horizontally.
  • Blood collection 1) Disinfect the blood collection fingers with alcohol disinfection tablets. After the alcohol is completely volatilized about half a minute later, use a disposable blood collection device to collect blood; 2) Gently squeeze the head of the disposable capillary and release it slowly. Fingertip blood is collected into the capillary tube under negative pressure, and air bubbles should be avoided when sampling.
  • Blood sample detection drop 1 drop (about 30 ⁇ L) of blood sample vertically into the sample hole in the form of a test card, and then add 3 drops (about 120 ⁇ L) of diluent to the sample hole. (If it is a general reagent strip, immerse the sample pad in the sample to be tested, take it out and wait for subsequent reading results)
  • IgG positive result the control line and IgG line are visible on the test strip. The test result was positive for IgG antibodies. This means that the patient is recovering from the infection.
  • IgM positive result Control line C line and IgM line are visible on the test strip. A positive IgM antibody test indicates that the patient is in the acute phase of infection.
  • IgM/IgG double positive result the control line, IgM line and IgG line are clearly visible on the test strip. Test positive for IgM and IgG antibodies. Indicates that the patient is in the acutely advanced stage of infection.
  • control line is the only visible line in the test strip. No IgG or IgM antibodies were detected. The result does not rule out infection. If the symptoms persist, a new sample should be taken from the patient within 3-5 days and the test should be repeated.
  • the detection amounts were 0.5 ⁇ g, 0.25 ⁇ g, 0.125 ⁇ g, 0.0625 ⁇ g, 0.031 ⁇ g, 0.016 ⁇ g, 0.0078 ⁇ g, 0.0039 ⁇ g, 0.0019 ⁇ g, 0.001 ⁇ g.
  • the sensitivity of the detection card Experiments show that the detection card prepared in Example 4 can reach the sensitivity of detecting 0.016 ⁇ g S protein IgG antibody by naked eye observation (see Figure 2 for details).
  • test card prepared in Example 4 can reach the sensitivity of naked eye detection of 0.031 ⁇ g S protein IgM antibody.
  • Figure 4 where 0.031 ⁇ g is visible to the naked eye but weaker, due to the difference between naked eyes and photos, It is not obvious in the photo, but 0.0625 ⁇ g is also more obvious in the photo).
