A SERS-based detection kit and detection method for the detection of novel coronavirus

Abstract

The present invention relates to a SERS-based detection kit and a detection method for the detection of novel coronavirus. The SERS-based detection kit for the detection of novel coronavirus includes: a virus lysis solution, magnetic beads modified with a specific polypeptide, a washing buffer, an elution buffer, and a SERS substrate; the amino acid sequence of the specific polypeptide used for modifying the magnetic beads is LVMGLNVWLRYSKAAALVMGLNVWLRYSKAAAHHHHHH; the elution buffer is an NaCl solution or a urea solution.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a novel coronavirus detection kit and detection method based on surface-enhanced Raman spectroscopy with specific polypeptide magnetic bead elution. Background Art

[0002] Among common diagnostic methods, enzyme-linked immunosorbent assay, real-time fluorescence quantitative reverse transcription polymerase chain reaction, and loop-mediated isothermal amplification are very important for detecting human coronaviruses. However, these methods also have limitations. For example, the real-time fluorescence quantitative reverse transcription polymerase chain reaction method requires skilled operators, good laboratory conditions, and takes more than 2 hours to complete the detection; enzyme-linked immunosorbent assay is a method based on antigen-antibody interaction. Although it has high sensitivity and simple operation, its reagent preparation process requires specific and highly affinity antibodies, and even expensive recombinant antibodies; the antigen detection (lateral flow immunoassay) currently used for large-scale population screening has a detection time of 15-30 minutes, but the detection sensitivity is low, and the detection limit is about Ct26. Therefore, there is an urgent need for a rapid, highly sensitive, and easy-to-operate detection method to inhibit the large-scale spread of the epidemic. Summary of the Invention

[0003] In view of the above problems, the object of the present invention is to provide a SERS-based detection kit and detection method for rapid, highly sensitive, and easy-to-operate detection of novel coronavirus. Among them, specific magnetic beads with high magnetic saturation intensity and small coercivity can complete the capture, enrichment purification, and rapid elution of analytes within 7 minutes. Compared with the currently commonly used nucleic acid / protein extraction process based on the magnetic bead method (more than 15 minutes), the time and efficiency of the analyte purification process in the present invention have been greatly improved. Combining the rapid magnetic separation and enriched protein purification process with the highly sensitive SERS detection technology can achieve rapid and highly sensitive detection of novel coronavirus (within 15 minutes).

[0004] Specifically, in the first aspect, the present invention provides a SERS-based detection kit for novel coronavirus detection, and the detection kit includes: virus lysate, magnetic beads modified with specific polypeptide, washing buffer, elution buffer, and SERS substrate;

[0005] The amino acid sequence of the specific polypeptide for modifying magnetic beads is LVMGLNVWLRYSKAAALVMGLNVWL-RYSKAAAHHHHHH;

[0006] The elution buffer is NaCl solution or urea solution.

[0007] Preferably, the formulation of the virus lysate comprises: 0.05 - 0.2 M Tris, 1 - 5 wt% polysorbate-20 (tween-20), 1 - 5 wt% polyvinylpyrrolidone (PVP), 0.001 - 0.01 M ethylenediaminetetraacetic acid (EDTA);

[0008] Alternatively, the formulation of the virus lysate comprises: 0.05 - 0.2 M Tris, 1 - 5 wt% tween-20, 1 - 5 wt% PVP, 0.001 - 0.01 M EDTA, 15 - 50 μg / mL glucose, 1 - 5 wt% bovine serum albumin, 10 - 50 μg / mL penicillin.

[0009] Preferably, the magnetic beads are amino-functionalized Fe3O4 magnetic particles; the amino-functionalized Fe3O4 magnetic particles are spheroid in shape and have a particle size of 20 - 50 nm.

[0010] Preferably, the method for preparing amino-functionalized Fe3O4 magnetic beads comprises: adding sodium acetate and polyetherimide (PEI) to an FeCl3·6H2O solution, and performing a hydrothermal reaction to obtain the amino-functionalized Fe3O4 magnetic beads;

[0011] wherein, the mass ratio of FeCl3·6H2O:sodium acetate:polyetherimide = (0.5 - 2):(2.0 - 8):(2 - 4).

[0012] Preferably, the 6 histidines in the polypeptide amino acid sequence are labeled at the C-terminus of the polypeptide amino acid sequence.

[0013] Preferably, the modification of the specific polypeptide on the surface of the magnetic beads is carried out by electrostatic binding or amide bond covalent binding.

[0014] Preferably, the step of electrostatically binding to link the specific polypeptide is: adding the specific polypeptide to the magnetic bead solution, and incubating the mixture at 4 - 25 °C for 2 - 5 hours.

[0015] Preferably, the step of covalently binding via an amide bond to link the specific polypeptide is: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the magnetic bead solution, adding the specific polypeptide after mixing, and incubating at 4 - 25 °C for 0.5 - 5 hours, preferably 2 - 5 hours.

[0016] Preferably, the washing buffer comprises phosphate buffered saline (DPBS) or / and deionized water.

[0017] Preferably, the solvent of the NaCl solution is deionized water, and the concentration of NaCl is 1 - 4 mol / L;

[0018] The urea solution has urea as the main component, an aqueous solution of Tris and NaH₂PO₄ as the buffer system, and the pH value is adjusted to 4 - 5.5 with HCl; the concentration of urea in the urea solution is 6 - 10 mol / L; preferably, the concentration of NaH₂PO₄ in the urea solution is 50 - 100 mM, and the concentration of Tris is 8 - 12 mM.

[0019] Preferably, the SERS substrate is a gold nanoparticle array, a gold nanocone array, or an SERS solid-state chip.

