WO2023221690A1 - Detection method, detection card and detection kit for sars-cov-2, and protective mask - Google Patents

Detection method, detection card and detection kit for sars-cov-2, and protective mask Download PDF

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Publication number
WO2023221690A1
WO2023221690A1 PCT/CN2023/087592 CN2023087592W WO2023221690A1 WO 2023221690 A1 WO2023221690 A1 WO 2023221690A1 CN 2023087592 W CN2023087592 W CN 2023087592W WO 2023221690 A1 WO2023221690 A1 WO 2023221690A1
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Prior art keywords
detection
cells
protein
substrate
indicator
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PCT/CN2023/087592
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French (fr)
Chinese (zh)
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张玥
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张玥
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Publication of WO2023221690A1 publication Critical patent/WO2023221690A1/en

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    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41D—OUTERWEAR; PROTECTIVE GARMENTS; ACCESSORIES
    • A41D13/00—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches
    • A41D13/05—Professional, industrial or sporting protective garments, e.g. surgeons' gowns or garments protecting against blows or punches protecting only a particular body part
    • A41D13/11—Protective face masks, e.g. for surgical use, or for use in foul atmospheres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the invention belongs to the field of virus detection technology, and specifically relates to a new coronavirus detection method and a detection card based on this method, a virus detection kit and a protective mask with a virus monitoring function.
  • virus detection is crucial.
  • the existing virus detection technologies are nucleic acid detection and antigen detection.
  • the sensitivity of antigen detection is low and there are The problem of high false positives and false negatives.
  • the protective mask can have the ability to identify the virus, it can quickly identify the infected person and some viruses within a detection cycle after the infected person has the ability to spread the virus. Contacts can avoid the spread of the virus to a certain extent and effectively achieve the epidemic prevention effect.
  • the new coronavirus mainly has four structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein).
  • the spike protein S protein has two subunits, S1 and S2.
  • the receptor binding site RBD is located on the S1 subunit.
  • the new coronavirus enters the cell through RBD binding to ACE2.
  • the new coronavirus also contains some non- Structural proteins such as 3-chymotrypsin-like protease (3CL or major protease Mpro hydrolase), papain-like protease (PLPro), gyrase Rdrp (RNA-dependent RNA polymerase) and accessory proteins.
  • the new coronavirus uses two viral proteases, Mpro and PLpro, to hydrolyze the viral multimeric protein into functional non-structural proteins.
  • pp1a and pp1ab form non-structural proteins nsp1-11 and nsp1-16 respectively.
  • PLpro encoded by nsp3
  • Mpro encoded by nsp5
  • Mpro is conserved among coronaviruses, and Mpro among different coronaviruses
  • the substrates have some common characteristics: the amino acids from the N-terminus to the C-terminus are numbered in a paired form (p4 ⁇ p3 ⁇ p2 ⁇ p1 ⁇ -p1′), and the cleavage site is between p1 and p1′.
  • Mpro has unique substrate selectivity for glutamine at the P1 site (Leu-Gln ⁇ (Ser, Ala, Gly)), a function not shared by closely related host proteases.
  • the conserved sequence of PLpro is Leu-Xaa-Gly-Gly ⁇ Xaa (the amino acid residue number of the substrate is P4-P3-P2-P1 ⁇ -P1', the downward arrow indicates the cleavage site).
  • Heme is also called ferrous heme, and its chemical name is ferrous protoporphyrin IX. It is the prosthetic group of hemoglobin with a molecular weight of 614D. Heme is composed of protoporphyrin IX and ferrous iron atoms and is the chromogenic group of hemoglobin. Hemoglobin is composed of heme and globin, and consists of four peptide chains, two alpha chains and two beta chains, each chain has a cyclic heme containing an iron atom.
  • Prothrombin (II, prothrombin) is one of the blood coagulation factors. Exists in plasma, also known as factor II. It is a zymogen containing 582 amino acid residues. It is cleaved by factor Xa at Arg-Thr and Arg-Ile, and the N-terminal 274 amino acid residues are removed. The remaining 308 amino acid residues are divided into two peptide chains, A and B. Connected by a disulfide bond, it is thrombin. Thrombin directly acts on the last step of the blood coagulation process, promoting the conversion of soluble fibrinogen in the plasma into insoluble fibrin, thereby achieving the purpose of quick hemostasis.
  • Fibrinogen is also known as the first factor and fibrinogen. Mainly produced in liver cells.
  • the fibrinogen molecule consists of three polypeptide chains called ⁇ (A) chain, ⁇ (B) chain, and ⁇ chain, each of which is paired and combined by S-S bonds to form a dimer.
  • One molecule of fibrinogen is expressed as [ ⁇ (A) ⁇ (B) ⁇ ]2 when blood coagulates.
  • Arg-Gly of ⁇ (A) chain and ⁇ (B) respectively, they are cleaved by thrombin, freeing fibrin peptide A and fibrin peptide B to form fibrin monomers.
  • CRISPR-Cas9 targeted gene modification (knockout, knock-in) technology is a recently developed and powerful molecular biology tool for genome editing. It is currently widely used in the genetics of individual or cell genomes of various animals and plants. Transformation. Using CRISPR technology, multiple genes can be knocked in and/or knocked out at the same time.
  • CRISPR technology mainly consists of Cas9 protein and single-stranded guide RNA (sgRNA).
  • the Cas9 protein plays the role of cutting the double strands of DNA, and the sgRNA plays a guiding role. Under the guidance of sgRNA, through the principle of complementary base pairing, the Cas9 protein can Different target sites are cut to achieve DNA double-strand breaks.
  • NHEJ non-homologous end joining
  • the repair template consists of the target gene to be imported and the homology sequences (homology arms) upstream and downstream of the target sequence. The length and position of the homology arms are determined by the size of the editing sequence.
  • the HDR repair mode occurs at a low frequency in cells, usually less than 10%.
  • the purpose of the present invention is to provide a new coronavirus detection method, detection card, detection kit and protective mask to solve the problems in the existing technology.
  • a novel coronavirus detection method which is characterized in that: the substrate protein synthesized by the indicator cell is contacted with the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection;
  • the indicator cells are prepared by the following method:
  • the genome sequence of the mother cell is modified through gene editing, so that the substrate protein with the following structure is expressed in the cell: Z-X1-X2-Y;
  • Z- is the indicator part: the indicator part reacts with itself or with external intervening substances, develops color or forms a gel;
  • X1-X2 is the connecting part: a polypeptide chain fragment containing a substrate cleavage site, and the substrate cleavage site can react with the enzyme protein in the new coronavirus and cause cleavage;
  • -Y is the control part: it has the function of covering Z or inhibiting the activity of Z. When connected, Z does not indicate, even if Z does not develop color and does not form a gel.
  • the enzyme protein of the new coronavirus reacts with the substrate cleavage site of the X1-X2 part on the substrate protein, causing the Y in the substrate protein to disconnect, losing its function of covering Z or inhibiting Z activity, thereby making Z It reacts with itself or with external intervening substances to develop color or form a gel.
  • the enzyme protein of the new coronavirus is Mpro hydrolase or PLPro protease.
  • the enzyme protein of the new coronavirus is Mpro hydrolase
  • the X1-X2 part on the substrate protein is Leu-Gln- (Ser, Ala, Gly)
  • the substrate cleavage site of Mpro hydrolase is Leu- Gln ⁇ (Ser,Ala,Gly);
  • the enzyme protein of the new coronavirus is PLPro protease
  • the X1-X2 part on the substrate protein is Leu-Xaa-Gly-Gly-Xaa
  • the substrate cleavage site of PLpro protease is Leu-Xaa-Gly-Gly ⁇ Xaa .
  • a new coronavirus detection card which is characterized by: including a detection card body, a sample hole is provided on the detection card body, and indicator cells are provided on the inner wall of the sample hole.
  • the detection card body is made of optically transparent polystyrene material.
  • a new coronavirus detection kit including a sampling tube and a detection card, which is characterized in that: the detection card is provided with a sample hole, and cultured cells are provided on the inner wall of the sample hole, so The storage solution of the sampling tube contains the substrate protein as claimed in claim 1.
  • the cultured cells are Vero cells.
  • a protective mask including a cover body, which is composed of an inner layer of non-woven microporous filter membrane, a melt-blown cloth layer and a non-woven microporous filter membrane from the inside to the outside.
  • the outer layer is characterized by: a layer of indicator cells is provided inside the melt-blown cloth layer.
  • the size of the micropores on the non-woven microporous filter membrane layer is between that of viruses and cells.
  • the advantages of the present invention are: in the method of the present invention, by cultivating live viruses, the content of the virus is increased, and the detection sensitivity is improved, and only one live virus can be detected; because the method of the present invention can detect the unique enzyme proteins of the new coronavirus, It has high selection specificity and its detection accuracy is higher than that of antigen detection.
  • the detection kit also has the advantage of self-testing, which avoids cross-infection during collective sampling and protects against infection.
  • Masks have real-time monitoring capabilities and can quickly identify infected people within a detection cycle after the infected person has the ability to spread the virus. They can also quickly identify some virus contacts, which can avoid the spread of the virus to a certain extent and effectively prevent the epidemic. Effect.
  • the invention discloses a novel coronavirus detection method: preparing targeted indicator cells, utilizing the substrate protein synthesized by the indicator cells to contact the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection; wherein, The indicator cells are prepared by the following method:
  • the genome sequence of the mother cell is modified through gene editing, so that the substrate protein with the following structure is expressed in the cell: Z-X1-X2-Y; where:
  • Z- is the indicator part: the indicator part reacts with itself or with external intervening substances, develops color or forms a gel;
  • X1-X2 is the connecting part: a polypeptide chain fragment containing a substrate cleavage site, and the substrate cleavage site can react with the enzyme protein in the new coronavirus and cause cleavage;
  • -Y is the control part: it has the function of covering Z or inhibiting the activity of Z. When connected, Z does not indicate, even if Z does not develop color and does not form a gel.
  • the substrate protein has two architectural forms, namely:
  • Z- is the indicated part: color development or gel formation
  • X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
  • -Y is the control part: in the connected state, Z is not indicated.
  • Z- is the indicator part: reacts with external intervening substances to develop color or form a gel
  • X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
  • -Y is the control part: in the connected state, Z is not indicated.
  • virus detection process using the above method is as follows:
  • Step 1 After the virus contacts the indicator cell, it enters the indicator cell through the ACE2 receptor on the indicator cell and uses the materials of the indicator cell to synthesize its own proteins, which include structural proteins, enzyme proteins and auxiliary proteins;
  • Step 2 The synthesized enzyme protein will catalyze a chemical reaction with the substrate protein on the indicator cell, disconnect Y, and release Z;
  • Step 3 When the substrate protein is structure 1, Z develops color or forms a gel; when the substrate protein is structure 2, Z reacts with external intervening substances, develops color or forms a gel;
  • Step 4 The color-developing or gel-forming substance is released from the indicator cells, and the indicator area becomes larger, thereby increasing the recognition degree;
  • Step 5 After the cells are lysed, a large amount of viruses and viral enzyme proteins are released from the indicator cells, catalyzing more chemical reactions of substrate proteins on the indicator cells, developing colors or forming gels, thereby further increasing Recognition.
  • the virus detection process using the above method is as follows:
  • Step 1 After the virus comes into contact with the cultured cells, it enters the cells through the ACE2 receptor on the cell membrane and uses the cell's materials to synthesize its own proteins, which include structural proteins, enzyme proteins and auxiliary proteins;
  • Step 2 After the synthesized enzyme protein comes into contact with the substrate protein, a chemical reaction will occur, breaking Y and releasing Z;
  • Step 3 When the substrate protein is Structure 1, Z develops color or forms a gel; when the substrate protein is Structure 2, Z reacts with external intervening substances, develops color or forms a gel.
  • Mpro hydrolase in the new coronavirus reacts with the substrate cleavage site on X1-X2, thereby disconnecting Y in the substrate protein.
  • Y loses its function of covering Z.
  • Z is exposed to develop color or form a gel
  • Y loses its function of inhibiting Z activity, and Z reacts with external intervening substances, and develops color or forms a gel.
  • the X1-X2 part on the substrate protein is Leu-Gln- (Ser, Ala, Gly), and the substrate cleavage site is Leu-Gln ⁇ (Ser, Ala, Gly);
  • the invention also discloses a protective mask, which includes a cover body, which is composed of an inner layer of non-woven microporous filter membrane, a melt-blown cloth layer and an outer layer of non-woven microporous filter membrane from the inside to the outside, wherein : A layer of indicator cells is provided on the inside of the melt-blown cloth layer, and the size of the micropores on the non-woven microporous filter membrane layer is between the virus and the cells.
  • the melt-blown cloth layer is made into two layers, the inner layer is a melt-blown cloth with a layer of indicator cells attached, and the outer layer is an electret melt-blown cloth without indicator cells, which is suitable for work in a virus environment. Personnel use;
  • Protective masks should be stored at 2 to 8°C, used for no more than 24 hours, and used at an ambient temperature below 40°C;
  • Preferred install a chip in the protective mask to report unqualified results to the Centers for Disease Control and Prevention, or provide a QR code from the Centers for Disease Control and Prevention to facilitate infected people reporting results;
  • a thermal insulation layer is added to the outside of the protective mask to make the protective mask applicable to a wider range of temperatures.
  • the invention also discloses a new coronavirus detection card.
  • the detection card is provided with a sample hole, and indicator cells are provided on the inner wall of the sample hole.
  • the detection card is made of optically transparent polystyrene material
  • the test card should be stored at 2-8°C;
  • a chip is installed on the test card to report unqualified results to the Centers for Disease Control and Prevention, or a QR code from the Centers for Disease Control and Prevention is provided to facilitate infected people reporting results.
  • the invention also discloses a virus detection kit, which includes a sampling tube and a detection card.
  • the preservation solution of the sampling tube contains substrate protein.
  • the detection card is provided with a sample hole, and the inner wall of the sample hole is provided with a Cultured cells.