  • 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 ⁇ g and the detection amount of IgM was 0.25 ⁇ g. The experimental results were consistent. See Figure 5.
  • test cards prepared in Example 5 into 3 groups, each with 10 pieces, and store them at 37°C for 2, 5, and 7 days, and then use them to detect five IgG/IgM standard samples, and store them at room temperature each time.
  • the test card is treated at 37°C for 0 days. The results showed that the storage at 37°C for 2 days, 5 days and 7 days did not affect the test results compared with the samples stored normally, as shown in Figure 9.

Abstract

一种检测新型冠状病毒的试剂盒和检测装置及其制备方法，其中是通过利用SARS-CoV-2刺突蛋白Spike、S-a1蛋白片段和S-a2蛋白片段，间接检测待测样本血清中是否含有抗新型冠状病毒的抗体。检测试剂的检测准确、检测效率高，检测结果稳定准确肉眼可识别，实验表明采用本发明的检测装置可以达到肉眼观察检测0.016μg S蛋白IgG抗体、0.031μg S蛋白IgM抗体的灵敏度。适用于医院、机场、海关、家庭等场所，能够在几分钟内判断结果，从而及早预防疫情扩散，具有十分重要的推广应用价值。

Description

一种检测新型冠状病毒的试剂盒和检测装置及其制备方法 技术领域
本发明涉及抗体的检测，具体的是一种检测SARS-CoV-2冠状病毒Spike蛋白抗体的胶体金层析试剂盒，及检测装置及其制备方法。
背景技术
新型冠状病毒肺炎(COVID-19)已成为国际关注的突发公共卫生事件，截至目前，全球累计确诊病例700万例，死亡人数超过40万人。对于新冠病毒疫情来说，加速疑似、密切接触者等的检测筛查确认，是极为有效的防控措施。
新型冠状病毒(SARS-CoV-2)是直径约为60～140nm，且有包膜的β属冠状病毒。SARS-CoV-2编码15种非结构蛋白(nsp1～nsp10、nsp12～nsp16)、4种结构蛋白(S、E、M和N)和8种辅助蛋白(3a、3b、p6、7a、7b、8b、9b和orf14)。新型冠状病毒的刺突蛋白有助于病毒进入靶细胞。刺突蛋白的受体结合域(S-RBD)与其受体血管紧张素转化酶2(ACE2)结合，这种结合有助于病毒附着至靶细胞的表面，进而侵染靶细胞。
世界各国的研究者们正在研究快速而准确的新型冠状病毒检测方法，目前主要方法有分子检测、细胞培养检测以及抗体检测。
一种是分子测试(PCR法)，其是检测患者样本中是否含有SARS-CoV-2特异性的RNA序列，来对COVID-19进行早期病原学诊断。但这一检测方法存在一定的假阴性，阳性率只有30％-50％。原因在于临床样本采集的部位与方法不同，导致SARS-CoV-2的载量过少，无法检测；另外，诊断试剂的可靠性及敏感性有限。同时该方法需要专业的人员操作，要求很高的实验工作条件；宏基因组测序(mNGS)是从患者的下呼吸道分泌物中提取新型冠状病毒的RNA，构建病毒cDNA文库，然后进行高通量测序，通过数据库比对分析，鉴定基因组序列是否与SARS-CoV-2高度同源。mNGS检测SARS-CoV-2的优势在于特异性和灵敏度高，缺点也较为明显，仪器配置要求高并且检测周期长；细胞培养法是一种无菌操作技术，需要无菌操作间、超净工作台、细胞培养箱、离心机、显微镜等，实验技术复杂，并需要有经验的专业技术人员操作，且实验周期很长，不适宜疾病的快速检测；抗体检测主要酶联免疫吸附反应(ELISA)：ELISA法要求抗原纯度高、特异性好，否则会出现非特异反应，操作程序较复杂，并需要反复洗涤，若洗涤次数不够或过多易造成假阳性和假阴性，并易造成操作者受害和环境污染，实验时间较长，需两小时以上得出检测结果。该法必须具备酶标仪和洗板机，这在基层实验室和小型门诊较难达到。其中，胶体金层析法检测病毒抗体的试剂，可应用于医院、机场、海关、家庭等场所，能够在几分钟内判断结果，从而及早预防疫情扩散；血液检测解决咽拭子取样不尽准确的问题。
但目前检测的新型冠状病毒的准确性等方面有待进一步提升，尤其是新型冠状病毒的变异快给检测带来更高的难度，因此基于目前严峻的防疫形势，尽快开发出一款高效、准确的检测方法是十分必要的。
发明内容
本发明的目的是克服了上述现有技术的缺点，提供了一种准确检测新型冠状病毒试剂盒或试剂条及其制备方法和应用，其可应用于医院、机场、海关、家庭等场所，能够在几分钟内判断结果，从而及早预防疫情扩散。