[0020] In a second aspect, the present invention provides a non-diagnostic and non-therapeutic detection method for the above SERS-based detection kit for novel coronavirus detection, including:

[0021] Put the collected nasal swab into the virus lysis solution;

[0022] Then, add a magnetic bead solution modified with a specific polypeptide, and mix well to make the magnetic beads bind to the target novel coronavirus S protein;

[0023] Under the action of an external magnetic field, enrich the magnetic beads bound with the S protein, and wash them successively with DPBS and deionized water;

[0024] Next, add an elution buffer to the enriched magnetic beads to dissociate the S protein from the polypeptide, enrich the magnetic beads again, and collect the supernatant;

[0025] Drop the supernatant onto the SERS substrate and dry it, collect signals using a Raman spectrometer, analyze the signals, and make a result determination.

[0026] Beneficial effects

[0027] (1) The present invention designs a surface-enhanced Raman spectroscopy-based kit and a complete detection process for novel coronavirus detection based on magnetic bead elution;

[0028] (2) The components of the lysis solution, washing buffer, and elution buffer involved in the detection kit are simple, with few Raman peaks, and do not affect the Raman peaks of the target protein; in addition, components such as bovine serum albumin, glucose, and penicillin are added to the lysis solution to protect the protein, which can not only protect the structural integrity of the protein to the greatest extent but also facilitate the transportation and preservation of the protein in scenarios where timely detection is not required;

[0029] (3) The magnetic beads modified with the polypeptide have strong magnetic saturation intensity, which is beneficial for rapid enrichment; small coercivity, representing excellent dispersibility of the magnetic beads, is beneficial for rapid dispersion and washing; in addition, the high specificity and strong binding force between the polypeptide and the analyte S protein can achieve rapid recognition and capture of the analyte;

[0030] (4) The full-process detection method can not only complete the highly sensitive detection of novel coronavirus within 15 minutes, but also achieve high-throughput detection with a sample volume of 120 samples / h. Description of the Drawings

[0031] Figure 1 It is a comparison chart of the Raman signals of three virus lysates. Among them, A is the Raman signal chart of the lysate with Triton as the main component, B is the Raman signal chart of the lysate with sodium dodecyl sulfate (SDS) as the main component, and C is the Raman signal chart of the lysate with polysorbate-20 as the main component;

[0032] Figure 2 It is a flow chart of the novel coronavirus detection by the SERS technology based on magnetic bead elution provided by the present invention;

[0033] Figure 3 It is a micrograph of the amino Fe3O4 magnetic beads provided by the present invention;

[0034] Figure 4 It is a characterization chart of the Raman signal of the S protein with an aqueous NaCl solution as the eluent in Example 2;

[0035] Figure 5 It is a characterization chart of the Raman signal of the S protein with a urea solution with a pH of 4.5 as the eluent in Example 3;

[0036] Figure 6 It is a characterization chart of the Raman signal of the S protein with a 1 wt% SDS solution as the eluent in Comparative Example 2;

[0037] Figure 7 It is a comparison chart of the Raman signals of the S protein with urea solution, NaCl solution, and 1 wt% SDS solution as the eluent. Detailed Embodiments

[0038] The present invention will be further described below through embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and do not limit the present invention.

[0039] Raman spectroscopy can provide "fingerprint spectrum" information for biological samples at the molecular level. Through Raman spectroscopy, the signal recognition of the specific S protein and RNA of the novel coronavirus can be achieved, thereby realizing the detection of the novel coronavirus. According to the above principle, in order to quickly and accurately detect the novel coronavirus, the present invention provides a SERS-based detection kit and detection method for novel coronavirus detection based on the surface-enhanced Raman spectroscopy (SERS) technology of specific magnetic bead elution.

[0040] The detection kit provided by the present invention includes: a virus lysate, magnetic beads modified with specific polypeptides, a washing buffer, an elution buffer, and a SERS substrate. The S protein after the lysis of the novel coronavirus can be specifically captured by the magnetic beads modified with polypeptides. After washing, enrichment, and elution, the purified and concentrated S protein can be subjected to Raman detection on the surface of the SERS-active substrate.

[0041] The formulation of the virus lysate 1 can be: 0.05 - 0.2 M Tris, 1 - 5 wt% polysorbate-20 (tween-20), 1 - 5 wt% polyvinylpyrrolidone PVP, 0.001 - 0.01 M ethylenediaminetetraacetic acid EDTA. In some embodiments, the formulation of the virus lysate 2 can be: 0.05 - 0.2 M Tris, 1 - 5 wt% tween-20, 1 - 5 wt% PVP, 0.001 - 0.01 M EDTA, 15 - 50 μg / mL glucose, 1 - 5 wt% bovine serum albumin, 10 - 50 μg / mL penicillin. Among them, by using the virus lysate with the above composition, not only can the structure of the S protein be stabilized and not be damaged, but also the effect of virus lysis can be ensured. Glucose, bovine serum albumin, and penicillin can maintain the originality of the sample to the greatest extent, which is beneficial to the transportation and preservation of the test sample. In addition, through the magnetic enrichment and washing steps described later in the present invention, most of the impurities in the lysate can be washed away.

[0042] The selection principle of the virus lysate is that it has no influence on the amino acid sequence structure of the S protein on the surface of the target novel coronavirus, has few Raman peaks, and does not interfere with the Raman peaks of the S protein. Figure 1 It is a comparative Raman signal diagram of three virus lysates. Among them, A is the Raman signal diagram of the lysate with Triton as the main component, B is the Raman signal diagram of the lysate with sodium dodecyl sulfate SDS as the main component, and C is the Raman signal diagram of the lysate with polysorbate-20 as the main component. It can be seen from the figure that the lysate with Tween as the main component has fewer Raman peaks. See Figure 1 C; compared with Figure 1 the lysate corresponding to B, Figure 1 the lysates in A and 1C have fewer Raman peaks. Figure 1 The surfactant in the lysate in A is prone to foaming during the oscillation process, which is not conducive to subsequent washing operations and detection.