  • the detection card is made of optically transparent polystyrene material
  • the cultured cells are Vero cells
  • the virus detection kit should be stored at 2-8°C;
  • the invention discloses a novel coronavirus detection method, which involves preparing targeted indicator cells and utilizing the substrate protein synthesized by the indicator cells to contact the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection; wherein,
  • the detailed preparation method of the indicator cells is as follows:
  • mother cells containing ACE2 receptors on their cell membranes are used as mother cells for preparing indicator cells;
  • Design substrate The structure of the substrate protein is Z-X1-X2-Y.
  • Z- is the indicated part: color development or gel formation
  • X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
  • -Y is the control part: in the connected state, Z is not indicated.
  • This substrate protein can be catalyzed and decomposed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y. Y loses its function of covering Z, causing Z-X1 to develop color or form a gel.
  • Z- is the indicator part: reacts with external intervening substances to develop color or form a gel
  • X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
  • -Y is the control part: in the connected state, Z is not indicated.
  • This substrate protein can be catalyzed and decomposed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y. Y loses its function of inhibiting Z activity. Z-X1 chemically reacts with external intervening substances to develop color or form a gel. ;
  • the mother cell is Vero cell
  • the substrate protein is structure 1, which is a protein formed by covalently connecting the hemoglobin ⁇ chain and the new coronavirus polyprotein nsp5-11 through a polypeptide chain (hereinafter referred to as hemoglobin ⁇ chain-nsp5-11),
  • the substrate protein is structure 2, and the substrate protein is a protein formed by covalently connecting the thrombin double chain and the new coronavirus polyprotein nsp5-11 through a polypeptide chain (hereinafter referred to as thrombin-nsp5-11);
  • the gene editing technology is CRISPR-Cas9 gene editing technology.
  • the virus detection method is a live virus culture method, which determines whether the test sample contains the virus through color development or gel formation.
  • the substrate protein does not need to be added artificially. If the test sample contains a virus, the virus will enter the indicator cell through the receptor on the indicator cell and use the materials of the indicator cell to synthesize its own proteins, including structural proteins, Enzyme proteins and auxiliary proteins; the synthesized enzyme proteins will catalyze the chemical reaction of the substrate protein on the indicator cell, break Y, and release Z; when the substrate protein is structure 1, Z will develop color or form a gel; When the substrate protein is structure 2, Z reacts with external intervening substances to develop color or form a gel; the substance that develops color or forms a gel is released from the indicator cells, and the indicator area becomes larger, thereby increasing the Recognition: After the cells are lysed, a large amount of viruses and viral enzyme proteins are released, which catalyze more chemical reactions with substrate proteins on the indicator cells, develop colors or form gels, thereby further increasing the recognition;
  • the substrate protein When the cultured cells are not indicator cells, the substrate protein needs to be added manually. If the test sample contains a virus, after the virus comes into contact with the cultured cells, it enters the cell through the ACE2 receptor on the cell membrane and uses the cell's materials to synthesize its own proteins.
  • the protein includes structural proteins, enzyme proteins and auxiliary proteins; when the synthesized enzyme protein comes into contact with the substrate protein, a chemical reaction will occur, breaking Y and releasing Z; when the substrate protein is structure 1, Z will develop color Or form a gel. When the substrate protein is structure 2, Z reacts with external intervening substances to develop color or form a gel;
  • test sample does not contain viruses, there will be no color change or gel formation.
  • the test sample is the gas exhaled through the mouth and nose or a sample collected by examination swab;
  • the cultured cells of the virus are indicator cells
  • the receptor is ACE2;
  • the enzyme protein is Mpro hydrolase
  • the substrate protein when the substrate protein is Structure 1, the substrate protein is hemoglobin ⁇ chain-nsp5-11.
  • the heme group of the substrate protein is blocked by nsp5-11 and does not develop color or appears light red.
  • the protein can be hydrolyzed by Mpro, releasing the hemoglobin ⁇ polypeptide chain and appearing red;
  • the substrate protein is Structure 2
  • the substrate protein is thrombin-nsp5-11.
  • the substrate protein is prothrombin, which can be hydrolyzed by Mpro to release the two chains of thrombin and make it Connected by a disulfide bond to generate activated thrombin.
  • the protective mask of the present invention is a mask made of melt-blown cloth with a layer of indicator cells, and includes a mask body, a nose clip and a mask belt; the mask body is composed of non-woven microporous filters from the inside to the outside.
  • the non-woven fabric is a non-woven fabric formed by air flow or mechanical meshing and reinforced by hydroentanglement, acupuncture or hot rolling;
  • the non-woven microporous filter membrane layer is made of polymer chemical materials and porogenic additives are specially treated and then applied to the non-woven fabric. Its pore size is between viruses and cells, allowing viruses to pass but preventing cells from passing through.
  • Through a layer of indicator cells is attached to the melt-blown cloth; the nose clip is made of bendable plastic material; and the mask strap is knitted from cotton thread and spandex thread.
  • the storage condition of protective masks is 2 ⁇ 8°C;
  • the use time of protective masks should not exceed 24 hours;
  • the protective mask used by virus environment workers has two layers of melt-blown cloth.
  • the inner layer is a melt-blown cloth with a layer of indicator cells attached
  • the outer layer is an electret melt-blown cloth without indicator cells.
  • the melt-blown cloth layer of the protective masks used by other personnel only contains one layer, which is a melt-blown cloth with a layer of indicator cells attached;
  • the protective mask should be used under environmental conditions not higher than 40°C.
  • the temperature is too low, a layer of insulation needs to be glued to the outside of the protective mask.
  • the test card described in this solution can be used as part of a virus detection kit.
  • the virus detection kit includes a test swab, a sampling tube, a test card and a dropper.
  • the test swab is made of a plastic rod (such as a polyethylene rod). Styrene) and a swab head (such as artificial fiber), used for sample collection;
  • the sampling tube contains a preservation solution, used to collect the samples collected by the inspection swab;
  • the test card is provided with a sample hole, and a sample hole arm A layer of indicator cells is attached; the dropper is used to drop the sample in the sampling tube into the sample hole of the detection card.
  • How to use the virus detection kit Take out the test swab, insert the swab head into the nostril or pharynx, turn it three times to the left, and then turn it to the right three times; insert the swab head into the preservation solution of the sampling tube, stir repeatedly, and then Squeeze the outer wall of the sampling tube with your hands, squeeze all the liquid on the swab head into the preservation solution in the sampling tube; take out the test card, drop the preservation solution in the sampling tube into the sample hole in the test card, and let it sit for 4 hours ; If the color changes or gel is formed, it is judged as positive; if the color does not change or gel is not formed, it is judged as negative. Used test kits should be placed in medical waste bags and discarded after uniform autoclaving.
  • the inspection swab is a cotton swab
  • the preservation solution in the sampling tube is DMEM liquid culture medium
  • the storage liquid level in the sampling tube is DMEM liquid medium containing calcium ions and fibrinogen;
  • the sampling tube also has the function of a dropper, replacing an ordinary dropper;
  • the virus detection kit should be stored at 2 to 8°C.
  • the detection kit described in this solution includes an inspection swab, a sampling tube, a test card, a lysate tube and a dropper.
  • the inspection swab is made of a plastic rod (such as polystyrene) and a swab head (such as artificial fiber) Composed of, used for sample collection;
  • the sampling tube contains a preservation solution for collecting samples collected by inspection swabs;
  • the detection card is provided with a sample hole, and a layer of cultured cells is attached to the sample hole arm;
  • the dropper It is used to drop the sample in the sampling tube into the sample hole of the detection card;
  • the lysis liquid tube contains lysis liquid, which is used to lyse the cultured cells and release the virus and viral proteins from the cultured cells.
  • How to use the virus detection kit Take out the test swab, insert the swab head into the nostril or pharynx, turn it three times to the left, and then turn it to the right three times; insert the swab head into the preservation solution of the sampling tube, stir repeatedly, and then Squeeze the outer wall of the sampling tube with your hands and squeeze all the liquid on the swab head into the preservation solution in the sampling tube; take out the test card, drop the preservation solution in the sampling tube into the sample hole in the test card, and let it sit for 2 hours.
  • test kits should be placed in medical waste bags and discarded after uniform autoclaving.
  • the cultured cells are Vero cells
  • the inspection swab is a cotton swab
  • the preservation solution in the sampling tube is DMEM liquid culture medium containing hemoglobin ⁇ chain-nsp5-11;
  • the storage liquid level in the sampling tube is DMEM liquid medium containing thrombin-nsp-5-11 and fibrinogen;
  • the sampling tube also has the function of a dropper, replacing an ordinary dropper;
  • the lysis solution is an improved version of RIPA lysis solution that does not contain SDS.
  • the virus detection kit should be stored at 2 to 8°C.
  • mother cells cells containing ACE2 receptors on their cell membranes are used as mother cells for preparing indicator cells.
  • Vero cells are used as mother cells;
  • Design substrate The structure of the substrate protein is Z-X1-X2-Y.
  • Z- indicates the part: colored protein or polypeptide
  • X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
  • -Y is the control part: in the connected state, Z is not indicated.
  • This substrate protein can be catalyzed and decomposed by the enzyme protein of the new coronavirus to generate Z-X1 and Ala-Leu-Gln-Gly-", the Y chain is the new coronavirus polyprotein nsp5-11;
  • CRISPR-Cas9 gene editing technology is used to modify the gene sequence of the promoter of the genome that synthesizes the hemoglobin ⁇ chain, and At the end of the genome, the gene sequence for synthesizing the "-Trp-Ala-Leu-Gln-Gly-" polypeptide chain fragment, the gene sequence for synthesizing the new coronavirus polyprotein nsp5-11 and the poly (A) tail are sequentially added to induce Vero cells. Synthetic hemoglobin ⁇ chain-nsp5-11.
  • the nsp5-11 of this protein blocks the heme group of the hemoglobin ⁇ chain so that it does not develop color or appears light red. Through verification, it is ensured that Mpro hydrolase can quickly hydrolyze the hemoglobin ⁇ chain- nsp5-11, releases the hemoglobin ⁇ polypeptide chain and appears red.
  • the gene sequence for synthesizing the X1-X2 chain can be modified to improve the ability of Mpro hydrolase to hydrolyze the hemoglobin ⁇ chain-nsp5-11.
  • the new cells after editing this gene are for indicator cells. Among them, CRISPR-Cas9 gene editing technology is an existing technology and will not be described in detail.
  • melt-blown cloth Take out the melt-blown cloth, wash it 3 times with PBS solution, then soak it in a protective solution containing bovine serum albumin, sucrose, MEM and other nutrients, soak it at 37°C for 2 hours, and then take out the melt-blown cloth. air dry;
  • protective masks Stack the inner layer of the sterile non-woven microporous filter membrane, the melt-blown cloth layer and the outer layer of the non-woven microporous filter membrane together, and make the main body of the mask through high-frequency welding. Cut into individual masks on the assembly line. Install a nose clip on one side of the mask, sew it by crimping, and then put on ear strings on the edge of the mask to obtain a protective mask; the melt-blown cloth layer is a melt-blown cloth prepared by the above method and attached with a layer of indicator cells. A layer of melt-blown cloth without indicator cells can be added to the outside of the above-mentioned melt-blown cloth for use by workers working in virus environments. This layer of oil-sprayed cloth can be electret with electret equipment, and a layer of glycerin protective agent can be applied on the inside.
  • the detection card carrier is made of optically transparent pure polystyrene material and is equipped with a sample hole;
  • the preservation solution in the sampling tube is DMEM liquid culture medium.
  • the color changes on the inside of the mask it indicates that the user is infected or potentially infected; if the color changes on the outside of the mask, it indicates that the user has been exposed to a virus environment; those who work in a virus environment for a long time can choose two layers of oil spray For cloth masks, the outer layer does not contain indicator cells, so only the inside of the mask will change color.
  • the user can collect the sample with a test swab, transfer the sample into the preservation solution of the sampling tube, prepare a sample solution, and then use a dropper to transfer the sample solution from the sampling tube. Drop it into the sample hole of the test card.
  • the virus in the sample solution will be adsorbed to the indicator cell, enter the cell through the receptor on the indicator cell, and synthesize its own protein in the cell, in which the virus synthesizes
  • the Mpro hydrolase will decompose the substrate protein in the instruction cell and release the hemoglobin ⁇ chain, resulting in a color change and recognition; if the user is not an infected person, the sample hole of the test card will not change color.
  • mother cells cells containing ACE2 receptors on their cell membranes are used as mother cells for preparing indicator cells.
  • Vero cells are used as mother cells;
  • Design substrate The structure of the substrate protein is Z-X1-X2-Y.
  • Z- is the indicator part: reacts with external intervening substances to form a gel
  • X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
  • -Y is the control part: in the connected state, Z is not indicated.
  • This substrate protein can be catalyzed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y.
  • Z-X1 reacts with external intervening substances to form a gel.
  • the Z chain in this example is the double chain of thrombin, and X
  • the chain is "-Trp-Ala-Leu-Gln-Gly-”
  • the Y chain is the new coronavirus polyprotein nsp5-11
  • the external intervening substance is fibrinogen isolated from the blood of livestock and poultry;
  • CRISPR-Cas9 gene editing technology is used to modify the gene sequence of the promoter of the genome that synthesizes prothrombin, and Modify the gene sequence corresponding to the action site "Arg-Thr" and "Arg-Ile” of factor
  • the gene sequence corresponding to the amino acid residues is modified to the gene sequence corresponding to the new coronavirus polyprotein nsp5-11, and a poly(A) tail is added to the end of the prothrombin genome, thereby inducing Vero cells to synthesize thrombin-nsp5-11.
  • the protein is prothrombin that can be activated by Mpro.
  • nsp5-11 Due to the presence of nsp5-11, it becomes an intracellular protein. Through verification, it is ensured that Mpro hydrolase can quickly hydrolyze thrombin-nsp5-11 and induce the synthesis of thrombin. If necessary, The gene sequence for synthesizing the X1-X2 chain can be modified to improve the ability of Mpro hydrolase to hydrolyze thrombin-nsp5-11.