为了实现上述目的，本发明的检测利用新型冠状病毒S蛋白及其特异性片段以检测新型冠状病毒刺突蛋白Spike的IgM与IgG抗体。其中，S蛋白是膜蛋白，其构成病毒的包颗粒，是病毒感染宿主细胞的主要成分。新型冠状病毒要感染细胞，首先要将其遗传物质RNA导入到宿主细胞中，这一过程主要是通过S蛋白来完成的。S蛋白的突起是其与受体相互作用和膜融合的关键部位。但S蛋白全长易发生变异，如果简单的针对该S蛋白进行检测，很可能不能将发生变异后的病毒。对此，发明人通过深入研究，寻找出S蛋白的特异性片段S-a1和S-a2，以便在S蛋白发生变异后，仍然还可以通过检测特异性片段S-a1，S-a2来检测目标待测物，不仅提高了本试剂盒的使用范围，还保证了本试剂盒的结果准确性。
因此，本发明提供一种检测新型冠状病毒试剂盒，其包括SARS-CoV-2冠状病毒的S蛋白，S-a1蛋白片段和S-a2蛋白 片段，其中，S-a1蛋白片段氨基酸序列如序列表SEQ ID NO:3所示，S-a2蛋白片段氨基酸序列如序列表SEQ ID NO:5所示。
进一步地，为了利用抗体检测法，即为了检测新型冠状病毒针对S蛋白抗体，所述试剂盒还包括小鼠IgG抗体，进一步还包括鼠抗人IgG抗体和/或鼠抗人IgM抗体，以及山羊抗鼠IgG抗体，进一步还包括作为标准品的S蛋白IgG抗体和/或作为S蛋白IgM抗体，更进一步包括样品处理液。
其中，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
在具体实施方式中，所述试剂盒是用于间接法检测待测样本血清中是否含有抗新型冠状病毒的抗体，优选包括所述试剂盒还包括用于间接法检测的相关试剂。
优选地，所述试剂盒是用于胶体金层析法进行检测，因而所述试剂盒包括检测装置和样品处理液，优选地所述样品稀释液为含有0.2％(体积)Tween 20和0.6％(体积)Casein的0.1MPBS溶液，所述检测装置试剂条或检测卡。在一个具体实施方式中，所述检测装置由下述方法制备得到：
(1)将鼠抗人IgG抗体、和/或鼠抗人IgM抗体、和/或山羊抗鼠IgG抗体分别喷到NC膜上；
(2)将胶体金加入S蛋白、S-a1蛋白片段与S-a2蛋白片段的混合蛋白中，加入小鼠IgG抗体，用标记洗涤液洗涤沉淀，再用金标抗体保存液溶解，然后均匀的铺在玻璃纤维膜(预先用金标结合垫封闭液预处理)，冷冻干燥为金标结合垫；
(3)将吸水纸、第(1)步获得的NC膜、金标结合垫以及样品垫交叠粘贴在胶板上，切条得到试剂条。
本发明另一方面提供一种检测新型冠状病毒的装置，其中所述装置包括依次相连的样品垫、金标结合垫、反应垫和吸水垫；其中所述金标结合垫上包被有胶体金标记的S蛋白、S-a1蛋白片段与S-a2蛋白片段的混合蛋白以及小鼠IgG抗体；所述反应垫上沿待检样品流动方向依次设有检测线和质控线，进一步地所述反应垫上沿待检样品流动方向依次设有鼠抗人IgG抗体或鼠抗人IgM抗体、以及山羊抗鼠IgG抗体分别记为T线(即G线或M线)、以及C线，优选地所述反应垫上沿待检样品流动方向依次设有鼠抗人IgG抗体、鼠抗人IgM抗体、以及山羊抗鼠IgG抗体分别记为G线、M线、和C线；所述装置为检测试剂条，更优选为带有样品孔的检测卡。
具体地，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
其中，当待检样本中含有人抗新型冠状病毒抗体时，抗体先和金标记的抗原结合，由于层析作用，反应复合物沿包被膜向前移动，当遇到包被抗抗体时，形成抗体--金标抗原--抗抗体复合物而富集在包被线上形成红色沉淀线，为阳性结果，从而快速诊断样本中是否含有新型冠状病毒(SARS-CoV-2)的IgM/IgG抗体。
本发明进一步提供制备上述的检测新型冠状病毒的装置的制备方法，其包括如下步骤：
(1)将鼠抗人IgG抗体、和/或鼠抗人IgM抗体，以及山羊抗鼠IgG抗体分别喷到NC膜上；
(2)将胶体金加入S蛋白、S-a1蛋白片段与S-a2蛋白片段的混合蛋白中，加入小鼠IgG抗体，用标记洗涤液洗涤沉淀，再用金标抗体保存液溶解，然后均匀的铺在预先用金标结合垫封闭液预处理的纤维膜上，冷冻干燥为金标结合垫；
(3)将吸水纸、第(1)步制得的NC膜、第(2)步制得的金标结合垫以及样品垫交叠粘贴在底板上即得，进一步地进行切条，组装得到试剂条或进一步制备成带有样品孔的检测卡。
具体地，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
在优选实施方式中，所述第(1)步是将鼠抗人IgG抗体、鼠抗人IgM抗体、山羊抗鼠IgG抗体分别喷到NC膜上，分别记为G线、M线、C线；所述第(2)步中混合蛋白是S蛋白、S-a1蛋白片段与S-a2蛋白片段等比例混合而成。
在一个具体实施方式中，其具体步骤如下：
(1)用0.01mol/L PBS稀释抗体，鼠抗人IgG抗体浓度为1.0mg/mL喷到NC膜上，记为G线；或者将鼠抗人IgM抗体浓度为1.0mg/mL喷到NC膜上，记为M线；或者将鼠抗人IgG抗体和鼠抗人IgM抗体分别喷到NC膜上，分别记为G线和M线；山羊抗鼠IgG抗体浓度为1.0mg/mL，喷到NC膜上，记为C线，完成后备用。
(2)用0.1M的K 2CO 3溶液调节胶体金pH值至9，按照12μg/mL加入前述S蛋白、S-a1蛋白片段与S-a2蛋白片段等比例混合的蛋白，同时加入12μg/mL小鼠IgG抗体，充分混匀，静置后离心弃上清，沉淀用金标洗涤液洗涤，弃上清，将沉淀用十分之一初始胶体金体积的金标抗体保存液溶解；将标记好的胶体金均匀的铺在玻璃纤维膜上，冷冻干燥为金标结合垫；