[0043] Among them, the amino acid sequence of the specific polypeptide for modifying magnetic beads provided by the present invention can be LVMGLNVWLRYSKAAALVMGLNVWLRYSKAAAHHHHHH, and six histidine tags are at the C-terminus of the polypeptide amino acid sequence. The magnetic beads are amino-functionalized Fe3O4 magnetic particles; the amino-functionalized Fe3O4 magnetic particles are spherical in shape, and the particle size is 20-50 nm. In some embodiments, the concentration ratio of the specific polypeptide to the magnetic beads can be controlled to be 10-15 μg / mL:50 μg / mL.

[0044] Among them, the preparation method of the amino-functionalized Fe3O4 magnetic beads can be as follows: Add 0.5-2 g of FeCl3·6H2O to 50 mL of ethylene glycol, and dissolve it by ultrasonic treatment to obtain solution 1; then, add 2.0-8 g of sodium acetate and 2-4 g of polyetherimide PEI to the solution 1, and stir at 40-70 °C for 20 minutes to obtain solution 2; transfer the solution 2 to a 100 mL reaction kettle, and hydrothermally react at 220-250 °C for 2-3 h to obtain a black product. After washing the black product with absolute ethanol and deionized water respectively, dry it in a vacuum oven at 45-60 °C for 8-12 hours to obtain the amino-functionalized Fe3O4 magnetic beads.

[0045] The polypeptide of the specific sequence can specifically bind to the S protein on the surface of the novel coronavirus to achieve specific capture of the target detection substance. At the same time, the histidine tagged at its C-terminus can electrostatically bind to the magnetic beads, thereby connecting the polypeptide to the surface of the magnetic beads to obtain specific magnetic beads. In addition, the surface of the magnetic beads can also be modified with amino groups. After activation with 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS, the connection between the amino groups on the surface of the magnetic beads and the carboxyl groups on the polypeptide can be achieved. Through the amide bond between the magnetic beads and histidine, the covalent binding between the polypeptide and the amino magnetic beads is realized.

[0046] Specifically, the modification of the polypeptide on the surface of the magnetic beads in the present invention can adopt the following two methods: electrostatic binding and covalent binding through amide bond.

[0047] The steps of electrostatically binding and connecting the polypeptide can be as follows: Add 20-40 μg of the polypeptide to 0.5-1.5 mL of a magnetic bead solution with a concentration of 0.1 mg / mL, mix by oscillation for 5 minutes, and then incubate at 4-25 °C for 2-5 hours; after the incubation, enrich the magnetic beads under the action of an external magnetic field, and wash away the excess unadsorbed polypeptide with 200-500 μL of phosphate buffer solution DPBS; after washing, disperse the magnetic beads in 2-3 mL of DPBS for use.

[0048] The step of covalently binding the amide bond to the linking polypeptide may be as follows: Add 20 - 30 μL of EDC with a concentration of 10 - 20 μg / mL and 20 - 30 μL of NHS with a concentration of 10 - 20 μg / mL to 0.5 - 1.5 mL of a magnetic bead solution with a concentration of 0.1 mg / mL, and mix evenly; then, add 20 - 40 μg of the polypeptide, and incubate at 4 - 25 °C for 0.5 - 5 hours, preferably 2 - 5 hours; after the incubation, enrich the magnetic beads under the action of an external magnetic field, and wash away the excess unadsorbed polypeptide with 200 - 500 μL of DPBS; after washing, disperse the magnetic beads in 2 - 3 mL of DPBS for use.

[0049] In some embodiments, the washing buffer may include DPBS or / and deionized water. Among them, DPBS can be selected for the first washing process to wash away the impurities in the lysis solution and other components after virus lysis. The DPBS does not contain CaCl2 and MgCl2, has a relatively simple composition, and can maintain the acid-base environment required for the S protein, which is beneficial to the stability of the protein properties; deionized water can be selected for the second washing to quickly wash away the residual impurities and DPBS, so that the S protein is in a more pure detection environment. On the basis of washing away the impurities, the Raman signal introduced by the washing solution is reduced as much as possible.

[0050] The elution step is an important part of the whole process detection. The eluent needs to have excellent elution effect, few Raman peaks, and does not affect the Raman signal of the target S protein. In some embodiments, the elution buffer may be an aqueous solution of deionized water with NaCl or a urea solution with a pH of 4 - 5.5. Among them, the concentration of the NaCl solution may be 1 - 4 mol / L. The concentration of the urea solution may be 6 - 10 mol / L. The formula of the urea solution may be: 50 - 100 mM of NaH2PO4, 8 - 12 mM of Tris, 6 - 10 M of urea, and the pH value of the solution can be adjusted to 4 - 5.5 with HCl.

[0051] For the eluent NaCl solution, the elution mechanism is as follows: The high-concentration salt solution will change the tertiary structure conformation of the S protein, destroy the affinity between ACE2 and the S protein, and the specific binding between the S protein and the polypeptide, so that the S protein dissociates from ACE2, achieving the purpose of purification in a complex lysis environment. If the concentration is too low, the effect of destroying the protein conformation cannot be achieved, and the elution efficiency is reduced; if the concentration is too high, the protein denaturation is serious, which will not only cause changes in the Raman spectrum, but also produce more crystals during the drying process, affecting Raman detection.