  • the new cells after editing the gene are indicator cells. Among them, CRISPR-Cas9 gene editing technology is an existing technology and will not be described in detail.
  • test card The culture medium for culturing the above indicator cells is rich in galactose, mannose, fucose, hexosamine, sialic acid, calcium ions, vitamin K, MEM, fetal bovine serum and two antibiotics
  • the culture medium of other substances is the same as in Example 1;
  • the preservation solution in the sampling tube is DMEM liquid culture medium containing calcium ions and fibrinogen.
  • the user can collect the sample with a test swab, transfer the sample into the preservation solution of the sampling tube, prepare a sample solution, and then use a dropper to drop the sample solution in the sampling tube.
  • the virus in the sample solution will be adsorbed to the indicator cell, enter the cell through the receptor on the indicator cell, and synthesize its own protein in the cell, among which the Mpro synthesized by the virus
  • the hydrolase will decompose the substrate protein in the indicator cell, generate and release activated thrombin.
  • the activated thrombin will convert soluble fibrinogen into insoluble fibrin, thereby forming a gel and being recognized; if the user is not infected Otherwise, the sample hole of the test card will not form gel.
  • an animal or plant cell or a microorganism such as a virus, bacteria, etc.
  • a microorganism such as a virus, bacteria, etc.
  • Vero cells are used as a carrier for preparing substrate proteins.
  • Design substrate The structure of the substrate protein is Z-X1-X2-Y.
  • Z- is the indicated part: forming gel
  • X is the connecting part: a polypeptide chain fragment containing the substrate cleavage site
  • Y is the control part: in the connected state, Z is not indicated.
  • This substrate protein can be catalyzed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y, releasing the insoluble protein Z, thereby forming a gel.
  • the Z chain is fibrin
  • the X1-X2 chain is " -Trp-Ala-Leu-Gln-Gly-”
  • the Y chain is fibrinopeptide A and fibrinopeptide B;
  • CRISPR-Cas9 gene editing technology is used to modify the synthesis of fibrinogen ⁇ (A) chain and ⁇ (B).
  • the gene sequence of the promoter of chain and gamma chain genomes, and the gene sequence of the action site for synthesizing thrombin was modified into the gene sequence for synthesizing "-Trp-Ala-Leu-Gln-Gly-".
  • the action site is located at "Arg-Gly" of fibrinogen ⁇ (A) chain and ⁇ (B) chain, thus inducing Vero cells to produce another type of fibrinogen.
  • This fibrinogen is a soluble protein that can be hydrolyzed by Mpro to generate fibers.
  • Protein peptide A, fibrinopeptide B and insoluble fibrin and have been verified to ensure that Mpro hydrolase can quickly hydrolyze the fibrinogen to release insoluble fibrin to form a gel.
  • the synthesis can be modified if necessary
  • the gene sequence of the X1-X2 chain optimizes the ability of Mpro hydrolase to hydrolyze fibrinogen.
  • the new cells after editing of this gene are indicator cells. Among them, CRISPR-Cas9 gene editing technology is an existing technology and will not be described in detail.
  • sampling tubes Add the fibrinogen prepared by the above method to DMEM liquid culture medium, distribute it into sterile plastic tubes, and cover the mouth of the tube with a cap to obtain a sampling tube;
  • test card Vero cells are selected as culture cells.
  • the culture medium of the culture cells is a culture medium rich in substances such as MEM, fetal bovine serum and two antibiotics. The rest is the same as in Example 1;
  • the prepared test card into the sealed bag and seal it. Put the sealed test card, inspection swab, sampling tube, dropper, lysate tube and instruction manual into the outer box to get the virus detection reagent. box, wherein the lysis solution in the lysis solution tube is a modified version of RIPA lysis solution that does not contain SDS.
  • the user can collect the sample with a test swab, transfer the sample into the preservation solution of the sampling tube, prepare a sample solution, and then use a dropper to drop the sample solution in the sampling tube.
  • a dropper to drop the sample solution in the sampling tube.
  • the sample hole of the test card add lysis solution dropwise after 2 hours, and let it sit for half an hour to observe the results. If the user is an infected person, after the lysis solution cleaves the cells, the virus and the proteins synthesized by the virus will be released, and the Mpro in it will be hydrolyzed.
  • the enzyme will decompose the fibrinogen in the sample solution, causing it to generate insoluble fibrin to form a gel; if the user is not an infected person, the sample hole of the test card will not form a gel.
  • the method of the present invention by cultivating live viruses, the content of the virus is increased, the detection sensitivity is improved, and only one live virus can be detected; because the method of the present invention has a higher sensitivity to the enzyme proteins unique to the new coronavirus
  • the selection specificity of the test kit is higher than that of the antigen test; in addition to high sensitivity and high accuracy, the test kit also has the advantage of self-testing to avoid cross-infection during gathering sampling, while the protective mask has real-time Monitoring capabilities can quickly identify infected people within a detection cycle after the infected person has the ability to spread the virus, and can also quickly identify some virus contacts, which can avoid the spread of the virus to a certain extent and effectively achieve the epidemic prevention effect.

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Abstract

Provided is a detection method for SARS-CoV-2, which achieves the purpose of detection by preparing a specific indication cell and utilizing the phenomenon of color development or gel formation after a substrate protein synthesized by the indication cell is in contact with an enzyme protein of SARS-CoV-2. In addition, also provided are a virus detection card and a virus detection kit prepared by using the indication cell or the substrate protein component, and a protective mask with virus monitoring functionality. By culturing live viruses, the present invention features increased viral load and improved detection sensitivity, as well as superior detection accuracy to that of antigen detections. In addition, the detection kit also has the advantage of self-testing, which avoids cross-infection during centralized sampling. The protective mask has real-time monitoring capability, which may effectively exert the epidemic control effect.

Description

一种新冠病毒检测方法、检测卡、检测试剂盒及防护口罩A new coronavirus detection method, detection card, detection kit and protective mask 技术领域Technical field
本发明属于病毒检测技术领域，具体涉及一种新冠病毒检测方法及基于此方法建立的检测卡、病毒检测试剂盒及具有病毒监测功能的防护口罩。The invention belongs to the field of virus detection technology, and specifically relates to a new coronavirus detection method and a detection card based on this method, a virus detection kit and a protective mask with a virus monitoring function.
背景技术Background technique
目前，世界各地均受到新冠病毒的影响，特别是奥密克戎变异毒株，R0值达到了9.5，其传染性强，传播速度特别快，且无症状者居多，这给了我们抗疫带来了很大难度。在防疫过程中，病毒的检测至关重要，目前现有的病毒检测技术为核酸检测和抗原检测，但核酸检测采样时可能存在聚集性交叉感染的风险，而抗原检测的灵敏度偏低，且存在假阳性和假阴性都偏高的问题。因此急需一种新的检测方法，既有较高的灵敏度，能快速识别感染者，又能避免因检测时带来的交叉感染。同时感染者大部分是通过口鼻传播病毒，若防护口罩能具有识别病毒能力，则可在感染者具有传播病毒能力后的一个检测周期内，迅速识别出感染者，又可以快速识别出部分病毒接触者，一定程度上可避免病毒传播，有效的起到防疫效果。At present, all parts of the world are affected by the new coronavirus, especially the Omicron mutant strain, with an R0 value of 9.5. It is highly contagious, spreads very quickly, and most people are asymptomatic. This gives us a new opportunity to fight against the epidemic. It came with great difficulty. In the process of epidemic prevention, virus detection is crucial. Currently, the existing virus detection technologies are nucleic acid detection and antigen detection. However, there may be a risk of clustered cross-infection when sampling for nucleic acid detection, and the sensitivity of antigen detection is low and there are The problem of high false positives and false negatives. Therefore, there is an urgent need for a new detection method that has high sensitivity, can quickly identify infected persons, and can avoid cross-infection caused by detection. At the same time, most infected people spread the virus through their mouth and nose. If the protective mask can have the ability to identify the virus, it can quickly identify the infected person and some viruses within a detection cycle after the infected person has the ability to spread the virus. Contacts can avoid the spread of the virus to a certain extent and effectively achieve the epidemic prevention effect.
通过研究发现，新冠病毒主要有四种结构蛋白：刺突蛋白（S蛋白）、核衣壳蛋白（N蛋白）、膜蛋白（M蛋白）、包膜蛋白（E蛋白）。刺突蛋白S蛋白有两个亚基，S1和S2，其中受体结合位点RBD位于S1亚基上，新冠病毒通过RBD与ACE2结合进入细胞内部，除了结构蛋白外，新冠病毒还含有一些非结构蛋白，如3-胰凝乳蛋白酶样蛋白酶（3CL或者主要蛋白酶Mpro水解酶）、木瓜蛋白酶样蛋白酶（PLPro）、旋转酶Rdrp（RNA依赖RNA聚合酶）和辅助蛋白。Through research, it was found that the new coronavirus mainly has four structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). The spike protein S protein has two subunits, S1 and S2. The receptor binding site RBD is located on the S1 subunit. The new coronavirus enters the cell through RBD binding to ACE2. In addition to structural proteins, the new coronavirus also contains some non- Structural proteins such as 3-chymotrypsin-like protease (3CL or major protease Mpro hydrolase), papain-like protease (PLPro), gyrase Rdrp (RNA-dependent RNA polymerase) and accessory proteins.
新型冠状病毒通过Mpro和PLpro这两种病毒蛋白酶将病毒多聚体蛋白水解为功能性非结构蛋白，pp1a和pp1ab分别形成非结构蛋白nsp1-11和nsp1-16。PLpro（由nsp3编码）负责切割nsp1/2，nsp2/3和nsp3/4边界，而Mpro（由nsp5编码）负责其余11个切割事件，Mpro在冠状病毒中是保守的，并且不同冠状病毒中Mpro的底物具有一些共同的特征：从n端到c端的氨基酸以配对的形式进行编号(p4‑p3‑p2‑p1↓-p1′)，裂解位点在p1和p1′之间。特别地，Mpro对P1位点（Leu-Gln↓（Ser，Ala，Gly））的谷氨酰胺有独特的底物选择性，是紧密相关的宿主蛋白酶所不具备的功能。PLpro的保守序列为Leu-Xaa-Gly-Gly↓Xaa（底物的氨基酸残基编号为P4-P3-P2-P1↓-P1'，向下的箭头表示裂解位点）。The new coronavirus uses two viral proteases, Mpro and PLpro, to hydrolyze the viral multimeric protein into functional non-structural proteins. pp1a and pp1ab form non-structural proteins nsp1-11 and nsp1-16 respectively. PLpro (encoded by nsp3) is responsible for cutting nsp1/2, nsp2/3 and nsp3/4 borders, while Mpro (encoded by nsp5) is responsible for the remaining 11 cleavage events. Mpro is conserved among coronaviruses, and Mpro among different coronaviruses The substrates have some common characteristics: the amino acids from the N-terminus to the C-terminus are numbered in a paired form (p4‑p3‑p2‑p1↓-p1′), and the cleavage site is between p1 and p1′. In particular, Mpro has unique substrate selectivity for glutamine at the P1 site (Leu-Gln↓(Ser, Ala, Gly)), a function not shared by closely related host proteases. The conserved sequence of PLpro is Leu-Xaa-Gly-Gly↓Xaa (the amino acid residue number of the substrate is P4-P3-P2-P1↓-P1', the downward arrow indicates the cleavage site).
血红素也称为亚铁血红素，化学命名为亚铁原卟啉Ⅸ，它是血红蛋白的辅基，分子量614D。血红素由原卟啉Ⅸ和亚铁原子组成，是血红蛋白的显色基团。血红蛋白由血红素和珠蛋白结合而成，由四条肽链组成，两条α链和两条β链，每一条链有一个包含铁原子的环状血红素。Heme is also called ferrous heme, and its chemical name is ferrous protoporphyrin IX. It is the prosthetic group of hemoglobin with a molecular weight of 614D. Heme is composed of protoporphyrin IX and ferrous iron atoms and is the chromogenic group of hemoglobin. Hemoglobin is composed of heme and globin, and consists of four peptide chains, two alpha chains and two beta chains, each chain has a cyclic heme containing an iron atom.
凝血酶原(Ⅱ，prothrombin)，血液凝固因子之一。存在于血浆中，亦称第Ⅱ因子。是含582氨基酸残基的酶原，被因子Xa在Arg-Thr及Arg-Ile处切开，切除N端274个氨基酸残基，余下308个氨基酸残基分成A、B两条肽链，由一个二硫键相连，即为凝血酶(thrombin)。凝血酶直接作用于血液凝固过程的最后一步，促使血浆中的可溶性纤维蛋白原转变成不溶的纤维蛋白，从而达到速效止血的目的。Prothrombin (II, prothrombin) is one of the blood coagulation factors. Exists in plasma, also known as factor II. It is a zymogen containing 582 amino acid residues. It is cleaved by factor Xa at Arg-Thr and Arg-Ile, and the N-terminal 274 amino acid residues are removed. The remaining 308 amino acid residues are divided into two peptide chains, A and B. Connected by a disulfide bond, it is thrombin. Thrombin directly acts on the last step of the blood coagulation process, promoting the conversion of soluble fibrinogen in the plasma into insoluble fibrin, thereby achieving the purpose of quick hemostasis.
血纤维蛋白原亦称为第一因子、纤维素原。主要在肝细胞中产生．血纤维蛋白原分子由称为α（A）链、β（B）链、γ链的三种多肽链，各自成对，以S-S键结合成二聚体。1分子血纤维蛋白原以[α（A）β（B）γ]2表示，血液凝固时．分别在α（A）链和β（B）的Arg-Gly处，被凝血酶切开，游离出血纤维蛋白肽A和血纤维蛋白肽B而形成血纤维蛋白单体。Fibrinogen is also known as the first factor and fibrinogen. Mainly produced in liver cells. The fibrinogen molecule consists of three polypeptide chains called α (A) chain, β (B) chain, and γ chain, each of which is paired and combined by S-S bonds to form a dimer. One molecule of fibrinogen is expressed as [α(A)β(B)γ]2 when blood coagulates. At the Arg-Gly of α (A) chain and β (B) respectively, they are cleaved by thrombin, freeing fibrin peptide A and fibrin peptide B to form fibrin monomers.