(3)将吸水纸、第(1)步制备得到的NC膜、第(2)步制备得到的金标结合垫，以及样品垫，依次交叠粘贴在聚氯乙烯底板或胶板上，切条，组装得到试剂条；或制备成带为样品孔的检测卡。
其中，优选地，所述样品垫是经过样品垫处理液浸渍处理的玻璃纤维膜或无纺布或滤纸，进一步优选地所述样品垫处理液包括如下组分：Tween 20、Triton x-405、Casein、BSA、PEG-20000和NaCl。
上述步骤，具体采用的金标洗涤液组分为：1％BSA，0.01mol/L PBS，pH9；所述金标抗体保存液：1％BSA，0.5％PEG20000，2％蔗糖，0.05％NaN 3的0.01mol/L PBS，pH9；所述金标结合垫封闭液：0.01M PBS，1％BSA，1％Tween-20，pH7.5。
本发明还提供包括SARS-CoV-2冠状病毒的S蛋白，S-a1蛋白片段和S-a2蛋白片段混合蛋白在制备检测新型冠状病毒的试剂中的应用，其中S-a1蛋白片段氨基酸序列如序列表SEQ ID NO:3所示，S-a2蛋白片段氨基酸序列如序列表SEQ ID NO:5所示。
在优选实施方式中，所述应用是通过胶体金层析法检测样品中是否存在含有抗新型冠状病毒的抗体，优选地，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒
在更具体应用中，其检测是的样品选自待检对象是恢复期人的样品及健康人的样品，所述样品优选为鼻咽拭子、痰液、肺泡灌洗液、血液、尿液中的至少一种。
本申请的检测装置如试剂条或检测卡可以同时检测IgM和IgG两种抗体。其中IgM是在病毒感染早期产生的，可以用于评价病毒的早期感染；IgG在感染的后期逐渐增加，主要发挥彻底清除病毒的作用，可用于评价患者体内免疫应答情况或疫苗免疫效果。本试剂盒是从免疫应答的角度检测病毒的感染，与目前通用的病毒核酸检测试剂盒恰好形成互补，可有效避免假阴性结果的产生。本发明的试剂采用胶体金免疫层析技术，选用纯化的新型冠状病毒(SARS-CoV-2)刺突蛋白Spike抗原及其两个特异性片段作为固相物，利用间接法检测血清中是否含有人抗新型冠状病毒抗体。其中，尤其是与现有技术相比，本发明的检测试剂条检测的待测物为S蛋白，避免了N蛋白检测假阳性高的问题，检测结果可靠、稳定性高；更尤其是，本发明的试剂条可以检测S蛋白全长，也可以检测S-a1蛋白片段和S-a2蛋白片段，因而可以在S蛋白发生变异时识别其高保守的蛋白片段S-a1、S-a2，提高了试剂盒的检测特异性，并且扩展了检测试剂盒的适用范围。同时可以测定S蛋白全长，也可识别特异性片段，大大提高了试剂盒的识别特异性和准确性。同时实验表明，本发明的试剂条可以达到肉眼观察检测0.016μg S蛋白IgG抗体、0.031μg S蛋白IgM抗体的灵敏度，检测结果稳定准确肉眼可识别，适用于医院、机场、海关、家庭等场所，能够在几分钟内判断结果，从而及早预防疫情扩散，具有十分重要的推广应用价值。
附图说明
图1为S-a1和S-a2与不同冠状病毒S蛋白序列比对结果；
图2为S蛋白的PAGE鉴定图；
图3为S-a1、S-a2以及S蛋白与康复期病人的血清中IgG抗体结合活性结果；
图4为实施例4制备的试剂条的灵敏度的检测结果示意图；
图5为实施例4制备的试剂条对阳性参考品的特异性检测结果示意图；
图6为实施例4制备的试剂条对13例经PCR鉴定阳性的血样特异性检测结果示意图；
图7为实施例4制备的试剂条对正常人血样特异性检测结果示意图；
图8为经DTT处理的特异性检测结果示意图；
图9为实施例4制备的试剂条的稳定性检测结果示意图；
图10为采用实施例4制备的试剂条的样品检测结果稳定性结果示意图；
图11为不同取样来源的样本稳定结果。
具体实施方式
为了能够更清楚地描述本发明的技术内容，下面结合具体实施例来进行进一步的描述。
下列实施例中仅对本发明内容涉及改进的部分进行详述，其他未提及步骤或原料来源同现有技术，在此不做赘述。
实施例1：S蛋白(spike蛋白)编码基因片段及强抗原性S蛋白片段的构建
1.S蛋白编码基因的合成
Spike蛋白全长碱基序列如SEQ ID NO:1所示，其编码基因的全长核苷酸序列如SEQ ID NO:2所示。通过常规方法合成Spike蛋白的全长编码基因。
根据上述全长序列设计下述上游引物和下游引物，引物序列如下：
引物 序列表编号 序列(5’-3’)
正向S-F SEQ ID NO:7 ggggggGCGGCCGCATGTTTGTTTTTCTTGTTTT
反向S-R SEQ ID NO:8 ggggggCCATGGTTATGTGTAATGTAA
以前面合成的S蛋白编码序列为模板，PCR扩增获得更多的Spike蛋白编码序列。其中PCR体系如下：
  体积(uL)
S-F(2μM) 5
S-R(2μM) 5
模板质粒(100ng/uL) 1
引物混合物(SEQ ID NO:7和SEQ ID NO:8) 5
水 34
总体积 50
按上表将反应溶液加入到PCR管中，混合均匀。将PCR反应管置于PCR仪中，设置反应条件：95℃变性5min；95℃30S，58℃30S，72℃4min；72℃10min；产物可4℃短暂保存，用于后续实验。