[0052] The main component in the urea solution is the urea component. The elution mechanism is similar to that of the NaCl solution. A high-concentration urea solution can also change the structure of the S protein, disrupt its specific binding with the polypeptide, and achieve the purpose of purification. Similarly, too low a concentration of urea will affect the elution efficiency, while too high a concentration of urea will cause strong crystallization, affecting Raman detection. In addition, urea has a characteristic peak in the range of 550 - 1650 cm -1 region. As the concentration of urea increases, the peak intensity also increases, while the relative intensity of the protein decreases, which will interfere with the Raman peak of the S protein. Therefore, the pH of the urea solution needs to be controlled within the range of 4 - 5.5 to maintain the stability of the buffer system. Too low a pH will cause serious damage to the structure of the S protein, while too high a pH will disrupt the ionization degree of NaH2PO4, destroy the buffer system, and affect the elution effect.

[0053] In some embodiments, the SERS substrate can be selected from a gold nanoparticle array, a gold nanocone array, or a SERS solid-state chip.

[0054] In the detection kit provided by the present invention, the magnetic beads modified with polypeptide have the characteristics of specifically capturing and enriching the target SARS-CoV-2 S protein. The introduction thereof can greatly simplify the test process; the interference of the lysate and other components can be minimized by simply washing with the washing buffer; the elution buffer is used to purify and concentrate the S protein and eliminate the influence of the magnetic beads on the performance of the SERS substrate; the SERS substrate can enhance the Raman signal of biomacromolecules by 10 orders of magnitude. The selected virus lysate, washing buffer, and elution buffer all have the characteristics of few Raman peaks and no influence on the amino acid sequence structure of the target S protein without affecting their corresponding lysis, washing, and elution functions.

[0055] Combined with the above kit, the present invention designs a whole-process detection of novel coronavirus based on the SERS technology of magnetic bead elution, which can achieve rapid and high-throughput detection of novel coronavirus. The following combines the attached Figure 2 , and exemplarily illustrates the detection method for non-diagnostic and non-therapeutic purposes of the SERS-based detection kit provided by the present invention. The detection method can be carried out by the following steps.

[0056] (1) Sampling. Collect the nasal swab of the person to be tested for nucleic acid, put it into 200 - 1000 μL of virus lysate, and perform virus lysis to obtain Solution 1.

[0057] (2) Binding. Add 50 - 250 μL of a magnetic bead solution modified with specific polypeptide with a concentration of 0.5 - 1.5 mg / mL to Solution 1 (lysate containing nasal swab), and mix by shaking or ultrasonic treatment for 90 s - 3 min to allow the magnetic beads to fully bind to the target S protein. The magnetic beads specifically bind to the target S protein through the polypeptide and capture the target S protein.

[0058] (3) Washing. Enrich the magnetic beads conjugated with the S protein under the action of an external magnetic field, then remove the supernatant, add 200 - 400 μL of DPBS, mix well by shaking for 20 - 60 s and then enrich, and remove the supernatant under the action of the external magnetic field; then, add 200 - 400 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0059] (4) Elution. Add 5 - 20 μL of elution buffer (such as an NaCl eluent with a concentration of 1 - 4 mol / L) to the magnetic beads enriched in step (3), mix well by shaking for 20 s, dissociate the S protein from the polypeptide, enrich the magnetic beads again and then collect the supernatant.

[0060] (5) Drying. Drop the supernatant collected in step (4) onto the SERS substrate, and place it in an oven at 45 °C for drying for 5 - 10 minutes.

[0061] (6) Raman detection. Use a Raman spectrometer to collect Raman signals from the dried SERS substrate, and the excitation wavelength for Raman detection is 785 nm.

[0062] (7) Result determination. Analyze the signals and determine the results of the collected Raman spectra by the method of AI intelligent recognition of the positive and negative of the sample.

[0063] The SERS-based detection kit provided by the present invention can achieve high-sensitivity detection of a single novel coronavirus sample within 15 minutes. At the same time, this kit can be further used in an automated device integrating sample processing and Raman detection such as magnetic bead extraction, drying, and Raman detection, and achieve high-throughput detection of novel coronavirus (120 samples / h) through prototype design.

[0064] The following further gives examples to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention, and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art's non-essential improvements and adjustments based on the above content of the present invention all fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range according to the description herein, rather than being limited to the specific values in the following examples. It should be noted that, in order to protect the safety of experimental operators, during the implementation process of the present invention, a mixture of a pseudovirus with a determined concentration and a negative nasal swab solution is used to simulate a positive nasal swab solution for detection.

[0065] In the following examples and comparative examples, unless otherwise specified, the components of Virus Lysis Solution 1 include: 0.05 - 0.2 M Tris, 1 - 5 wt% polysorbate - 20 (tween - 20), 1 - 5 wt% polyvinylpyrrolidone (PVP), 0.001 - 0.01 M ethylenediaminetetraacetic acid (EDTA). The components of Virus Lysis Solution 2 include: 0.05 - 0.2 M Tris, 1 - 5 wt% tween - 20, 1 - 5 wt% PVP, 0.001 - 0.01 M EDTA, 15 - 50 μg / mL glucose, 1 - 5 wt% bovine serum albumin, 10 - 50 μg / mL penicillin.

[0066] In the following examples and comparative examples, unless otherwise specified, the preparation process of amino - Fe3O4 magnetic beads is as follows: Add 0.5 - 2 g of FeCl3·6H2O to 50 mL of ethylene glycol, and dissolve it by ultrasonic to obtain Solution 1; then, add 2.0 - 8 g of sodium acetate and 2 - 4 g of polyetherimide (PEI) to Solution 1, stir at 40 - 70 °C for 20 minutes to obtain Solution 2; transfer Solution 2 to a 100 - mL reaction kettle, perform hydrothermal treatment at 220 - 250 °C for 2 - 3 h; after washing the obtained black product with absolute ethanol and deionized water respectively, dry it in a vacuum oven at 45 - 60 °C for 8 - 12 hours to obtain the amino - Fe3O4 magnetic beads. The microscopic morphology of the amino magnetic beads is shown in Figure 3 .