CRISPR-Cas9靶向基因改造（敲除、敲入）技术是最新发展起来的一种强有力的用于基因组编辑的分子生物学工具，目前广泛应用于各类动植物个体或细胞基因组的遗传学改造。采用CRISPR技术，可同时对多个基因进行基因敲入和/或敲除。CRISPR技术主要由Cas9蛋白和单链导向RNA（sgRNA）组成，其中Cas9蛋白起切割DNA双链的作用，sgRNA起导向的作用，在sgRNA的导向下，通过碱基互补配对原则，Cas9蛋白可对不同的靶部位进行切割，实现DNA双链的断裂。通常情况下，当细胞出现DNA断裂的情况时，会采用高效的非同源性末端接合的方式（NHEJ）对断裂的DNA双链进行修复，在修复的过程，通常会发生碱基的插入或者缺失的错配现象，造成移码突变，使原来编码某种肽链的基因变成编码另一种完全不同的肽链序列，实现基因敲除。CRISPR-Cas9 targeted gene modification (knockout, knock-in) technology is a recently developed and powerful molecular biology tool for genome editing. It is currently widely used in the genetics of individual or cell genomes of various animals and plants. Transformation. Using CRISPR technology, multiple genes can be knocked in and/or knocked out at the same time. CRISPR technology mainly consists of Cas9 protein and single-stranded guide RNA (sgRNA). The Cas9 protein plays the role of cutting the double strands of DNA, and the sgRNA plays a guiding role. Under the guidance of sgRNA, through the principle of complementary base pairing, the Cas9 protein can Different target sites are cut to achieve DNA double-strand breaks. Normally, when cells experience DNA breaks, they will use highly efficient non-homologous end joining (NHEJ) to repair the broken DNA double strands. During the repair process, base insertion or insertion usually occurs. The missing mismatch phenomenon causes frameshift mutations, causing the gene that originally encoded a certain peptide chain to encode another completely different peptide chain sequence, achieving gene knockout.
当DNA双链断裂后，如果有DNA修复模板进入到细胞中，基因组断裂部分会依据修复模板进行同源重组修复（HDR），从而实现基因敲入。修复模板由需要导入的目标基因和靶序列上下游的同源性序列（同源臂）组成，同源臂的长度和位置由编辑序列的大小决定。HDR修复模式在细胞中发生率较低，通常小于10%。为了增加基因敲入的成功率，目前有很多科学家致力于提高HDR效率，将编辑的细胞同步至HDR最活跃的细胞分裂时期，促进修复方式以HDR进行；或者利用化学方法抑制基因进行NHEJ，提高HDR的效率。When a DNA double-strand break occurs, if a DNA repair template enters the cell, the broken part of the genome will undergo homologous recombination repair (HDR) based on the repair template, thereby achieving gene knock-in. The repair template consists of the target gene to be imported and the homology sequences (homology arms) upstream and downstream of the target sequence. The length and position of the homology arms are determined by the size of the editing sequence. The HDR repair mode occurs at a low frequency in cells, usually less than 10%. In order to increase the success rate of gene knock-in, many scientists are currently working on improving the HDR efficiency, synchronizing the edited cells to the most active cell division period of HDR, and promoting the repair method to proceed with HDR; or using chemical methods to inhibit genes for NHEJ to improve HDR efficiency.
发明内容Contents of the invention
本发明的目的是：提供一种新冠病毒检测方法、检测卡、检测试剂盒及防护口罩，以解决现有技术中的问题。The purpose of the present invention is to provide a new coronavirus detection method, detection card, detection kit and protective mask to solve the problems in the existing technology.
本发明是通过如下技术方案实现的：The present invention is achieved through the following technical solutions:
一种新冠病毒检测方法，其特征在于：利用指示细胞合成的底物蛋白与新冠病毒的酶类蛋白接触后显色或者形成凝胶达到检测的目的；A novel coronavirus detection method, which is characterized in that: the substrate protein synthesized by the indicator cell is contacted with the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection;
其中，所述指示细胞通过如下方法制备：Wherein, the indicator cells are prepared by the following method:
选取细胞膜上含有ACE2受体的细胞作为制备指示细胞的母细胞；Select cells containing ACE2 receptors on their cell membranes as mother cells for preparing indicator cells;
通过基因编辑方式修改母细胞的基因组序列，从而在该细胞中表达出如下结构的底物蛋白：Z-X1-X2-Y；The genome sequence of the mother cell is modified through gene editing, so that the substrate protein with the following structure is expressed in the cell: Z-X1-X2-Y;
其中：in:
Z-为指示部分：指示部分自身或与外部介入物质反应，显色或形成凝胶；Z- is the indicator part: the indicator part reacts with itself or with external intervening substances, develops color or forms a gel;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段，且该底物裂解位点可与新冠病毒中的酶类蛋白反应并发生断裂；X1-X2 is the connecting part: a polypeptide chain fragment containing a substrate cleavage site, and the substrate cleavage site can react with the enzyme protein in the new coronavirus and cause cleavage;
-Y为控制部分：具有遮盖Z或抑制Z活性的作用，连接状态下，使Z不指示，即使Z不显色和不形成凝胶。-Y is the control part: it has the function of covering Z or inhibiting the activity of Z. When connected, Z does not indicate, even if Z does not develop color and does not form a gel.
进一步的：新冠病毒的酶类蛋白与底物蛋白上的X1-X2部分的底物裂解位点反应，使底物蛋白中的Y断开，失去遮盖Z或抑制Z活性的作用，从而使Z自身或与外部介入物质反应，显色或形成凝胶。Further: the enzyme protein of the new coronavirus reacts with the substrate cleavage site of the X1-X2 part on the substrate protein, causing the Y in the substrate protein to disconnect, losing its function of covering Z or inhibiting Z activity, thereby making Z It reacts with itself or with external intervening substances to develop color or form a gel.
进一步的：所述新冠病毒的酶类蛋白为Mpro水解酶或者PLPro蛋白酶。Further: the enzyme protein of the new coronavirus is Mpro hydrolase or PLPro protease.
进一步的：当新冠病毒的酶类蛋白为Mpro水解酶时，底物蛋白上的X1-X2部分为Leu-Gln-（Ser，Ala，Gly），Mpro水解酶的底物裂解位点为Leu-Gln↓（Ser，Ala，Gly）；Further: When the enzyme protein of the new coronavirus is Mpro hydrolase, the X1-X2 part on the substrate protein is Leu-Gln- (Ser, Ala, Gly), and the substrate cleavage site of Mpro hydrolase is Leu- Gln↓(Ser,Ala,Gly);
当新冠病毒的酶类蛋白为PLPro蛋白酶时，底物蛋白上的X1-X2部分为Leu-Xaa-Gly-Gly-Xaa，PLpro蛋白酶的底物裂解位点为Leu-Xaa-Gly-Gly↓Xaa。When the enzyme protein of the new coronavirus is PLPro protease, the X1-X2 part on the substrate protein is Leu-Xaa-Gly-Gly-Xaa, and the substrate cleavage site of PLpro protease is Leu-Xaa-Gly-Gly↓Xaa .
本发明的另一技术方案是：一种新冠病毒检测卡，其特征在于：包括检测卡本体，在检测卡本体上设置有样品孔，在样品孔的内壁上设置有指示细胞。Another technical solution of the present invention is: a new coronavirus detection card, which is characterized by: including a detection card body, a sample hole is provided on the detection card body, and indicator cells are provided on the inner wall of the sample hole.
进一步的：所述检测卡本体为采用光学透明聚苯乙烯材料制备。Further: the detection card body is made of optically transparent polystyrene material.
本发明的另一技术方案是：一种新冠病毒检测试剂盒，包括采样管和检测卡，其特征在于：所述检测卡上设置有样品孔，在样品孔的内壁上设置有培养细胞，所述采样管的保存液内含有如权利要求1所述的底物蛋白。Another technical solution of the present invention is: a new coronavirus detection kit, including a sampling tube and a detection card, which is characterized in that: the detection card is provided with a sample hole, and cultured cells are provided on the inner wall of the sample hole, so The storage solution of the sampling tube contains the substrate protein as claimed in claim 1.
进一步的：培养细胞为Vero细胞。Further: the cultured cells are Vero cells.
本发明的另一技术方案是：一种防护口罩，包括罩体，所述罩体由内到外依次为无纺布微孔滤膜内层、熔喷布层和无纺布微孔滤膜外层，其特征在于：在熔喷布层内侧设置有一层指示细胞。Another technical solution of the present invention is: a protective mask, including a cover body, which is composed of an inner layer of non-woven microporous filter membrane, a melt-blown cloth layer and a non-woven microporous filter membrane from the inside to the outside. The outer layer is characterized by: a layer of indicator cells is provided inside the melt-blown cloth layer.
进一步的：所述无纺布微孔滤膜层上的微孔孔径大小在病毒和细胞之间。Further: the size of the micropores on the non-woven microporous filter membrane layer is between that of viruses and cells.
本发明的优点是：本发明方法中，通过对活病毒的培养，增加病毒的含量，提高检测灵敏度，仅需一个活病毒就可以被检出；由于本发明方法对新冠病毒特有的酶类蛋白质具有较高的选择特异性，其检测的准确率较抗原检测更高；除了具有高灵敏度和高准确率以外，检测试剂盒还具有可自测的优点，避免聚集采样时的交叉感染，而防护口罩具有实时监测能力，可在感染者具有传播病毒能力后的一个检测周期内，迅速识别出感染者，又可以快速识别出部分病毒接触者，一定程度上可避免病毒传播，有效的起到防疫效果。The advantages of the present invention are: in the method of the present invention, by cultivating live viruses, the content of the virus is increased, and the detection sensitivity is improved, and only one live virus can be detected; because the method of the present invention can detect the unique enzyme proteins of the new coronavirus, It has high selection specificity and its detection accuracy is higher than that of antigen detection. In addition to its high sensitivity and accuracy, the detection kit also has the advantage of self-testing, which avoids cross-infection during collective sampling and protects against infection. Masks have real-time monitoring capabilities and can quickly identify infected people within a detection cycle after the infected person has the ability to spread the virus. They can also quickly identify some virus contacts, which can avoid the spread of the virus to a certain extent and effectively prevent the epidemic. Effect.
具体实施方式Detailed ways
下面通过使用方法及原理对本发明方案做进一步说明。The solution of the present invention will be further explained below through the usage method and principle.
本发明公开了一种新冠病毒检测方法：制备具有针对性的指示细胞，利用指示细胞合成的底物蛋白与新冠病毒的酶类蛋白接触后显色或者形成凝胶达到检测的目的；其中，所述指示细胞通过如下方法制备：The invention discloses a novel coronavirus detection method: preparing targeted indicator cells, utilizing the substrate protein synthesized by the indicator cells to contact the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection; wherein, The indicator cells are prepared by the following method:
选取细胞膜上含有ACE2受体的细胞作为制备指示细胞的母细胞，Select cells containing ACE2 receptors on their cell membranes as mother cells for preparing indicator cells.
通过基因编辑方式修改母细胞的基因组序列，从而在该细胞中表达出如下结构的底物蛋白：Z-X1-X2-Y；其中：The genome sequence of the mother cell is modified through gene editing, so that the substrate protein with the following structure is expressed in the cell: Z-X1-X2-Y; where:
Z-为指示部分：指示部分自身或与外部介入物质反应，显色或形成凝胶；Z- is the indicator part: the indicator part reacts with itself or with external intervening substances, develops color or forms a gel;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段，且该底物裂解位点可与新冠病毒中的酶类蛋白反应并发生断裂；X1-X2 is the connecting part: a polypeptide chain fragment containing a substrate cleavage site, and the substrate cleavage site can react with the enzyme protein in the new coronavirus and cause cleavage;
-Y为控制部分：具有遮盖Z或抑制Z活性的作用，连接状态下，使Z不指示，即使Z不显色和不形成凝胶。-Y is the control part: it has the function of covering Z or inhibiting the activity of Z. When connected, Z does not indicate, even if Z does not develop color and does not form a gel.
根据Z-以及-Y的不同，该底物蛋白共有两种架构形式，分别为：According to the difference between Z- and -Y, the substrate protein has two architectural forms, namely:
架构1：Z-为指示部分：显色或形成凝胶；Structure 1: Z- is the indicated part: color development or gel formation;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段；X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
-Y为控制部分：连接状态下，使Z不指示。-Y is the control part: in the connected state, Z is not indicated.
架构2：Z-为指示部分：与外部介入物质反应，显色或形成凝胶；Structure 2: Z- is the indicator part: reacts with external intervening substances to develop color or form a gel;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段；X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
-Y为控制部分：连接状态下，使Z不指示。-Y is the control part: in the connected state, Z is not indicated.
当病毒的培养细胞为指示细胞时，底物蛋白无需人工添加，上述方法的病毒的检测过程如下：When the virus culture cells are indicator cells, the substrate protein does not need to be added manually. The virus detection process using the above method is as follows:
步骤1，病毒与指示细胞接触后，通过指示细胞上的ACE2受体进入指示细胞内，利用指示细胞的物质合成自身的蛋白质，该蛋白质包括结构蛋白、酶类蛋白和辅助蛋白；Step 1: After the virus contacts the indicator cell, it enters the indicator cell through the ACE2 receptor on the indicator cell and uses the materials of the indicator cell to synthesize its own proteins, which include structural proteins, enzyme proteins and auxiliary proteins;
步骤2，合成的酶类蛋白会催化指示细胞上的底物蛋白发生化学反应，断开Y，释放出Z；Step 2: The synthesized enzyme protein will catalyze a chemical reaction with the substrate protein on the indicator cell, disconnect Y, and release Z;
步骤3，当底物蛋白为架构1时，Z显色或者形成凝胶；当底物蛋白为架构2时，Z与外部介入物质反应，显色或者形成凝胶；Step 3: When the substrate protein is structure 1, Z develops color or forms a gel; when the substrate protein is structure 2, Z reacts with external intervening substances, develops color or forms a gel;
步骤4，显色或者形成凝胶的物质从指示细胞中被释放出来，指示区域也随之变大，从而增加了识别度；Step 4: The color-developing or gel-forming substance is released from the indicator cells, and the indicator area becomes larger, thereby increasing the recognition degree;
步骤5，细胞被裂解后，大量的病毒及病毒的酶类蛋白从指示细胞中被释放出来，催化更多指示细胞上的底物蛋白发生化学反应，显色或者形成凝胶，从而进一步的增加识别度。Step 5: After the cells are lysed, a large amount of viruses and viral enzyme proteins are released from the indicator cells, catalyzing more chemical reactions of substrate proteins on the indicator cells, developing colors or forming gels, thereby further increasing Recognition.