2.S-a1和S-a2片段的编码序列的合成
经Protein knowledgebase分析比对，筛选Spike中高免疫原性的特异序列。两条序列与不同冠状病毒Spike蛋白序列比对结果见图1。经分析，确定了位于RBD不同区域的两条序列S-a1与S-a2经数据库分析得到了肽段a1与a2，同时也经过相关实验验证效果不低于S蛋白，对最终得到的检测试剂卡效果好起到重要作用。
其中第一保守区域是S-a1片段，其氨基酸序列如SEQ ID NO:3所示，其编码的核苷酸序列如SEQ ID NO:4所示；另一个保守区域S-a2片段，其氨基酸序列如SEQ ID NO:5所示，其编码的核苷酸序列如SEQ ID NO:6所示。同样采用常规方法合成，S-a1片段和S-a2片段的编码序列。
其中：
Spike蛋白全长碱基序列如下所示(SEQ ID NO:1)
Figure PCTCN2020102212-appb-000001
Figure PCTCN2020102212-appb-000002
Spike蛋白编码基因的全长序列如下所示：(SEQ ID NO:2)
Figure PCTCN2020102212-appb-000003
S-a1氨基酸序列如下所示：(SEQ ID NO:3)
Figure PCTCN2020102212-appb-000004
S-a1碱基序列如下所示：(SEQ ID NO:4)
Figure PCTCN2020102212-appb-000005
S-a2氨基酸序列如下所示：(SEQ ID NO:5)
Figure PCTCN2020102212-appb-000006
Figure PCTCN2020102212-appb-000007
S-a2碱基序列如下所示：(SEQ ID NO:6)
Figure PCTCN2020102212-appb-000008
根据上述序列分别针对S-a1与S-a2片段的编码序列设计上游引物和下游引物，引物序列如下：
引物 序列表编号 序列(5’-3’)
正向S-a1-F SEQ ID NO:9 ggggggGCGGCCGCGTCCAACCAACAGAATCTAT
反向S-a1-R SEQ ID NO:10 ggggggCCATGGAAAATCATCTGGTAATTT
正向S-a2-F SEQ ID NO:11 ggggggGCGGCCGCACAGGCTGCGTTATAGCTTGGA
反向S-a1-R SEQ ID NO:12 ggggggCCATGGGAAATTGACACATTTTT
以前面合成的S-a1片段的碱基序列为模板，PCR的方法来扩增S-a1碱基序列。其中，PCR体系如下：
  体积(uL)
S-a1-F(2μM) 5
S-a1-R(2μM) 5
模板质粒(100ng/uL) 1
引物混合物(SEQ ID NO:9和SEQ ID NO:10) 5
水 34
总体积 50
按上表将反应溶液加入到PCR管中，混合均匀。将PCR反应管置于PCR仪中，设置反应条件：95℃变性5min；95℃ 30S，58℃ 30S，72℃ 4min；72℃ 10min；产物可4℃短暂保存，用于后续实验。
以前面合成的S-a2片段的碱基序列为模板，通过PCR方法扩增S-a2碱基序列。其中，PCR体系如下：
  体积(uL)
S-a2-F(2μM) 5
S-a2-R(2μM) 5
模板质粒(100ng/uL) 1
引物混合物(SEQ ID NO:11和SEQ ID NO:12) 5
水 34
总体积 50
按上表将反应溶液加入到PCR管中，混合均匀。将PCR反应管置于PCR仪中，设置反应条件：95℃变性5min；95℃ 30S，58℃ 30S，72℃ 4min；72℃ 10min；产物可4℃短暂保存，用于后续实验。
3.S蛋白、S-a1及S-a2表达质粒的构建
3.1.pCDNA载体的预处理
酶切体系如下：
pCDNA 50μg
NotI-HF 5μL
NcoI-HF 5μL
CutSmart 10× 20μL
ddH2O up 200μL
总计 200μL
37℃水浴酶切5h，酶切结束后进行DNA电泳。首先使用纯水清洗电泳槽，更换新的1×TAE buffer。酶切后的体系加25μL 10×loading buffer，混匀点样，每孔点样50μL，150V电泳30min。在切胶仪上将约4000bp左右的目的片段切下来，转移至预先称重的5mL离心管中，称重，得到胶块的重量，使用天根通用型DNA纯化回收试剂盒回收目的片段。得到的产物用Nano-300测定DNA浓度，并稀释至50ng/μL。
3.2.连接
连接体系如下：
  体积(μL)
上述预处理的pCDNA(NcoI/NotI) 0.5
S蛋白基因全长或S-a1或S-a2片段 8
T4 Ligase 0.5
T4 Ligase Buffer 1
总体积 10
按照上述连接体系分别对S蛋白基因全长或S-a1或S-a2片段连接到pCDNA载体上的连接反应，16℃连接30min，使用TOP10感受态进行转化涂板，第二天挑菌送测。比对测序结果，分别获得三种正确序列的表达质粒，称为pCDNA-S，pCDNA-S-a1，pCDNA-S-a2，用于进行后续试验。
实施例2
本实施例与实施例1的区别仅在于，本实施例的对S蛋白编码基因扩增的引物不同，根据S蛋白序列的合成的下述上游引物和下游引物：
引物 序列表编号 序列(5’-3’)