[0067] Example 1

[0068] The detection process of novel coronavirus using Virus Lysis Solution 1 as the lysis solution and NaCl aqueous solution as the eluent:

[0069] Step 1: First, put the nasal swab of the person to be tested for nucleic acid into 250 μL of Virus Lysis Solution 1, and add 200 μL of virus with a virus load of 1000 copies / mL. The virus is lysed therein to obtain Solution 1.

[0070] Step 2: Take 50 μL of magnetic bead solution with a concentration of 1 mg / mL and put it into the above - mentioned Solution 1, oscillate and mix evenly for 90 s to enable the magnetic beads modified with polypeptide to specifically capture the S protein.

[0071] Step 3: After sufficient binding, the magnetic beads are enriched under the action of an external magnetic field. After removing the supernatant, add 200 μL of DPBS, oscillate and mix evenly for 20 s and then enrich. Remove the supernatant under the action of the external magnetic field; then, add 200 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0072] Step 4: Add 10 μL of NaCl eluent with a concentration of 2 mol / L to the enriched magnetic beads, oscillate and mix evenly for 20 S, and then enrich the magnetic beads again and collect the supernatant.

[0073] Step 5: Drop the supernatant onto the SERS substrate, place it in an oven at 45 °C and dry for 5 minutes, then collect the Raman signal. The excitation wavelength for Raman detection is 785 nm.

[0074] Step 6: Analyze the collected Raman spectrum to accurately determine the result.

[0075] Example 2

[0076] Novel coronavirus detection process using virus lysate 2 as the lysate and aqueous NaCl solution as the eluent:

[0077] Step 1: First, place the nasal swab of the person to be tested for nucleic acid into 250 μL of virus lysate 2, add 200 μL of virus with a virus load of 1000 copies / mL, and lyse it therein to obtain Solution 1. After placing Solution 1 at 4 °C for 48 hours, then conduct the detection.

[0078] Step 2: Take 50 μL of the magnetic bead solution with a concentration of 1 mg / mL and put it into the above Solution 1, oscillate and mix well for 90 S to enable the magnetic beads modified with polypeptides to specifically capture the S protein.

[0079] Step 3: Enrich the fully combined magnetic beads under the action of an external magnetic field. After removing the supernatant, add 200 μL of DPBS, oscillate and mix well for 20 S and then enrich. Remove the supernatant under the action of the external magnetic field; then, add 200 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0080] Step 4: Add 10 μL of the NaCl eluent with a concentration of 2 mol / L to the enriched magnetic beads, oscillate and mix well for 20 S, then enrich the magnetic beads again and collect the supernatant.

[0081] Step 5: Drop the supernatant onto the SERS substrate, place it in an oven at 45 °C and dry for 5 minutes, then collect the Raman signal. The excitation wavelength for Raman detection is 785 nm.

[0082] Step 6: Analyze the collected Raman spectrum to accurately determine the result.

[0083] Figure 4 It is a Raman signal characterization diagram of the S protein with the aqueous NaCl solution as the eluent in Example 2. It can be seen from the figure that the obtained Raman signal belongs to the S protein and the signal intensity is relatively high. It can be seen from this example that using virus lysate 2 to lyse the virus and preserve the test sample, after the sample is placed at 4 °C for 48 hours, it still does not affect the detection performance.

[0084] Example 3

[0085] Novel coronavirus detection process using virus lysate 2 as the lysate and urea solution with pH 4.5 as the eluent:

[0086] Step 1: First, place the nasal swab of the person to be tested for nucleic acid into 250 μL of virus lysate 2, and add 200 μL of virus with a virus load of 1000 copies / mL. The virus is lysed therein to obtain Solution 1.

[0087] Step 2: Take 50 μL of magnetic bead solution with a concentration of 1 mg / mL and put it into the above Solution 1, shake and mix well for 90S to enable the magnetic beads modified with polypeptide to specifically capture the S protein.

[0088] Step 3: After sufficient binding, the magnetic beads are enriched under the action of an external magnetic field. After removing the supernatant, add 200 μL of DPBS, shake and mix well for 20S and then enrich. Remove the supernatant under the action of the external magnetic field; then, add 200 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0089] Step 4: Add 10 μL of urea solution with pH 4.5 (100 mM NaH2PO4, 10 mM Tris, 8 M urea) to the enriched magnetic beads, shake and mix well for 20S, and then enrich the magnetic beads again and collect the supernatant.

[0090] Step 5: Drop the supernatant onto the SERS substrate, place it in an oven at 45 °C and dry for 5 minutes, and then collect the Raman signal. The excitation wavelength for Raman detection is 785 nm.

[0091] Step 6: Analyze the collected Raman spectrum to accurately determine the result.

[0092] Figure 5 It is the Raman signal characterization diagram of the S protein with urea solution with pH 4.5 as the eluent in Example 3. It can be seen from the figure that the obtained Raman signal can be attributed to the S protein and the signal intensity is relatively high. Through this example, it can be seen that using urea solution as the eluent can also achieve the dissociation between the S protein and the polypeptide, and realize the detection of novel coronavirus with a virus load of 1000 copies / mL.

[0093] Example 4

[0094] In this Example 4, the novel coronavirus detection process using virus lysate 2 as the lysate and NaCl aqueous solution as the eluent refers to Example 2, and the difference is only in: Step 4: Add 10 μL of NaCl eluent with a concentration of 1 mol / L to the enriched magnetic beads, shake and mix well for 20S, and then enrich the magnetic beads again and collect the supernatant. By using the above concentration of eluent, the dissociation between the S protein and the polypeptide can also be achieved, and the detection of novel coronavirus with a virus load of 1000 copies / mL can be realized.