当病毒的培养细胞不是指示细胞时，底物蛋白需人工添加，上述方法的病毒的检测过程如下：When the virus culture cells are not indicator cells, the substrate protein needs to be added manually. The virus detection process using the above method is as follows:
步骤1，病毒与培养细胞接触后，通过细胞膜上的ACE2受体进入细胞内，利用该细胞的物质合成自身的蛋白质，该蛋白质包括结构蛋白、酶类蛋白和辅助蛋白；Step 1: After the virus comes into contact with the cultured cells, it enters the cells through the ACE2 receptor on the cell membrane and uses the cell's materials to synthesize its own proteins, which include structural proteins, enzyme proteins and auxiliary proteins;
步骤2，合成的酶类蛋白与底物蛋白接触后，会发生化学反应，断开Y，释放出Z；Step 2: After the synthesized enzyme protein comes into contact with the substrate protein, a chemical reaction will occur, breaking Y and releasing Z;
步骤3，当底物蛋白为架构1时，Z显色或者形成凝胶；当底物蛋白为架构2时，Z与外部介入物质反应，显色或者形成凝胶。Step 3: When the substrate protein is Structure 1, Z develops color or forms a gel; when the substrate protein is Structure 2, Z reacts with external intervening substances, develops color or forms a gel.
优选的：所述新冠病毒中的Mpro水解酶与X1-X2上的底物裂解位点反应，从而使底物蛋白中的Y断开，底物蛋白为架构1时，Y失去遮盖Z的作用，Z暴露显色或者形成凝胶；底物蛋白为架构2时，Y失去抑制Z活性的作用，Z与外部介入物质反应，显色或者形成凝胶。Preferably: Mpro hydrolase in the new coronavirus reacts with the substrate cleavage site on X1-X2, thereby disconnecting Y in the substrate protein. When the substrate protein is structure 1, Y loses its function of covering Z. , Z is exposed to develop color or form a gel; when the substrate protein is structure 2, Y loses its function of inhibiting Z activity, and Z reacts with external intervening substances, and develops color or forms a gel.
优选的：底物蛋白上的X1-X2部分为Leu-Gln-（Ser，Ala，Gly），底物裂解位点为Leu-Gln↓（Ser，Ala，Gly）；Preferred: The X1-X2 part on the substrate protein is Leu-Gln- (Ser, Ala, Gly), and the substrate cleavage site is Leu-Gln↓ (Ser, Ala, Gly);
本发明还公开了一种防护口罩，包括罩体，所述罩体由内到外依次为无纺布微孔滤膜内层、熔喷布层和无纺布微孔滤膜外层，其中：在熔喷布层内侧设置有一层指示细胞，所述无纺布微孔滤膜层上的微孔孔径大小在病毒和细胞之间。The invention also discloses a protective mask, which includes a cover body, which is composed of an inner layer of non-woven microporous filter membrane, a melt-blown cloth layer and an outer layer of non-woven microporous filter membrane from the inside to the outside, wherein : A layer of indicator cells is provided on the inside of the melt-blown cloth layer, and the size of the micropores on the non-woven microporous filter membrane layer is between the virus and the cells.
优选的；熔喷布层设为两层，内层为附有一层指示细胞的熔喷布，外层为被驻极后的不含指示细胞的熔喷布，适用于处在病毒环境的工作人员使用；Preferably, the melt-blown cloth layer is made into two layers, the inner layer is a melt-blown cloth with a layer of indicator cells attached, and the outer layer is an electret melt-blown cloth without indicator cells, which is suitable for work in a virus environment. Personnel use;
优选的：防护口罩应储存于2～8℃条件下，使用时长不应超过24h，需在低于40℃的环境温度下使用；Preferred: Protective masks should be stored at 2 to 8°C, used for no more than 24 hours, and used at an ambient temperature below 40°C;
优选的；在防护口罩安装芯片，用于向疾控中心报告不合格结果，或者提供疾控中心的二维码，便于感染者上报结果；Preferred; install a chip in the protective mask to report unqualified results to the Centers for Disease Control and Prevention, or provide a QR code from the Centers for Disease Control and Prevention to facilitate infected people reporting results;
优选的；防护口罩外侧添加一层保温层，使防护口罩的使用温度更广泛。Preferably, a thermal insulation layer is added to the outside of the protective mask to make the protective mask applicable to a wider range of temperatures.
本发明还公开了一种新冠病毒检测卡，在检测卡上设置有样品孔，在样品孔的内壁上设置有指示细胞。The invention also discloses a new coronavirus detection card. The detection card is provided with a sample hole, and indicator cells are provided on the inner wall of the sample hole.
优选的：所述检测卡采用光学透明聚苯乙烯材料制备；Preferably: the detection card is made of optically transparent polystyrene material;
优选的：所述检测卡应储存于2～8℃条件下；Preferably: the test card should be stored at 2-8°C;
优选的；在检测卡上安装芯片，用于向疾控中心报告不合格结果，或者提供疾控中心的二维码，便于感染者上报结果。Preferably, a chip is installed on the test card to report unqualified results to the Centers for Disease Control and Prevention, or a QR code from the Centers for Disease Control and Prevention is provided to facilitate infected people reporting results.
本发明还公开了一种病毒检测试剂盒，包括采样管和检测卡，所述采样管的保存液内含有底物蛋白，所述检测卡上设置有样品孔，在样品孔的内壁上设置有培养细胞。The invention also discloses a virus detection kit, which includes a sampling tube and a detection card. The preservation solution of the sampling tube contains substrate protein. The detection card is provided with a sample hole, and the inner wall of the sample hole is provided with a Cultured cells.
优选的：所述检测卡采用光学透明聚苯乙烯材料制备；Preferably: the detection card is made of optically transparent polystyrene material;
优选的：所述培养细胞为Vero细胞；Preferably: the cultured cells are Vero cells;
优选的：所述病毒检测试剂盒应储存于2～8℃条件下；Preferably: the virus detection kit should be stored at 2-8°C;
下面通过检测原理对本方案进一步说明。This solution will be further explained below through the detection principle.
本发明公开了一种新冠病毒检测方法，制备具有针对性的指示细胞，利用指示细胞合成的底物蛋白与新冠病毒的酶类蛋白接触后显色或者形成凝胶达到检测的目的；其中，所述指示细胞的详细制备方法如下：The invention discloses a novel coronavirus detection method, which involves preparing targeted indicator cells and utilizing the substrate protein synthesized by the indicator cells to contact the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection; wherein, The detailed preparation method of the indicator cells is as follows:
1，母细胞的选择：以细胞膜上含有ACE2受体的细胞作为制备指示细胞的母细胞；1. Selection of mother cells: cells containing ACE2 receptors on their cell membranes are used as mother cells for preparing indicator cells;
2，设计底物：底物蛋白的结构为Z-X1-X2-Y，2. Design substrate: The structure of the substrate protein is Z-X1-X2-Y.
架构1：Z-为指示部分：显色或形成凝胶；Structure 1: Z- is the indicated part: color development or gel formation;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段；X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
-Y为控制部分：连接状态下，使Z不指示。-Y is the control part: in the connected state, Z is not indicated.
该底物蛋白可被新冠病毒的酶类蛋白催化分解，生成Z-X1和X2-Y，Y失去了遮盖Z的作用，使Z-X1显色或者形成凝胶。This substrate protein can be catalyzed and decomposed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y. Y loses its function of covering Z, causing Z-X1 to develop color or form a gel.
架构2：Z-为指示部分：与外部介入物质反应，显色或形成凝胶；Structure 2: Z- is the indicator part: reacts with external intervening substances to develop color or form a gel;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段；X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
-Y为控制部分：连接状态下，使Z不指示。-Y is the control part: in the connected state, Z is not indicated.
该底物蛋白可被新冠病毒的酶类蛋白催化分解，生成Z-X1和X2-Y，Y失去了抑制Z活性的作用，Z-X1与外部介入物质发生化学反应，显色或者形成凝胶；This substrate protein can be catalyzed and decomposed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y. Y loses its function of inhibiting Z activity. Z-X1 chemically reacts with external intervening substances to develop color or form a gel. ;
3，通过基因编辑技术修改母细胞的基因组序列，使上述底物蛋白能在该细胞中被充分表达。3. Modify the genome sequence of the mother cell through gene editing technology so that the above substrate protein can be fully expressed in the cell.
优选的：所述母细胞为Vero细胞；Preferably: the mother cell is Vero cell;
优选的：底物蛋白为架构1，所述底物蛋白为通过一段多肽链，将血红蛋白α链与新冠病毒多蛋白nsp5-11，以共价键形式连接在一起形成的蛋白质（以下称为血红蛋白α链-nsp5-11）,Preferably: the substrate protein is structure 1, which is a protein formed by covalently connecting the hemoglobin α chain and the new coronavirus polyprotein nsp5-11 through a polypeptide chain (hereinafter referred to as hemoglobin α chain-nsp5-11),
优选的：底物蛋白为架构2，所述底物蛋白为通过一段多肽链，将凝血酶双链与新冠病毒多蛋白nsp5-11，以共价键形式连接在一起形成的蛋白质（以下称为凝血酶-nsp5-11）；Preferably: the substrate protein is structure 2, and the substrate protein is a protein formed by covalently connecting the thrombin double chain and the new coronavirus polyprotein nsp5-11 through a polypeptide chain (hereinafter referred to as thrombin-nsp5-11);
优选的，所述基因编辑技术为CRISPR-Cas9基因编辑技术。Preferably, the gene editing technology is CRISPR-Cas9 gene editing technology.
所述病毒检测方法是一种活病毒培养法，系通过显色或者形成凝胶来判断检测样品是否含有病毒。The virus detection method is a live virus culture method, which determines whether the test sample contains the virus through color development or gel formation.
当培养细胞为指示细胞时，底物蛋白无需人工添加，检测样品若含有病毒，则病毒会通过指示细胞上的受体进入指示细胞内，利用指示细胞的物质合成自身的蛋白质，包括结构蛋白、酶类蛋白和辅助蛋白；合成的酶类蛋白会催化指示细胞上的底物蛋白发生化学反应，断开Y，释放出Z；当底物蛋白为架构1时，Z显色或者形成凝胶；当底物蛋白为架构2时，Z与外部介入物质反应，显色或者形成凝胶；显色或者形成凝胶的物质从指示细胞中被释放出来，指示区域也随之变大，从而增加了识别度；细胞被裂解后，大量的病毒及病毒的酶类蛋白被释放出来，催化更多指示细胞上的底物蛋白发生化学反应，显色或者形成凝胶，从而进一步的增加识别度；When the cultured cells are indicator cells, the substrate protein does not need to be added artificially. If the test sample contains a virus, the virus will enter the indicator cell through the receptor on the indicator cell and use the materials of the indicator cell to synthesize its own proteins, including structural proteins, Enzyme proteins and auxiliary proteins; the synthesized enzyme proteins will catalyze the chemical reaction of the substrate protein on the indicator cell, break Y, and release Z; when the substrate protein is structure 1, Z will develop color or form a gel; When the substrate protein is structure 2, Z reacts with external intervening substances to develop color or form a gel; the substance that develops color or forms a gel is released from the indicator cells, and the indicator area becomes larger, thereby increasing the Recognition: After the cells are lysed, a large amount of viruses and viral enzyme proteins are released, which catalyze more chemical reactions with substrate proteins on the indicator cells, develop colors or form gels, thereby further increasing the recognition;
当培养细胞不是指示细胞时，底物蛋白需人工添加，检测样品若含有病毒，则病毒与培养细胞接触后，通过细胞膜上的ACE2受体进入细胞内，利用该细胞的物质合成自身的蛋白质，该蛋白质包括结构蛋白、酶类蛋白和辅助蛋白；合成的酶类蛋白与底物蛋白接触后，会发生化学反应，断开Y，释放出Z；当底物蛋白为架构1时，Z显色或者形成凝胶，当底物蛋白为架构2时，Z与外部介入物质反应，显色或者形成凝胶；When the cultured cells are not indicator cells, the substrate protein needs to be added manually. If the test sample contains a virus, after the virus comes into contact with the cultured cells, it enters the cell through the ACE2 receptor on the cell membrane and uses the cell's materials to synthesize its own proteins. The protein includes structural proteins, enzyme proteins and auxiliary proteins; when the synthesized enzyme protein comes into contact with the substrate protein, a chemical reaction will occur, breaking Y and releasing Z; when the substrate protein is structure 1, Z will develop color Or form a gel. When the substrate protein is structure 2, Z reacts with external intervening substances to develop color or form a gel;
若检测样品不含病毒，则不会发生颜色变化或者形成凝胶。If the test sample does not contain viruses, there will be no color change or gel formation.