正向S-F SEQ ID NO:13 cgtccggCAGTGCGTGAACCTGACCACCCGCACCCAGCTGCCGCCGGCGTATgc
反向S-R SEQ ID NO:14 ggccgcGGTATAATGCAGTTTCACGCCTTTCAGCACccg
实施例3：S蛋白、S-a1和S-a2蛋白片段的表达纯化
1、细胞转染：
i.将293F细胞培养到密度约为1x10 6/mL；
ii.1mL新鲜培养基重悬50μL PEI，1mL新鲜培养基分别重悬pCDNA-S，pCDNA-S-a1，pCDNA-S-a2三种质粒10μg，混匀，室温静置5min；
iii.将PEI预混液与质粒预混液混匀后室温静置20min；
iv.将PEI-质粒复合物逐滴滴加到20ml 293F细胞中，静止5min后放于CO2摇床中培养，培养三天后收上清。
2、蛋白的纯化及鉴定：
i.收集上清到50mL离心管中，10000rpm离心10min，取上清至一干净的离心管中；
ii.将预装有2mL ProteinA填料的柱子用10倍柱床体积的去离子水清洗介质；
iii.30mL PBS以平衡介质；
iv.将上清转移至柱子中，调节流速使液体以2s每滴的速度流出；
v.用30mL PBS清洗样品；
vi.10mL的洗脱缓冲液以10s每滴的速度进行洗脱，洗脱液每EP管收集1.5mL，同时滴加20μL中和缓冲液，连续收集7支；
vii.用Nano-300测定每管抗体浓度，将浓度>0.1mg/mL的收集液合并放入透析袋中，加入PBS透析；
viii.将抗体溶液用0.22μm的过滤膜过滤收集到无菌离心管中，保存备用；
ix.取样品5μg用1×PBS稀释至20μL，加入6μL 4×LDS Sample Buffer及4μL DTT，沸水煮10min，12000rpm，离心1min。上样20μL，PAGE胶150V，60min跑胶并使用InstantBlue蛋白电泳染色30min，将胶块置于水中，脱色约30min后观察拍照，结果见图2(其中重点显示的是S蛋白全长的结果，而由于S-a1与S-a2只有110个氨基酸左右，实验室现有PAGE胶比较难显示分子量10KD或更小的条带，故没有提供显示结果)。
实施例4：检测S-a1、S-a2以及S蛋白与康复期病人的血清中IgG抗体结合活性
用ELISA检测S-a1、S-a2以及S蛋白与康复期病人的血清中IgG抗体结合。具体步骤如下：
1.稀释S-a1、S-a2以及S蛋白，浓度为1μg/ml，将稀释好的蛋白100μL/孔加入96-well plate中，密封，4℃包被过夜；
2.弃去上清，每孔200μL PBST(TWEEN-20 0.1％)洗涤3次；
3.每孔200μL 2％BSA封闭液，室温封闭1小时，封闭后的酶标板弃液体，每孔200μL PBST(TWEEN-20 0.1％)洗3次；
4.每孔加入1μg/mL anti-human IgG-HRP抗体100μL，室温孵育2小时；
5.弃上清，使用吸水纸吸干上清，100μL Wash Buffer洗涤5次；
6.每孔加入100μL 1X TMB，避光室温摇动显色10min；
7.每孔加入100μL终止液；
8.使用酶标仪读取450nm的吸光值；
实验结果见图3所示，其中可以发现S-a1、S-a2的结合活性要略高于Spike蛋白。
实施例5：检测装置的制备及使用
使用上述制备得到的SARS-CoV-2的Spike、S-a1与S-a2混合物作为固相物，利用间接法原理检测血清中是否含有抗新型冠状病毒的抗体。具体步骤如下：
1.包被膜的制备：0.01mol/L PBS稀释抗体，鼠抗人IgG抗体浓度为1.0mg/mL喷到NC膜上，记为G线；鼠抗人IgM抗体浓度为1.0mg/mL喷到NC膜上，记为M线；山羊抗鼠IgG抗体浓度为1.0mg/mL，喷到NC膜上，记为C线，完成后备用。
2.金标Spike、S-a1与S-a2混合物的制备：0.1M的K 2CO 3溶液调节胶体金pH值至9，按照12μg/mL加入前述实施例制备得到的Spike、S-a1与S-a2按重量计等比例(即Spike、S-a1与S-a2的最终浓度分别为12μg/mL)混合的蛋白，同时加入12μg/mL小鼠IgG抗体，充分混匀，静置30分钟，13000rpm离心45分钟，弃上清，沉淀用金标洗涤液洗涤两次，最后一次弃上清，将沉淀用十分之一初始胶体金体积的金标抗体保存液溶解。将标记好的胶体金均匀的铺在玻璃纤维膜作为金标结合垫(预先使用金标结合垫封闭液封闭30分钟)，并于室温干燥，每毫升溶液铺3平方厘米，冷冻干燥为金标结合垫，封袋，4℃保存备用。
3.装配：将吸水纸、第(1)步制备的NC膜、第(2)步制备的金标结合垫以及样品垫，依次交叠粘贴在胶板上，切条组装，生成成品检测试剂条，或制备成带为样品孔的检测卡。
其中，金标洗涤液：1％BSA，0.01mol/L PBS，pH9；金标抗体保存液：1％BSA，0.5％PEG20000，2％蔗糖，0.05％NaN3的0.01mol/L PBS，pH9；金标结合垫封闭液：0.01M PBS，1％BSA，1％Tween-20，pH7.5。
下面以血液样品为例介绍本发明检测试剂条的使用方法：
1.检测卡准备:从检测试剂盒中取出检测卡并平衡到室温。撕开箔袋，将测试卡水平放置。
2.采血：1)用酒精消毒片对采血手指进行消毒，约半分钟后待酒精完全挥发后，使用一次性采血器进行采血；2)轻轻挤压一次性毛细管头部，再缓慢释放，在负压作用下指尖血被采集入毛细管中，取样时应避免气泡产生。