[0095] Example 5

[0096] In this Example 5, taking virus lysate 2 as the lysate and NaCl aqueous solution as the eluent, the detection process of novel coronavirus is shown in Example 2, with the difference only in: Step 4: Add 10 μL of NaCl eluent with a concentration of 4 mol / L to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and then collect the supernatant. By using the eluent with the above concentration, the dissociation between the S protein and the polypeptide can also be achieved, and the detection of novel coronavirus with a virus load of 1000 copies / mL can be realized.

[0097] Example 6

[0098] In this Example 6, taking virus lysate 2 as the lysate and urea solution as the eluent, the detection process of novel coronavirus is shown in Example 3, with the difference only in: Step 4: Add 10 μL of urea solution with a pH of 5.5 (100 mM NaH2PO4, 10 mM Tris, 8 M urea, adjust the pH value to 5.5 with HCl) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and then collect the supernatant. By using the eluent with the above concentration, the dissociation between the S protein and the polypeptide can also be achieved, and the detection of novel coronavirus with a virus load of 1000 copies / mL can be realized.

[0099] Example 7

[0100] In this Example 7, taking virus lysate 2 as the lysate and urea solution as the eluent, the detection process of novel coronavirus is shown in Example 3, with the difference only in: Step 4: Add 10 μL of urea solution with a pH of 4 (100 mM NaH2PO4, 10 mM Tris, 8 M urea, adjust the pH value to 4 with HCl) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and then collect the supernatant. By using the eluent with the above concentration, the dissociation between the S protein and the polypeptide can also be achieved, and the detection of novel coronavirus with a virus load of 1000 copies / mL can be realized.

[0101] Example 8

[0102] In Example 8, the detection process of novel coronavirus using virus lysate 2 as the lysate and urea solution as the eluent is as described in Example 3, with the only difference being: Step 4: Add 10 μL of urea solution with a pH of 4.5 (100 mM NaH2PO4, 10 mM Tris, 6 M urea) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and collect the supernatant. By using the eluent with the above concentration, the dissociation between the S protein and the polypeptide can also be achieved, realizing the detection of novel coronavirus with a virus load of 1000 copies / mL.

[0103] Example 9

[0104] In Example 9, the detection process of novel coronavirus using virus lysate 2 as the lysate and urea solution as the eluent is as described in Example 3, with the only difference being: Step 4: Add 10 μL of urea solution with a pH of 4.5 (100 mM NaH2PO4, 10 mM Tris, 10 M urea) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and collect the supernatant. By using the eluent with the above concentration, the dissociation between the S protein and the polypeptide can also be achieved, realizing the detection of novel coronavirus with a virus load of 1000 copies / mL.

[0105] Comparative Example 1

[0106] The detection process of novel coronavirus using virus lysate 1 as the lysate and aqueous NaCl solution as the eluent:

[0107] Step 1: First, place the nasal swab of the person to be tested for nucleic acid into 250 μL of virus lysate 1, and add 200 μL of virus with a virus load of 1000 copies / mL, which is lysed therein to obtain Solution 1. After placing Solution 1 at 4 °C for 48 hours, then perform the detection.

[0108] Step 2: Take 50 μL of magnetic bead solution with a concentration of 1 mg / mL and put it into the above Solution 1, shake and mix evenly for 90 s to enable the magnetic beads modified with polypeptide to specifically capture the S protein.

[0109] Step 3: After the magnetic beads are fully bound, they are enriched under the action of an external magnetic field. After removing the supernatant, add 200 μL of DPBS, shake and mix evenly for 20 s and then enrich, and remove the supernatant under the action of the external magnetic field; then, add 200 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0110] Step 4: Add 10 μL of NaCl eluent with a concentration of 2 mol / L to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and collect the supernatant.

[0111] Step 5: Drop the supernatant onto the SERS substrate, place it in an oven at 45 °C and dry for 5 minutes, then collect the Raman signal. The excitation wavelength for Raman detection is 785 nm.

[0112] Step 6: Analyze the collected Raman spectra.

[0113] It can be seen from Comparative Example 1 that due to the failure to effectively preserve the protein, the protein conformation changes, the binding ability of the S protein to the specific magnetic beads becomes weaker, and the S protein is lost during the washing process. Therefore, using virus lysate 1 to lyse the virus and preserve the test sample, after the sample is placed at 4 °C for 48 hours, the detection performance will be affected, and the detection limit can only reach 10 4 copies / mL.

[0114] Comparative Example 2

[0115] The detection process of novel coronavirus using virus lysate 2 as the lysate and 1 wt% sodium dodecyl sulfate (SDS) solution as the eluent:

[0116] Step 1: First, place the nasal swab of the nucleic acid testee into 250 μL of virus lysate 2, add 200 μL of virus with a virus load of 10,000 copies / mL, and lyse it to obtain Solution 1.

[0117] Step 2: Take 50 μL of the magnetic bead solution with a concentration of 1 mg / mL and put it into the above Solution 1, shake and mix well for 90 s to allow the polypeptide-modified magnetic beads to specifically capture the S protein.

[0118] Step 3: Enrich the fully bound magnetic beads under the action of an external magnetic field. After removing the supernatant, add 200 μL of DPBS, shake and mix well for 20 s, and then remove the supernatant under the action of the external magnetic field; then, add 200 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0119] Step 4: Add 10 μL of the SDS solution with a concentration of 1 wt% to the enriched magnetic beads, shake and mix well for 20 s, then enrich the magnetic beads again and collect the supernatant.

[0120] Step 5: Drop the supernatant onto the SERS substrate, place it in an oven at 45 °C and dry for 5 minutes, then collect the Raman signal. The excitation wavelength for Raman detection is 785 nm.

[0121] Step 6: Analyze the collected Raman spectra, and the obtained Raman signal intensity is weak.