优选的，所述检测样品为通过口鼻呼出的气体或通过检查拭子收集到的样品；Preferably, the test sample is the gas exhaled through the mouth and nose or a sample collected by examination swab;
优选的，所述病毒的培养细胞为指示细胞；Preferably, the cultured cells of the virus are indicator cells;
优选的，所述受体为ACE2；Preferably, the receptor is ACE2;
优选的，所述酶类蛋白为Mpro水解酶；Preferably, the enzyme protein is Mpro hydrolase;
优选的，底物蛋白为架构1时，所述底物蛋白为血红蛋白α链-nsp5-11，该底物蛋白的血红素基团被nsp5-11挡住而不显色或显浅红色，该底物蛋白可被Mpro水解，释放出血红蛋白α多肽链而显红色；Preferably, when the substrate protein is Structure 1, the substrate protein is hemoglobin α chain-nsp5-11. The heme group of the substrate protein is blocked by nsp5-11 and does not develop color or appears light red. The protein can be hydrolyzed by Mpro, releasing the hemoglobin α polypeptide chain and appearing red;
优选的，底物蛋白为架构2时，所述底物蛋白为凝血酶-nsp5-11，该底物蛋白为凝血酶原，可被Mpro水解，释放出凝血酶的两条链，并使其由一个二硫键相连，生成活化的凝血酶。Preferably, when the substrate protein is Structure 2, the substrate protein is thrombin-nsp5-11. The substrate protein is prothrombin, which can be hydrolyzed by Mpro to release the two chains of thrombin and make it Connected by a disulfide bond to generate activated thrombin.
本发明所述的防护口罩，是用附有一层指示细胞的熔喷布制成的口罩，包括口罩体、鼻夹和口罩带；所述口罩体由内到外依次为无纺布微孔滤膜内层、熔喷布层和无纺布微孔滤膜外层，所述无纺布是通过气流或者机械成网，经过水刺、针刺或者热轧加固形成的无编织布料；所述无纺布微孔滤膜层是利用高分子化学材料，致孔添加剂经特殊处理后涂抹在无纺布上制作而成，其孔径大小介于病毒和细胞之间，允许病毒通过但会禁止细胞通过；所述熔喷布上附有一层指示细胞；所述鼻夹由可弯折的塑性材料制成；所述口罩带由棉纶线和氨纶线针织而成。The protective mask of the present invention is a mask made of melt-blown cloth with a layer of indicator cells, and includes a mask body, a nose clip and a mask belt; the mask body is composed of non-woven microporous filters from the inside to the outside. The inner layer of the membrane, the melt-blown cloth layer and the outer layer of the non-woven microporous filter membrane. The non-woven fabric is a non-woven fabric formed by air flow or mechanical meshing and reinforced by hydroentanglement, acupuncture or hot rolling; The non-woven microporous filter membrane layer is made of polymer chemical materials and porogenic additives are specially treated and then applied to the non-woven fabric. Its pore size is between viruses and cells, allowing viruses to pass but preventing cells from passing through. Through; a layer of indicator cells is attached to the melt-blown cloth; the nose clip is made of bendable plastic material; and the mask strap is knitted from cotton thread and spandex thread.
优选的，防护口罩的存储条件为2~8℃；Preferably, the storage condition of protective masks is 2~8℃;
优选的，防护口罩的使用时长不应超过24h；Preferably, the use time of protective masks should not exceed 24 hours;
优选的，病毒环境工作人员使用的防护口罩的熔喷布层共有两层，内层为附有一层指示细胞的熔喷布，外层为经驻极后的不含指示细胞的熔喷布，其他人员使用的防护口罩的熔喷布层仅含有一层，为附有一层指示细胞的熔喷布；Preferably, the protective mask used by virus environment workers has two layers of melt-blown cloth. The inner layer is a melt-blown cloth with a layer of indicator cells attached, and the outer layer is an electret melt-blown cloth without indicator cells. The melt-blown cloth layer of the protective masks used by other personnel only contains one layer, which is a melt-blown cloth with a layer of indicator cells attached;
优选的，防护口罩应在不高于40℃的环境条件下使用，温度过低时，需在防护口罩外侧粘一层保温层。Preferably, the protective mask should be used under environmental conditions not higher than 40°C. When the temperature is too low, a layer of insulation needs to be glued to the outside of the protective mask.
本方案所述的检测卡，可做为病毒检测试剂盒的一部分，所述的病毒检测试剂盒包括检查拭子，采样管、检测卡和滴管，所述检查拭子由塑料杆（如聚苯烯）和拭子头（如人造纤维）组成，用于样品的采集；所述采样管内含有保存液，用于收集检查拭子采集的样品；所述检测卡设有样品孔，样品孔臂上附有一层指示细胞；所述滴管，用于将采样管中的样品滴入检测卡的样品孔中。The test card described in this solution can be used as part of a virus detection kit. The virus detection kit includes a test swab, a sampling tube, a test card and a dropper. The test swab is made of a plastic rod (such as a polyethylene rod). Styrene) and a swab head (such as artificial fiber), used for sample collection; the sampling tube contains a preservation solution, used to collect the samples collected by the inspection swab; the test card is provided with a sample hole, and a sample hole arm A layer of indicator cells is attached; the dropper is used to drop the sample in the sampling tube into the sample hole of the detection card.
病毒检测试剂盒的使用方法：取出检查拭子，将拭子头插入鼻孔或者咽部，左旋转三圈，再右旋转三圈；将拭子头插入采样管的保存液中，反复搅拌，然后用手挤压采样管外壁，将拭子头上的液体全部挤进采样管中的保存液中；取出检测卡，将采样管中的保存液滴入检测卡中的样品孔中，静置4h；若颜色发生变化或者形成凝胶，则判定为阳性，若颜色未改变或者未形成凝胶，则判定为阴性。使用后的试剂盒应放入医疗废弃垃圾袋中，经统一高压灭菌后丢弃。How to use the virus detection kit: Take out the test swab, insert the swab head into the nostril or pharynx, turn it three times to the left, and then turn it to the right three times; insert the swab head into the preservation solution of the sampling tube, stir repeatedly, and then Squeeze the outer wall of the sampling tube with your hands, squeeze all the liquid on the swab head into the preservation solution in the sampling tube; take out the test card, drop the preservation solution in the sampling tube into the sample hole in the test card, and let it sit for 4 hours ; If the color changes or gel is formed, it is judged as positive; if the color does not change or gel is not formed, it is judged as negative. Used test kits should be placed in medical waste bags and discarded after uniform autoclaving.
优选的：所述检查拭子为棉拭子；Preferably: the inspection swab is a cotton swab;
优选的：底物蛋白为血红蛋白α链-nsp5-11时，所述采样管内的保存液为DMEM液体培养基；Preferably: when the substrate protein is hemoglobin α chain-nsp5-11, the preservation solution in the sampling tube is DMEM liquid culture medium;
优选的：底物蛋白为凝血酶-nsp5-11时，所述采样管内的保存液位为含有钙离子、纤维蛋白原的DMEM液态培养基；Preferably: when the substrate protein is thrombin-nsp5-11, the storage liquid level in the sampling tube is DMEM liquid medium containing calcium ions and fibrinogen;
优选的，所述采样管兼有滴管功能，替代普通滴管；Preferably, the sampling tube also has the function of a dropper, replacing an ordinary dropper;
优选的，所述病毒检测试剂盒应在2~8℃条件下储存。Preferably, the virus detection kit should be stored at 2 to 8°C.
本方案所述的检测试剂盒，包括检查拭子，采样管、检测卡、裂解液管和滴管，所述检查拭子由塑料杆（如聚苯烯）和拭子头（如人造纤维）组成，用于样品的采集；所述采样管内含有保存液，用于收集检查拭子采集的样品；所述检测卡设有样品孔，样品孔臂上附有一层培养细胞；所述滴管，用于将采样管中的样品滴入检测卡的样品孔中；所述裂解液管内含有裂解液，用于裂解培养细胞，将病毒及病毒蛋白从培养细胞中释放出来。The detection kit described in this solution includes an inspection swab, a sampling tube, a test card, a lysate tube and a dropper. The inspection swab is made of a plastic rod (such as polystyrene) and a swab head (such as artificial fiber) Composed of, used for sample collection; the sampling tube contains a preservation solution for collecting samples collected by inspection swabs; the detection card is provided with a sample hole, and a layer of cultured cells is attached to the sample hole arm; the dropper, It is used to drop the sample in the sampling tube into the sample hole of the detection card; the lysis liquid tube contains lysis liquid, which is used to lyse the cultured cells and release the virus and viral proteins from the cultured cells.
病毒检测试剂盒的使用方法：取出检查拭子，将拭子头插入鼻孔或者咽部，左旋转三圈，再右旋转三圈；将拭子头插入采样管的保存液中，反复搅拌，然后用手挤压采样管外壁，将拭子头上的液体全部挤进采样管中的保存液中；取出检测卡，将采样管中的保存液滴入检测卡中的样品孔中，静置2h；向样品孔中滴加裂解液，再静置0.5h；若颜色发生变化或者形成凝胶，则判定为阳性，若颜色未改变或者未形成凝胶，则判定为阴性。使用后的试剂盒应放入医疗废弃垃圾袋中，经统一高压灭菌后丢弃。How to use the virus detection kit: Take out the test swab, insert the swab head into the nostril or pharynx, turn it three times to the left, and then turn it to the right three times; insert the swab head into the preservation solution of the sampling tube, stir repeatedly, and then Squeeze the outer wall of the sampling tube with your hands and squeeze all the liquid on the swab head into the preservation solution in the sampling tube; take out the test card, drop the preservation solution in the sampling tube into the sample hole in the test card, and let it sit for 2 hours. ; Drop the lysis solution into the sample well and let it stand for 0.5 hours; if the color changes or a gel is formed, it is judged as positive; if the color does not change or a gel is not formed, it is judged as negative. Used test kits should be placed in medical waste bags and discarded after uniform autoclaving.
优选的：所述培养细胞为Vero细胞；Preferably: the cultured cells are Vero cells;
优选的：所述检查拭子为棉拭子；Preferably: the inspection swab is a cotton swab;
优选的：底物蛋白为架构1时，所述采样管内的保存液为含有血红蛋白α链-nsp5-11的DMEM液体培养基；Preferably: when the substrate protein is Structure 1, the preservation solution in the sampling tube is DMEM liquid culture medium containing hemoglobin α chain-nsp5-11;
优选的：底物蛋白为架构2时，所述采样管内的保存液位为含有凝血酶-nsp-5-11和纤维蛋白原的DMEM液态培养基；Preferably: when the substrate protein is Structure 2, the storage liquid level in the sampling tube is DMEM liquid medium containing thrombin-nsp-5-11 and fibrinogen;
优选的：所述采样管兼有滴管功能，替代普通滴管；Preferably: the sampling tube also has the function of a dropper, replacing an ordinary dropper;
优选的：所述裂解液为不含SDS的改良版 RIPA 裂解液。Preferably: the lysis solution is an improved version of RIPA lysis solution that does not contain SDS.
优选的，所述病毒检测试剂盒应在2~8℃条件下储存。Preferably, the virus detection kit should be stored at 2 to 8°C.
实施例Example
一、指示细胞的制备1. Preparation of indicator cells
1，母细胞的选择：以细胞膜上含有ACE2受体的细胞作为制备指示细胞的母细胞，本次示例以Vero细胞作为母细胞；1. Selection of mother cells: cells containing ACE2 receptors on their cell membranes are used as mother cells for preparing indicator cells. In this example, Vero cells are used as mother cells;
2，设计底物：底物蛋白的结构为Z-X1-X2-Y，2. Design substrate: The structure of the substrate protein is Z-X1-X2-Y.
其中：Z-为指示部分：有色蛋白或者多肽；Among them: Z- indicates the part: colored protein or polypeptide;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段；X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
-Y为控制部分：连接状态下，使Z不指示。-Y is the control part: in the connected state, Z is not indicated.
该底物蛋白可被新冠病毒的酶类蛋白催化分解，生成Z-X1和X2-Y，释放出有色蛋白而发生颜色变化，本示例的Z链为血红蛋白α链，X链为“-Trp-Ala-Leu-Gln-Gly-”，Y链为新冠病毒多蛋白nsp5-11；This substrate protein can be catalyzed and decomposed by the enzyme protein of the new coronavirus to generate Z-X1 and Ala-Leu-Gln-Gly-", the Y chain is the new coronavirus polyprotein nsp5-11;
3，通过基因编辑技术修改母细胞的基因组序列，使上述底物蛋白在该细胞中被充分表达，本示例通过CRISPR-Cas9基因编辑技术修改合成血红蛋白α链的基因组的启动子的基因序列，并在该基因组的末端依次添加合成“-Trp-Ala-Leu-Gln-Gly-”多肽链片段的基因序列、合成新冠病毒多蛋白nsp5-11的基因序列和poly（A）尾，从而诱导Vero细胞合成血红蛋白α链-nsp5-11，该蛋白的nsp5-11挡住了血红蛋白α链的血红素基团而使其不显色或显浅红色，通过验证，确保Mpro水解酶可以快速水解血红蛋白α链-nsp5-11，释放出血红蛋白α多肽链而显红色，必要时可修改合成X1-X2链的基因序列，提高Mpro水解酶水解血红蛋白α链-nsp5-11的能力，该基因编辑后的新细胞即为指示细胞。其中，CRISPR-Cas9基因编辑技术为现有技术，不再进行详细叙述。3. Modify the genome sequence of the mother cell through gene editing technology so that the above-mentioned substrate protein is fully expressed in the cell. In this example, CRISPR-Cas9 gene editing technology is used to modify the gene sequence of the promoter of the genome that synthesizes the hemoglobin α chain, and At the end of the genome, the gene sequence for synthesizing the "-Trp-Ala-Leu-Gln-Gly-" polypeptide chain fragment, the gene sequence for synthesizing the new coronavirus polyprotein nsp5-11 and the poly (A) tail are sequentially added to induce Vero cells. Synthetic hemoglobin α chain-nsp5-11. The nsp5-11 of this protein blocks the heme group of the hemoglobin α chain so that it does not develop color or appears light red. Through verification, it is ensured that Mpro hydrolase can quickly hydrolyze the hemoglobin α chain- nsp5-11, releases the hemoglobin α polypeptide chain and appears red. If necessary, the gene sequence for synthesizing the X1-X2 chain can be modified to improve the ability of Mpro hydrolase to hydrolyze the hemoglobin α chain-nsp5-11. The new cells after editing this gene are for indicator cells. Among them, CRISPR-Cas9 gene editing technology is an existing technology and will not be described in detail.