3.血样检测：垂直滴加1滴(大约30μL)血样到检测卡的形式的样品孔中，然后加入稀释液3滴(大约120μL)到样品孔中。(如果是一般的试剂条，则将样品垫浸于待测样品中，取出等待后续读取结果)
4.读取结果：加入样本后十分钟之后半小时之内读取结果。视窗C为控制线，G为IgG线，M为IgM线。
结果解释：
1.IgG阳性结果：控制线和IgG线在测试条上可见。检测结果为IgG抗体阳性。这表示病人处于感染的恢复期。
2.IgM阳性结果：控制线C线和IgM线在测试条上可见。IgM抗体检测呈阳性，表明患者处于感染的急性期。
3.IgM/IgG双阳性结果：控制线、IgM线、IgG线在测试条上清晰可见。检测IgM和IgG抗体阳性。表明患者处于感染的急性晚期。
4.阴性结果：控制线是测试条中唯一可见的线。未检测到IgG或IgM抗体。结果并不排除感染。如果症状持续存在，应在3-5天内从患者身上提取新的样本，然后重新进行测试。
5.无效结果：如果控制线没有出现在测试条中，那么无论测试条中是否存在其它可见线，测试结果都是无效的。需更换检测卡重新测试。
实施例6：灵敏度实验
使用S蛋白IgG抗体作为标准品，检测量分别为0.5μg、0.25μg、0.125μg、0.0625μg、0.031μg、0.016μg、0.0078μg、0.0039μg、0.0019μg、0.001μg，检测实施例5制备得到的检测卡的灵敏度，通过实验表明，实施例4制备得到的检测卡可以达到肉眼观察检测0.016μg S蛋白IgG抗体的灵敏度(具体见图2)。
使用S蛋白IgM抗体作为标准品，检测量分别为0.5μg、0.25μg、0.125μg、0.0625μg、0.031μg、0.016μg、0.0078μg、0.0039μg、0.0019μg、0.001μg，检测检测卡的灵敏度，通过实验表明，实施例4制备的检测卡可以达到肉眼观察检测0.031μg S蛋白IgM抗体的灵敏度，具体结果见图4(其中由于在0.031μg时肉眼可见但较弱，由于肉眼观察与照片存在差异，照片中显示不太明显，但在0.0625μg在照片中也比较明显)。
实施例7：特异性实验
采用实施例5制备的检测卡检测SARS-CoV-2 IgG与IgM阳性参考品各十个，实验结果表明，IgG的检测量为0.125μg，IgM检测量为0.25μg，实验结果一致性好，结果见图5。
13例经PCR鉴定阳性的恢复期样本，经实施例4制备的检测卡检测，结果均呈阳性，结果见图6。10例健康人的血样，经实施例4制备的检测卡检测，结果均呈阴性，结果见图7。
取5例阳性参考品、5例经PCR鉴定阳性的恢复期样本经0.01M的DTT，37℃水浴处理30min后进行检测。IgM标准品使用5个实施例4制备的检测卡进行重复测试，每个的检测量为0.5μg，处理样本后，再次进行检测，检测量为0.5μg，检测检测卡IgM特异性。经实施例4制备的检测卡检测，经DTT处理后，IgM检测结果减弱，IgG检测不受影响，如图8所示。
实施例8：稳定性实验
1、试剂稳定性
将实施例5制备的检测卡分为3组，每组10个，放置于37℃ 2天、5天与7天时，再用于分别检测IgG/IgM标准品各 五例，每次加室温储存的检测卡做对照，即为37℃处理0天。结果表明，37℃保存2天、5天与7天，与正常储存的样品相比，不影响检测结果，如图9。
2、样本检测结果稳定性
将3份恢复期样本分为四组，分别置于37℃下保存0小时、1小时、2小时与5小时，每组样品采用实施例4制备的检测卡平行检测两次，与0小时样本相比，37℃放置5小时不影响样本的检测，结果见图10。
3、不同取样来源的样本稳定性
取经PCR鉴定阳性的恢复期全血、血清以及血浆样本各3例采用实施例4制备的检测卡进行测试，同时设置平行阳性参考品和阴性对照各3例，三种形式的样本检测结果类似，结果如图11所示。
在此说明书中，本发明已参照其特定的实施例作了描述。但是，很显然仍可以作出各种修改和变换而不背离本发明的精神和范围。因此，说明书应被认为是说明性的而非限制性的。

Claims (16)

  1. 一种检测新型冠状病毒试剂盒，其特征在于，其包括SARS-CoV-2冠状病毒的S蛋白，S-a1蛋白片段和S-a2蛋白片段，进一步包括小鼠IgG抗体，其中，S-a1蛋白片段氨基酸序列如序列表SEQ ID NO:3所示，S-a2蛋白片段氨基酸序列如序列表SEQ ID NO:5所示。
  2. 如权利要求1所述的试剂盒，其特征在于，其还包括鼠抗人IgG抗体，和/或鼠抗人IgM抗体，以及山羊抗鼠IgG抗体，进一步还包括作为标准品的S蛋白IgG抗体和/或作为S蛋白IgM抗体，更进一步包括样品处理液。
  3. 如权利要求1所述的试剂盒，其特征在于，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
  4. 如权利要求3所述的试剂盒，其特征在于，所述试剂盒是用于间接法检测待测样本血清中是否含有抗新型冠状病毒的抗体，优选包括所述试剂盒还包括用于间接法检测的相关试剂。
  5. 如权利要求1至4所述的试剂盒，其特征在于，所述试剂盒是用于胶体金层析法进行检测，所述试剂盒包括检测装置和样品处理液，优选地所述样品稀释液为含有0.2％(体积)Tween 20和0.6％(体积)Casein的0.1MPBS溶液，所述检测装置试剂条或检测卡。