[0122] Figure 6 It is the Raman signal characterization diagram of the S protein with 1 wt% SDS solution as the eluent in Comparative Example 2; Figure 7Raman signal comparison chart of S protein with urea solution, NaCl solution, and 1wt% SDS solution as eluents. It can be seen from the figure that when using 1wt% SDS solution as the eluent, the elution efficiency of SDS eluent for S protein is lower than that of NaCl solution and urea solution. Compared with NaCl solution and urea solution, SDS solution has less impact on the structure of S protein, poorer ability to disrupt the binding between S protein and polypeptide, and lower elution efficiency.

[0123] Comparative Example 3

[0124] In this comparative example, the polypeptide used in the polypeptide-modified magnetic beads has a sequence similar to that of the polypeptide in Example 2, but a non-repetitive sequence is adopted: LVMGLNVWLRYSKAAAHHHHHH. By comparing the two polypeptides, it is explored whether the repetition of the polypeptide sequence can increase the affinity between the polypeptide and S protein.

[0125] Novel coronavirus detection process with virus lysate 2 as the lysate and NaCl aqueous solution as the eluent:

[0126] Step 1: First, place the nasal swab of the person to be tested for nucleic acid into 250 μL of virus lysate 2, and add 200 μL of virus with a virus load of 10,000 copies / mL. The virus is lysed therein to obtain Solution 1.

[0127] Step 2: Take 50 μL of magnetic bead solution with a concentration of 1 mg / mL and put it into the above Solution 1, shake and mix well for 90S to enable the polypeptide-modified magnetic beads to specifically capture S protein.

[0128] Step 3: Enrich the fully bound magnetic beads under the action of an external magnetic field. After removing the supernatant, add 200 μL of DPBS, shake and mix well for 20S and then enrich. Remove the supernatant under the action of an external magnetic field; then, add 200 μL of deionized water, repeat the above washing steps, and collect the enriched magnetic beads.

[0129] Step 4: Add 10 μL of NaCl eluent with a concentration of 2 mol / L to the enriched magnetic beads, shake and mix well for 20S, then enrich the magnetic beads again and collect the supernatant.

[0130] Step 5: Drop the supernatant on the SERS substrate, place it in an oven at 45 °C and dry for 5 minutes, then collect Raman signals. The excitation wavelength for Raman detection is 785 nm.

[0131] Step 6: Analyze the collected Raman spectra.

[0132] Comparing Example 2 and Comparative Example 3, the repetition of this polypeptide sequence can improve the affinity between S protein and ACE2. The detection sensitivity of the non-repetitive sequence polypeptide for pseudovirus is 10 4copies / mL。

[0133] Comparative Example 4

[0134] In this Comparative Example 4, the detection process of the novel coronavirus using virus lysate 2 as the lysate and an aqueous NaCl solution as the eluent is shown in Example 2, with the only difference being: Step 4: Add 10 μL of an NaCl eluent with a concentration of 0.5 mol / L to the enriched magnetic beads. After shaking and mixing evenly for 20 S, the magnetic beads are enriched again and the supernatant is collected. Since the concentration of NaCl is too low, the elution purpose cannot be achieved, so the Raman peak of the desired S protein cannot be obtained, and only the basal peak of the aqueous NaCl solution can be obtained (see Figure 4 the basal peak of the "high-concentration salt solution" in

[0135] Comparative Example 5

[0136] In this Comparative Example 5, the detection process of the novel coronavirus using virus lysate 2 as the lysate and an aqueous NaCl solution as the eluent is shown in Example 2, with the only difference being: Step 4: Add 10 μL of an NaCl eluent with a concentration of 5 mol / L to the enriched magnetic beads. After shaking and mixing evenly for 20 S, the magnetic beads are enriched again and the supernatant is collected. Since the concentration of NaCl is too high, more and larger particles of crystals are generated on the basal surface, preventing the incidence of Raman laser, so the signal of the desired S protein cannot be obtained, and only the basal peak of the aqueous NaCl solution can be obtained (see Figure 4 the basal peak of the "high-concentration salt solution" in

[0137] Comparative Example 6

[0138] In this Comparative Example 6, the detection process of the novel coronavirus using virus lysate 2 as the lysate and a urea solution as the eluent is shown in Example 3, with the only difference being: Step 4: Add 10 μL of a urea solution with a pH of 4.5 (100 mM NaH2PO4, 10 mM Tris, 3 M urea) to the enriched magnetic beads. After shaking and mixing evenly for 20 S, the magnetic beads are enriched again and the supernatant is collected. Since the concentration of urea is too low, the elution purpose cannot be achieved, so the Raman peak of the desired S protein cannot be obtained, and only the basal peak of the urea solution can be obtained (see Figure 5 the basal peak of the "urea solution with pH = 4.5" in

[0139] Comparative Example 7

[0140] In Comparative Example 7, the detection process of novel coronavirus using virus lysate 2 as the lysate and urea solution as the eluent is the same as that in Example 3, except that: Step 4: Add 10 μL of urea solution with a pH of 4.5 (100 mM NaH2PO4, 10 mM Tris, 11 M urea) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and collect the supernatant. Due to the too high concentration of urea, the signal of the urea base peak is strong, which interferes with the Raman signal of the S protein, resulting in a weak signal intensity. Due to the interference of the urea base peak, the detection limit of novel coronavirus is reduced, and the lowest can only detect 10 5 copies / mL of novel coronavirus.

[0141] Comparative Example 8

[0142] In Comparative Example 8, the detection process of novel coronavirus using virus lysate 2 as the lysate and urea solution as the eluent is the same as that in Example 3, except that: Step 4: Add 10 μL of urea solution with a pH of 3.5 (100 mM NaH2PO4, 10 mM Tris, 8 M urea) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and collect the supernatant. Due to the too low pH, the structure of the protein is damaged, and the obtained Raman signal cannot be attributed to the Raman signal of the S protein. In the machine learning stage, the peak shape cannot be determined as positive, so the urea solution based on this pH value cannot be used for this process.