二、防护口罩的制备2. Preparation of protective masks
1，指示细胞的培养：将上述指示细胞，在培养瓶中，在富含有铁离子、血红素、MEM、胎牛血清和两种抗生素等物质的培养基中培养，37℃培养72小时，传代1次，再培养72小时；1. Culture of indicator cells: Cultivate the above indicator cells in a culture flask in a medium rich in iron ions, heme, MEM, fetal bovine serum and two antibiotics, and culture at 37°C for 72 hours. Passage once and culture for another 72 hours;
2，用PBS溶液冲洗3次，将胰酶加入培养瓶，3分钟内去掉胰酶，放入37℃培养箱消化，每隔10秒左右轻轻晃动培养瓶，直到观察到有流沙状细胞滑动；2. Rinse 3 times with PBS solution, add trypsin to the culture bottle, remove the trypsin within 3 minutes, put it into a 37°C incubator for digestion, and gently shake the culture bottle every 10 seconds or so until quicksand-like cells are observed. ;
3，指示细胞消化好后，转移至富含有铁离子、血红素、MEM、胎牛血清和两种抗生素等物质的培养基中，将熔喷布浸泡在上述培养基中，37℃培养24小时；3. After the indicator cells are digested, transfer them to a medium rich in iron ions, heme, MEM, fetal bovine serum and two antibiotics. Soak the melt-blown cloth in the above medium and incubate at 37°C for 24 Hour;
4，取出熔喷布，用PBS溶液清洗3次，再将其浸泡至含有牛血清白蛋白、蔗糖、MEM等营养物的保护液中，37℃浸泡2小时，再将上述熔喷布取出，风干；4. Take out the melt-blown cloth, wash it 3 times with PBS solution, then soak it in a protective solution containing bovine serum albumin, sucrose, MEM and other nutrients, soak it at 37°C for 2 hours, and then take out the melt-blown cloth. air dry;
5，防护口罩的制备：将无菌的无纺布微孔滤膜内层、熔喷布层和无纺布微孔滤膜外层堆叠在一起，通过高频焊接制作成口罩主体，随之在流水线上裁剪成单个口罩。在口罩的一侧安装鼻夹，通过卷边缝合，然后在口罩边缘再穿上耳绳，即得防护口罩；所述熔喷布层为上述方法制备的附有一层指示细胞的熔喷布，可在上述熔喷布外侧，再添加一层不含指示细胞的熔喷布，供病毒环境的工作人员使用，该层油喷布可用驻极设备驻极，内侧涂一层甘油保护剂。5. Preparation of protective masks: Stack the inner layer of the sterile non-woven microporous filter membrane, the melt-blown cloth layer and the outer layer of the non-woven microporous filter membrane together, and make the main body of the mask through high-frequency welding. Cut into individual masks on the assembly line. Install a nose clip on one side of the mask, sew it by crimping, and then put on ear strings on the edge of the mask to obtain a protective mask; the melt-blown cloth layer is a melt-blown cloth prepared by the above method and attached with a layer of indicator cells. A layer of melt-blown cloth without indicator cells can be added to the outside of the above-mentioned melt-blown cloth for use by workers working in virus environments. This layer of oil-sprayed cloth can be electret with electret equipment, and a layer of glycerin protective agent can be applied on the inside.
三、病毒检测试剂盒的制备3. Preparation of virus detection kits
1，采用光学透明纯聚苯乙烯材料制备检测卡载体，设有样品孔；1. The detection card carrier is made of optically transparent pure polystyrene material and is equipped with a sample hole;
2，将上述制备好的指示细胞溶液加入样品孔中，37℃培养24小时；2. Add the indicator cell solution prepared above into the sample well and incubate at 37°C for 24 hours;
3，将样品孔内的液体拍干，用PBS溶液清洗3次，加入含有牛血清白蛋白、蔗糖、MEM等营养物的保护液，37℃恒温孵育2小时，再拍干，即为检测卡；3. Pat the liquid in the sample well dry, wash it 3 times with PBS solution, add a protective solution containing bovine serum albumin, sucrose, MEM and other nutrients, incubate at 37°C for 2 hours, pat dry again, and it is the test card. ;
4，将制备好的检测卡放入密封袋内密封，将密封好的检测卡与检查拭子，采样管、滴管和使用说明书放入外包装盒内，即得病毒检测试剂盒，其中，所述采样管内的保存液为DMEM液体培养基。4. Put the prepared test card into the sealed bag and seal it. Put the sealed test card, inspection swab, sampling tube, dropper and instruction manual into the outer box to get the virus detection kit. Among them, The preservation solution in the sampling tube is DMEM liquid culture medium.
本实施例的工作原理如下：The working principle of this embodiment is as follows:
1，病毒检测口罩的工作原理：感染者呼气时将病毒呼出，部分病毒会吸附到口罩的指示细胞上，经指示细胞上的ACE2受体进入细胞，在细胞内合成自身的蛋白，其中病毒合成的Mpro水解酶将指示细胞上的底物蛋白分解，释放出血红蛋白α链，从而发生颜色变化而被识别。若口罩内侧发生颜色变化，则表明该使用者为感染者或潜在感染者；若口罩外侧发生颜色变化，则表明该使用者接触过病毒环境；长期在病毒环境工作的人员可以选择两层油喷布的口罩，外层不含指示细胞，则仅有口罩内侧会发生颜色变化。1. Working principle of virus detection masks: When an infected person exhales the virus, some of the viruses will be adsorbed to the indicator cells of the mask, enter the cells through the ACE2 receptors on the indicator cells, and synthesize their own proteins in the cells. Among them, the virus The synthesized Mpro hydrolase will decompose the substrate protein on the instruction cell and release the hemoglobin α chain, resulting in a color change and recognition. If the color changes on the inside of the mask, it indicates that the user is infected or potentially infected; if the color changes on the outside of the mask, it indicates that the user has been exposed to a virus environment; those who work in a virus environment for a long time can choose two layers of oil spray For cloth masks, the outer layer does not contain indicator cells, so only the inside of the mask will change color.
2，病毒检测试剂盒的工作原理：使用者按照说明书，可自行用检查拭子收集样品，将样品传入采样管的保存液中，制备出样品溶液，再用滴管将采样管的样品溶液滴入检测卡的样品孔中，若使用者为感染者，则样品溶液中的病毒会吸附到指示细胞上，经指示细胞上的受体进入细胞，在细胞内合成自身的蛋白，其中病毒合成的Mpro水解酶将指示细胞内的底物蛋白分解，释放出血红蛋白α链，从而发生颜色变化而被识别；若使用者不是感染者，则检测卡的样品孔不会发生颜色变化。2. Working principle of the virus detection kit: According to the instructions, the user can collect the sample with a test swab, transfer the sample into the preservation solution of the sampling tube, prepare a sample solution, and then use a dropper to transfer the sample solution from the sampling tube. Drop it into the sample hole of the test card. If the user is an infected person, the virus in the sample solution will be adsorbed to the indicator cell, enter the cell through the receptor on the indicator cell, and synthesize its own protein in the cell, in which the virus synthesizes The Mpro hydrolase will decompose the substrate protein in the instruction cell and release the hemoglobin α chain, resulting in a color change and recognition; if the user is not an infected person, the sample hole of the test card will not change color.
实施例Example
一．指示细胞的制备one. Preparation of indicator cells
1，母细胞的选择：以细胞膜上含有ACE2受体的细胞作为制备指示细胞的母细胞，本次示例以Vero细胞作为母细胞；1. Selection of mother cells: cells containing ACE2 receptors on their cell membranes are used as mother cells for preparing indicator cells. In this example, Vero cells are used as mother cells;
2，设计底物：底物蛋白的结构为Z-X1-X2-Y，2. Design substrate: The structure of the substrate protein is Z-X1-X2-Y.
其中：Z-为指示部分：与外部介入物质反应，形成凝胶；Among them: Z- is the indicator part: reacts with external intervening substances to form a gel;
X1-X2为连接部分：一段含有底物裂解位点的多肽链片段；X1-X2 is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
-Y为控制部分：连接状态下，使Z不指示。-Y is the control part: in the connected state, Z is not indicated.
该底物蛋白可被新冠病毒的酶类蛋白催化分解，生成Z-X1和X2-Y，Z-X1与外部介入物质反应，形成凝胶，本示例的Z链为凝血酶的双链，X链为“-Trp-Ala-Leu-Gln-Gly-”，Y链为新冠病毒多蛋白nsp5-11，外部介入物质为从畜禽血液里分离得到的纤维蛋白原；This substrate protein can be catalyzed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y. Z-X1 reacts with external intervening substances to form a gel. The Z chain in this example is the double chain of thrombin, and X The chain is "-Trp-Ala-Leu-Gln-Gly-", the Y chain is the new coronavirus polyprotein nsp5-11, and the external intervening substance is fibrinogen isolated from the blood of livestock and poultry;
3，通过基因编辑技术修改母细胞的基因组序列，使上述底物蛋白在该细胞中被充分表达，本示例通过CRISPR-Cas9基因编辑技术修改合成凝血酶原的基因组的启动子的基因序列，并把因子Xa的作用位点“Arg-Thr”及“Arg-Ile”对应的基因序列修改成为合成“-Trp-Ala-Leu-Gln-Gly-”的基因序列，并把因子Xa切掉的274个氨基酸残基对应的基因序列，修改成新冠病毒多蛋白nsp5-11对应的基因序列，并在凝血酶原基因组尾部添加poly（A）尾，从而诱导Vero细胞合成凝血酶-nsp5-11，该蛋白为可被Mpro活化的凝血酶原，由于nsp5-11的存在，使其成为胞内蛋白，通过验证，确保Mpro水解酶可以快速水解凝血酶-nsp5-11，诱导合成凝血酶，必要时，可修改合成X1-X2链的基因序列，提高Mpro水解酶水解凝血酶-nsp5-11的能力，该基因编辑后的新细胞即为指示细胞。其中，CRISPR-Cas9基因编辑技术为现有技术，不再进行详细叙述。3. Modify the genome sequence of the mother cell through gene editing technology so that the above substrate protein is fully expressed in the cell. In this example, CRISPR-Cas9 gene editing technology is used to modify the gene sequence of the promoter of the genome that synthesizes prothrombin, and Modify the gene sequence corresponding to the action site "Arg-Thr" and "Arg-Ile" of factor The gene sequence corresponding to the amino acid residues is modified to the gene sequence corresponding to the new coronavirus polyprotein nsp5-11, and a poly(A) tail is added to the end of the prothrombin genome, thereby inducing Vero cells to synthesize thrombin-nsp5-11. The protein is prothrombin that can be activated by Mpro. Due to the presence of nsp5-11, it becomes an intracellular protein. Through verification, it is ensured that Mpro hydrolase can quickly hydrolyze thrombin-nsp5-11 and induce the synthesis of thrombin. If necessary, The gene sequence for synthesizing the X1-X2 chain can be modified to improve the ability of Mpro hydrolase to hydrolyze thrombin-nsp5-11. The new cells after editing the gene are indicator cells. Among them, CRISPR-Cas9 gene editing technology is an existing technology and will not be described in detail.
二、病毒检测试剂盒的制备2. Preparation of virus detection kits
1，检测卡的制备：培养上述指示细胞的培养基为富含有半乳糖、甘露糖、岩藻糖、氨基已糖、唾液酸、钙离子、维生素K、MEM、胎牛血清和两种抗生素等物质的培养基，其余与实施例1相同；1. Preparation of test card: The culture medium for culturing the above indicator cells is rich in galactose, mannose, fucose, hexosamine, sialic acid, calcium ions, vitamin K, MEM, fetal bovine serum and two antibiotics The culture medium of other substances is the same as in Example 1;
2，将制备好的检测卡放入密封袋内密封，将密封好的检测卡与检查拭子，采样管、滴管和使用说明书放入外包装盒内，即得病毒检测试剂盒，其中，所述采样管内的保存液为含有钙离子、纤维蛋白原的DMEM液体培养基。2. Put the prepared test card into the sealed bag and seal it. Put the sealed test card, inspection swab, sampling tube, dropper and instruction manual into the outer packaging box to get the virus detection kit. Among them, The preservation solution in the sampling tube is DMEM liquid culture medium containing calcium ions and fibrinogen.
本实施例的工作原理如下：The working principle of this embodiment is as follows:
病毒检测试剂盒的工作原理：使用者按照说明书，可自行用检查拭子收集样品，将样品传入采样管的保存液中，制备出样品溶液，再用滴管将采样管的样品溶液滴入检测卡的样品孔中，若使用者为感染者，则样品溶液中的病毒会吸附到指示细胞上，经指示细胞上的受体进入细胞，在细胞内合成自身的蛋白，其中病毒合成的Mpro水解酶将指示细胞内的底物蛋白分解，生成并释放出活化的凝血酶，活化的凝血酶将可溶性纤维蛋白原转变成不溶的纤维蛋白，从而形成凝胶而被识别；若使用者不是感染者，则检测卡的样品孔不会形成凝胶。The working principle of the virus detection kit: According to the instructions, the user can collect the sample with a test swab, transfer the sample into the preservation solution of the sampling tube, prepare a sample solution, and then use a dropper to drop the sample solution in the sampling tube. In the sample hole of the test card, if the user is an infected person, the virus in the sample solution will be adsorbed to the indicator cell, enter the cell through the receptor on the indicator cell, and synthesize its own protein in the cell, among which the Mpro synthesized by the virus The hydrolase will decompose the substrate protein in the indicator cell, generate and release activated thrombin. The activated thrombin will convert soluble fibrinogen into insoluble fibrin, thereby forming a gel and being recognized; if the user is not infected Otherwise, the sample hole of the test card will not form gel.
实施例Example
一、载体的选择1. Selection of carriers
选取一种动植物细胞或者一种微生物（如病毒、细菌等）作为制备底物蛋白的载体，本示例以Vero细胞作为制备底物蛋白的载体。Select an animal or plant cell or a microorganism (such as a virus, bacteria, etc.) as a carrier for preparing substrate proteins. In this example, Vero cells are used as a carrier for preparing substrate proteins.
二、指示细胞的制备2. Preparation of indicator cells
1，设计底物：底物蛋白的结构为Z-X1-X2-Y，1. Design substrate: The structure of the substrate protein is Z-X1-X2-Y.