  6. 如权利要求5所述的试剂盒，其特征在于，所述试剂条由下述方法制备得到：
    (1)将鼠抗人IgG抗体和/或鼠抗人IgM抗体，以及山羊抗鼠IgG抗体分别喷到NC膜上；
    (2)将胶体金加入S蛋白、S-a1蛋白片段与S-a2蛋白片段的混合蛋白中，加入小鼠IgG抗体，用标记洗涤液洗涤沉淀，再用金标抗体保存液溶解，然后均匀的铺在玻璃纤维膜，冷冻干燥为金标结合垫；
    (3)将吸水纸、第(1)步获得的NC膜、金标结合垫以及样品垫交叠粘贴在胶板上，切条得到试剂条。
  7. 一种检测新型冠状病毒的装置，其特征在于，所述装置包括依次相连的样品垫、金标结合垫、反应垫和吸水垫；其中所述金标结合垫上包被有胶体金标记的S蛋白、S-a1蛋白片段与S-a2蛋白片段的混合蛋白，以及小鼠IgG抗体；所述反应垫上沿待检样品流动方向依次设有检测线和质控线，进一步地所述反应垫上沿待检样品流动方向依次设有鼠抗人IgG抗体或鼠抗人IgM抗体、山羊抗鼠IgG抗体分别记为G线或M线、C线，优选地所述反应垫上沿待检样品流动方向依次设有鼠抗人IgG抗体、或鼠抗人IgM抗体、山羊抗鼠IgG抗体分别记为G线、M线、C线；所述装置为检测试剂条，更优选为带样品孔的检测卡。
  8. 如权利要求7所述的装置，其特征在于，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
  9. 制备如权利要求7或8所述的检测新型冠状病毒的装置的制备方法，其包括如下步骤：
    (1)将鼠抗人IgG抗体、和/或鼠抗人IgM抗体、以及山羊抗鼠IgG抗体分别喷到NC膜上；
    (2)将胶体金加入S蛋白、S-a1蛋白片段与S-a2蛋白片段的混合蛋白中，加入小鼠IgG抗体，用标记洗涤液洗涤沉淀，再用金标抗体保存液溶解，然后均匀的铺在纤维膜，冷冻干燥为金标结合垫；其中纤维膜预先用金标结合垫封闭液预处理；
    (3)将吸水纸、第(1)步制得的NC膜、第(2)步制得的金标结合垫以及样品垫交叠粘贴在底板上。
  10. 如权利要求9所述的制备方法，其特征在于，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
  11. 如权利要求10所述的制备方法，其特征在于，所述第(1)步是将鼠抗人IgG抗体、鼠抗人IgM抗体、山羊抗鼠IgG抗体分别喷到NC膜上，分别记为G线、M线、C线；所述第(2)步中混合蛋白是S蛋白、S-a1蛋白片段与S-a2蛋白片段等比例混合而成。
  12. 如权利要求11所述的制备方法，其特征在于，其具体步骤如下：
    (1)用0.01mol/L PBS稀释抗体，将鼠抗人IgG抗体浓度为1.0mg/mL喷到NC膜上，记为G线，或者将鼠抗人IgM抗 体浓度为1.0mg/mL喷到NC膜上，记为M线；或者将鼠抗人IgG抗体和鼠抗人IgM抗体分别喷到NC膜上，分别记为G线和M线；将山羊抗鼠IgG抗体浓度为1.0mg/mL，喷到NC膜上，记为C线，完成后备用；
    (2)用0.1M的K 2CO 3溶液调节胶体金pH值至9，按照12μg/mL加入前述S蛋白、S-a1蛋白片段与S-a2蛋白片段等比例混合的蛋白，同时加入12μg/mL小鼠IgG抗体，充分混匀，静置后离心弃上清，沉淀用金标洗涤液洗涤，弃上清，将沉淀用十分之一初始胶体金体积的金标抗体保存液溶解；将标记好的胶体金均匀的铺在预先用金标结合垫封闭液预处理的玻璃纤维膜上，冷冻干燥为金标结合垫；
    (3)将吸水纸、第(1)步制备得到的NC膜、第(2)步制备得到的金标结合垫，以及样品垫，依次交叠粘贴在聚氯乙烯底板或胶板上即成所述装置，进一步地进行切条，组装得到试剂条或进一步制备成带有样品孔的检测卡；
    其中，优选地，所述样品垫是经过样品垫处理液浸渍处理的玻璃纤维膜或无纺布或滤纸，进一步优选地所述样品垫处理液包括如下组分：Tween 20、Triton x-405、Casein、BSA、PEG-20000和NaCl。
  13. 如权利要求12所述的制备方法，其特征在于，其中，所述金标洗涤液组分为：1％BSA，0.01mol/L PBS，pH9；所述金标抗体保存液：1％BSA，0.5％PEG20000，2％蔗糖，0.05％NaN 3的0.01mol/L PBS，pH9；所述金标结合垫封闭液：0.01M PBS，1％BSA，1％Tween-20，pH7.5。
  14. 包括SARS-CoV-2冠状病毒的S蛋白，S-a1蛋白片段和S-a2蛋白片段混合蛋白在制备检测新型冠状病毒的试剂中的应用，其中S-a1蛋白片段氨基酸序列如序列表SEQ ID NO:3所示，S-a2蛋白片段氨基酸序列如序列表SEQ ID NO:5所示。
  15. 如权利要求14所述的应用，其特征在于，其是通过胶体金层析法检测样品中是否存在含有抗新型冠状病毒的抗体，优选地，所述新型冠状病毒是β冠状病毒，尤其是SARS-CoV-2冠状病毒，SARS病毒，MERS病毒，更优选是SARS-CoV-2冠状病毒。
  16. 如权利要求15所述的应用，其检测是的样品选自待检对象是恢复期人的样品及健康人的样品，所述样品优选为鼻咽拭子、痰液、肺泡灌洗液、血液、尿液中的至少一种。
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