[0143] Comparative Example 9

[0144] In Comparative Example 9, the detection process of novel coronavirus using virus lysate 2 as the lysate and urea solution as the eluent is the same as that in Example 3, except that: Step 4: Add 10 μL of urea solution with a pH of 6 (100 mM NaH2PO4, 10 mM Tris, 8 M urea) to the enriched magnetic beads. After shaking and mixing evenly for 20 s, enrich the magnetic beads again and collect the supernatant. Due to the too high pH, the buffer system of the eluent is damaged, the elution effect of the urea solution becomes poor, the intensity of the obtained Raman signal decreases, and the detection limit of novel coronavirus is 10 6 copies / mL.

[0145] Comparative Example 10

[0146] In Comparative Example 10, the detection process of the novel coronavirus using virus lysate 2 as the lysate and NaCl solution as the eluent is as described in Example 2, with the only difference being that the preparation method of the amino magnetic beads used is as follows: 1 g of FeCl3·6H2O is added to 50 mL of ethylene glycol and dissolved by ultrasonic treatment to obtain Solution 1; then, 4 g of sodium acetate and 1 g of polyetherimide PEI are added to Solution 1, and after stirring at 60 °C for 20 minutes, Solution 2 is obtained; Solution 2 is transferred to a 100 mL reaction kettle and hydrothermally treated at 220 °C for 2 h; the black product obtained above is washed twice with absolute ethanol and deionized water respectively, and then dried in a vacuum oven at 60 °C for 12 hours.

[0147] Since the PEI content of the magnetic beads prepared above is too low, the confinement effect of PEI during the growth of the magnetic beads is weakened, resulting in too large magnetic bead particles with a particle size of about 100 nm, corresponding to a decrease in superparamagnetism and a decline in dispersion performance. Due to the low dispersion of the magnetic beads, some impurities are wrapped inside the magnetic bead particles and cannot be completely removed during the cleaning process, and finally there are many impurity peaks in the obtained Raman spectrum. Interfered by the impurity peaks, the detection limit of the novel coronavirus decreases, and the detection sensitivity is 10 5 copies / mL.

[0148] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A SERS-based detection kit for the detection of novel coronavirus, characterized in that, The detection kit includes: a virus lysis solution, magnetic beads modified with a specific polypeptide, a washing buffer, an elution buffer, and a SERS substrate; The amino acid sequence of the specific polypeptide used for modifying the magnetic beads is LVMGLNVWLRYSKAAALVMGLNVWL-RYSKAAAHHHHHH; The elution buffer is an NaCl solution or a urea solution.

2. The detection kit according to claim 1, wherein The formulation of the virus lysis solution includes: 0.05 - 0.2 M Tris, 1 - 5 wt% polysorbate-20, 1 - 5 wt% polyvinylpyrrolidone, 0.001 - 0.01 M ethylenediaminetetraacetic acid; Alternatively, the formulation of the virus lysis solution includes: 0.05 - 0.2 M Tris, 1 - 5 wt% polysorbate-20, 1 - 5 wt% polyvinylpyrrolidone, 0.001 - 0.01 M ethylenediaminetetraacetic acid, 15 - 50 μg / mL glucose, 1 - 5 wt% bovine serum albumin, 10 - 50 μg / mL penicillin.

3. The detection kit according to claim 1, characterized in that The magnetic beads are amino-functionalized Fe3O4 magnetic particles; the amino-functionalized Fe3O4 magnetic particles are spherical in shape, and the particle size is 20 - 50 nm.

4. The detection kit according to claim 3, wherein The preparation method of the amino-functionalized Fe3O4 magnetic particles includes: adding sodium acetate and polyetherimide to an FeCl3·6H2O solution, and performing a hydrothermal reaction to obtain the amino-functionalized Fe3O4 magnetic particles; Wherein, the mass ratio of FeCl3·6H2O:sodium acetate:polyetherimide = (0.5 - 2):(2.0 - 8):(2 - 4).

5. The detection kit according to claim 1, wherein Six histidines in the amino acid sequence of the polypeptide are labeled at the C-terminus of the polypeptide amino acid sequence.

6. The detection kit according to claim 1, wherein, The modification of the specific polypeptide on the surface of the magnetic beads is carried out by electrostatic binding or amide bond covalent binding.

7. The detection kit according to claim 6, wherein The steps for electrostatic binding to connect the specific polypeptide are: adding the specific polypeptide to the magnetic bead solution, and incubating the mixture at 4 - 25 °C for 2 - 5 hours; The steps for amide bond covalent binding to connect the specific polypeptide are: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the magnetic bead solution, adding the specific polypeptide after mixing, and incubating at 4 - 25 °C for 0.5 - 5 hours.

8. According to the detection kit described in claim 7, in the steps for amide bond covalent binding to connect the specific polypeptide, incubate at 4 - 25 °C for 2 - 5 hours.

9. The detection kit according to claim 1, wherein, The washing buffer includes a phosphate buffer solution or / and deionized water.

10. The detection kit according to claim 1, wherein, The solvent of the NaCl solution is deionized water, and the concentration of NaCl is 1 - 4 mol / L; The urea solution has urea as the main component, an aqueous solution of Tris and NaH2PO4 as the buffer system, and the pH value is adjusted to 4 - 5.5 with HCl; the concentration of urea in the urea solution is 6 - 10 mol / L, the concentration of NaH2PO4 is 50 - 100 mM, and the concentration of Tris is 8 - 12 mM.

11. The detection kit according to claim 1, characterized in that, The SERS substrate is a gold nanoparticle array, a gold nanocone array, or a SERS solid-state chip.

Images