其中：Z-为指示部分：形成凝胶；Among them: Z- is the indicated part: forming gel;
X为连接部分：一段含有底物裂解位点的多肽链片段；X is the connecting part: a polypeptide chain fragment containing the substrate cleavage site;
Y为控制部分：连接状态下，使Z不指示。Y is the control part: in the connected state, Z is not indicated.
该底物蛋白可被新冠病毒的酶类蛋白催化分解，生成Z-X1和X2-Y，释放出不溶蛋白Z，从而形成凝胶，本示例的Z链为纤维蛋白，X1-X2链为“-Trp-Ala-Leu-Gln-Gly-”，Y链为血纤维蛋白肽A和血纤维蛋白肽B；This substrate protein can be catalyzed by the enzyme protein of the new coronavirus to generate Z-X1 and X2-Y, releasing the insoluble protein Z, thereby forming a gel. In this example, the Z chain is fibrin, and the X1-X2 chain is " -Trp-Ala-Leu-Gln-Gly-", the Y chain is fibrinopeptide A and fibrinopeptide B;
2，通过基因编辑技术修改Vero细胞的基因组序列，使上述底物蛋白在该细胞中被充分表达，本示例通过CRISPR-Cas9基因编辑技术修改合成纤维蛋白原α（A）链、β（B）链、γ链基因组的启动子的基因序列，并把合成凝血酶的作用位点的基因序列，修改成为合成“-Trp-Ala-Leu-Gln-Gly-”的基因序列，该作用位点位于纤维蛋白原α（A）链和β（B）链的“Arg-Gly”处，从而诱导Vero细胞生成另一种纤维蛋白原，该纤维蛋白原为可溶蛋白，可被Mpro水解，生成纤维蛋白肽A、纤维蛋白肽B及不可溶的纤维蛋白，并通过验证，确保Mpro水解酶可以快速水解该纤维蛋白原，使其释放出不溶的纤维蛋白，从而形成凝胶，必要时可修改合成X1-X2链的基因序列，优化Mpro水解酶的水解纤维蛋白原的能力，该基因编辑后的新细胞即为指示细胞。其中，CRISPR-Cas9基因编辑技术为现有技术，不再进行详细叙述。2. Modify the genome sequence of Vero cells through gene editing technology so that the above substrate proteins are fully expressed in the cells. In this example, CRISPR-Cas9 gene editing technology is used to modify the synthesis of fibrinogen α (A) chain and β (B). The gene sequence of the promoter of chain and gamma chain genomes, and the gene sequence of the action site for synthesizing thrombin was modified into the gene sequence for synthesizing "-Trp-Ala-Leu-Gln-Gly-". The action site is located at "Arg-Gly" of fibrinogen α (A) chain and β (B) chain, thus inducing Vero cells to produce another type of fibrinogen. This fibrinogen is a soluble protein that can be hydrolyzed by Mpro to generate fibers. Protein peptide A, fibrinopeptide B and insoluble fibrin, and have been verified to ensure that Mpro hydrolase can quickly hydrolyze the fibrinogen to release insoluble fibrin to form a gel. The synthesis can be modified if necessary The gene sequence of the X1-X2 chain optimizes the ability of Mpro hydrolase to hydrolyze fibrinogen. The new cells after editing of this gene are indicator cells. Among them, CRISPR-Cas9 gene editing technology is an existing technology and will not be described in detail.
三、底物蛋白的制备3. Preparation of substrate protein
1，指示细胞的培养：将上述指示细胞，在培养瓶中，在富含有MEM、胎牛血清和两种抗生素等物质的培养基中培养，37℃培养72小时，传代1次，再培养72小时；1. Culture of indicator cells: Cultivate the above indicator cells in a culture bottle in a medium rich in MEM, fetal bovine serum and two antibiotics, culture at 37°C for 72 hours, passage once, and then culture. 72 hours;
2，收集指示细胞培养液，并分离提纯纤维蛋白原，分离纤维蛋白原的方法为现有技术，不再进行详细叙述。2. Collect the indicator cell culture fluid, and separate and purify fibrinogen. The method of separating fibrinogen is an existing technology and will not be described in detail.
四、病毒检测试剂盒的制备4. Preparation of virus detection kits
1，采样管的制备：将上述方法制备好的纤维蛋白原加入DMEM液体培养基中，分装至无菌塑料管中，并用盖子盖住管口，即得采样管；1. Preparation of sampling tubes: Add the fibrinogen prepared by the above method to DMEM liquid culture medium, distribute it into sterile plastic tubes, and cover the mouth of the tube with a cap to obtain a sampling tube;
2，检测卡的制备：选取Vero细胞作为培养细胞，该培养细胞的培养基为富含有MEM、胎牛血清和两种抗生素等物质的培养基，其余与实施例1相同；2. Preparation of test card: Vero cells are selected as culture cells. The culture medium of the culture cells is a culture medium rich in substances such as MEM, fetal bovine serum and two antibiotics. The rest is the same as in Example 1;
3，将制备好的检测卡放入密封袋内密封，将密封好的检测卡与检查拭子，采样管、滴管、裂解液管和使用说明书放入外包装盒内，即得病毒检测试剂盒，其中所述裂解液管中的裂解液为不含SDS的改良版 RIPA 裂解液。3. Put the prepared test card into the sealed bag and seal it. Put the sealed test card, inspection swab, sampling tube, dropper, lysate tube and instruction manual into the outer box to get the virus detection reagent. box, wherein the lysis solution in the lysis solution tube is a modified version of RIPA lysis solution that does not contain SDS.
本实施例的工作原理如下：The working principle of this embodiment is as follows:
病毒检测试剂盒的工作原理：使用者按照说明书，可自行用检查拭子收集样品，将样品传入采样管的保存液中，制备出样品溶液，再用滴管将采样管的样品溶液滴入检测卡的样品孔中，2小时后滴加裂解液，静置半小时观察结果，若使用者为感染者，则裂解液裂解细胞后，病毒及病毒合成的蛋白质会释放出来，其中的Mpro水解酶将分解样品溶液中的纤维蛋白原，使其生成不溶的纤维蛋白而形成凝胶；若使用者不是感染者，则检测卡的样品孔不会形成凝胶。The working principle of the virus detection kit: According to the instructions, the user can collect the sample with a test swab, transfer the sample into the preservation solution of the sampling tube, prepare a sample solution, and then use a dropper to drop the sample solution in the sampling tube. In the sample hole of the test card, add lysis solution dropwise after 2 hours, and let it sit for half an hour to observe the results. If the user is an infected person, after the lysis solution cleaves the cells, the virus and the proteins synthesized by the virus will be released, and the Mpro in it will be hydrolyzed. The enzyme will decompose the fibrinogen in the sample solution, causing it to generate insoluble fibrin to form a gel; if the user is not an infected person, the sample hole of the test card will not form a gel.
综上，本发明方法中，通过对活病毒的培养，增加病毒的含量，提高检测灵敏度，仅需一个活病毒就可以被检出；由于本发明方法对新冠病毒特有的酶类蛋白质具有较高的选择特异性，其检测的准确率较抗原检测更高；除了具有高灵敏度和高准确率以外，检测试剂盒还具有可自测的优点，避免聚集采样时的交叉感染，而防护口罩具有实时监测能力，可在感染者具有传播病毒能力后的一个检测周期内，迅速识别出感染者，又可以快速识别出部分病毒接触者，一定程度上可避免病毒传播，有效的起到防疫效果。In summary, in the method of the present invention, by cultivating live viruses, the content of the virus is increased, the detection sensitivity is improved, and only one live virus can be detected; because the method of the present invention has a higher sensitivity to the enzyme proteins unique to the new coronavirus The selection specificity of the test kit is higher than that of the antigen test; in addition to high sensitivity and high accuracy, the test kit also has the advantage of self-testing to avoid cross-infection during gathering sampling, while the protective mask has real-time Monitoring capabilities can quickly identify infected people within a detection cycle after the infected person has the ability to spread the virus, and can also quickly identify some virus contacts, which can avoid the spread of the virus to a certain extent and effectively achieve the epidemic prevention effect.

Claims (10)

  1.  一种新冠病毒检测方法，其特征在于：利用指示细胞合成的底物蛋白与新冠病毒的酶类蛋白接触后显色或者形成凝胶达到检测的目的；A novel coronavirus detection method, characterized by: utilizing a substrate protein synthesized by an indicator cell to contact the enzyme protein of the novel coronavirus to develop color or form a gel to achieve the purpose of detection;
    其中，所述指示细胞通过如下方法制备：Wherein, the indicator cells are prepared by the following method:
    选取细胞膜上含有ACE2受体的细胞作为制备指示细胞的母细胞；Select cells containing ACE2 receptors on their cell membranes as mother cells for preparing indicator cells;
    通过基因编辑方式修改母细胞的基因组序列，从而在该细胞中表达出如下结构的底物蛋白：Z-X1-X2-Y；The genome sequence of the mother cell is modified through gene editing, so that the substrate protein with the following structure is expressed in the cell: Z-X1-X2-Y;
    其中：in:
    Z-为指示部分：指示部分自身或与外部介入物质反应，显色或形成凝胶；Z- is the indicator part: the indicator part reacts with itself or with external intervening substances, develops color or forms a gel;
    X1-X2为连接部分：一段含有底物裂解位点的多肽链片段，且该底物裂解位点可与新冠病毒中的酶类蛋白反应并发生断裂；X1-X2 is the connecting part: a polypeptide chain fragment containing a substrate cleavage site, and the substrate cleavage site can react with the enzyme protein in the new coronavirus and cause cleavage;
    -Y为控制部分：具有遮盖Z或抑制Z活性的作用，连接状态下，使Z不指示，即使Z不显色和不形成凝胶。-Y is the control part: it has the function of covering Z or inhibiting the activity of Z. When connected, Z does not indicate, even if Z does not develop color and does not form a gel.
  2.  根据权利要求1所述的新冠病毒检测方法，其特征在于：新冠病毒的酶类蛋白与底物蛋白上的X1-X2部分的底物裂解位点反应，使底物蛋白中的Y断开，失去遮盖Z或抑制Z活性的作用，从而使Z自身或与外部介入物质反应，显色或形成凝胶。The new coronavirus detection method according to claim 1, characterized in that: the enzyme protein of the new coronavirus reacts with the substrate cleavage site of the X1-X2 part on the substrate protein to break the Y in the substrate protein, It loses its function of covering Z or inhibiting its activity, causing Z itself or to react with external intervening substances to develop color or form a gel.
  3.  根据权利要求1或2所述的新冠病毒检测方法，其特征在于：所述新冠病毒的酶类蛋白为Mpro水解酶或者PLPro蛋白酶。The novel coronavirus detection method according to claim 1 or 2, characterized in that: the enzyme protein of the novel coronavirus is Mpro hydrolase or PLPro protease.
  4.  根据权利要求3所述的新冠病毒检测方法，其特征在于：当新冠病毒的酶类蛋白为Mpro水解酶时，底物蛋白上的X1-X2部分为Leu-Gln-（Ser，Ala，Gly），Mpro水解酶的底物裂解位点为Leu-Gln↓（Ser，Ala，Gly）；The new coronavirus detection method according to claim 3, characterized in that: when the enzyme protein of the new coronavirus is Mpro hydrolase, the X1-X2 part on the substrate protein is Leu-Gln-(Ser, Ala, Gly) , the substrate cleavage site of Mpro hydrolase is Leu-Gln↓ (Ser, Ala, Gly);
    当新冠病毒的酶类蛋白为PLPro蛋白酶时，底物蛋白上的X1-X2部分为Leu-Xaa-Gly-Gly-Xaa，PLpro蛋白酶的底物裂解位点为Leu-Xaa-Gly-Gly↓Xaa。When the enzyme protein of the new coronavirus is PLPro protease, the X1-X2 part on the substrate protein is Leu-Xaa-Gly-Gly-Xaa, and the substrate cleavage site of PLpro protease is Leu-Xaa-Gly-Gly↓Xaa .
  5. 一种新冠病毒检测卡，其特征在于：包括检测卡本体，在检测卡本体上设置有样品孔，在样品孔的内壁上设置有指示细胞。A novel coronavirus detection card is characterized by: including a detection card body, a sample hole is provided on the detection card body, and indicator cells are provided on the inner wall of the sample hole.
  6.  根据权利要求5所述的新冠病毒检测卡，其特征在于：所述检测卡本体为采用光学透明聚苯乙烯材料制备。The new coronavirus detection card according to claim 5, characterized in that: the detection card body is made of optically transparent polystyrene material.
  7. 一种新冠病毒检测试剂盒，包括采样管和检测卡，其特征在于：所述检测卡上设置有样品孔，在样品孔的内壁上设置有培养细胞，所述采样管的保存液内含有如权利要求1所述的底物蛋白。A new coronavirus detection kit, including a sampling tube and a detection card, characterized in that: the detection card is provided with a sample hole, and cultured cells are provided on the inner wall of the sample hole, and the preservation solution of the sampling tube contains such as The substrate protein of claim 1.
  8.  根据权利要求7所述的新冠病毒检测试剂盒，其特征在于：培养细胞为Vero细胞。The new coronavirus detection kit according to claim 7, characterized in that: the cultured cells are Vero cells.
  9.  一种防护口罩，包括罩体，所述罩体由内到外依次为无纺布微孔滤膜内层、熔喷布层和无纺布微孔滤膜外层，其特征在于：在熔喷布层内侧设置有一层指示细胞。A protective mask, including a cover body, which is composed of an inner layer of non-woven microporous filter membrane, a melt-blown cloth layer and an outer layer of non-woven microporous filter membrane from the inside to the outside. It is characterized in that: A layer of indicator cells is provided inside the spray layer.
  10.  根据权利要求9所述的防护口罩，其特征在于：所述无纺布微孔滤膜层上的微孔孔径大小在病毒和细胞之间。The protective mask according to claim 9, characterized in that: the size of the micropores on the non-woven microporous filter membrane layer is between viruses and cells.
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