CN117604067A - Novel coronavirus detection method, detection card, detection kit and protective mask - Google Patents
Novel coronavirus detection method, detection card, detection kit and protective mask Download PDFInfo
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- CN117604067A CN117604067A CN202210546332.4A CN202210546332A CN117604067A CN 117604067 A CN117604067 A CN 117604067A CN 202210546332 A CN202210546332 A CN 202210546332A CN 117604067 A CN117604067 A CN 117604067A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a novel coronavirus detection method, which achieves the detection purpose by preparing targeted indicator cells and utilizing the phenomenon that substrate proteins synthesized by the indicator cells are colored or gel is formed after contacting enzyme proteins of the novel coronavirus. In addition, a virus detection card, a virus detection kit and a protective mask with a virus monitoring function prepared by using the indicator cell or the substrate protein component are also disclosed. According to the invention, the content of viruses is increased by culturing the live viruses, so that the detection sensitivity is improved; meanwhile, the detection accuracy is higher than that of antigen; in addition, the detection kit provided by the invention has the advantage of self-testing, cross infection during aggregation sampling is avoided, and the protective mask has the capability of monitoring in real time, so that an epidemic prevention effect is effectively achieved.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a novel coronavirus detection method, a detection card and a virus detection kit established based on the method, and a protective mask with a virus monitoring function.
Background
At present, the world is affected by novel coronaviruses in many places, particularly the Omikovia variant strain, the R0 value reaches 9.5, the infectivity is strong, the transmission speed is extremely high, and most asymptomatic people exist, which brings great difficulty to the epidemic prevention of people. In the epidemic prevention process, virus detection is crucial, and the existing virus detection technology is nucleic acid detection and antigen detection at present, but the risk of aggregation cross infection possibly exists during nucleic acid detection sampling, and the antigen detection sensitivity is low, and the problems of high false positive and false negative exist. Therefore, a new detection method is urgently needed, which has higher sensitivity, can rapidly identify the infected person and can avoid cross infection caused by detection. Meanwhile, most of the infected persons transmit viruses through the mouth and nose, if the protective mask can have the virus transmission capacity, the infected persons can be rapidly identified in a detection period after the infected persons have the virus transmission capacity, partial virus contact persons can be rapidly identified, the virus transmission can be avoided to a certain extent, and an epidemic prevention effect is effectively achieved.
Through research, the novel coronavirus mainly has four structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), envelope protein (E protein). The spike protein S protein has two subunits, S1 and S2, with the receptor binding site RBD located on the S1 subunit, and the novel coronavirus binds ACE2 through RBD into the cell interior, and contains, in addition to structural proteins, some non-structural proteins such as 3-chymotrypsin-like protease (3 CL or the major protease Mpro hydrolase), papain-like protease (PLPro), gyrase Rdrp (RNA-dependent RNA polymerase) and accessory proteins.
The novel coronavirus proteolytically hydrolyzes the viral multimer into functional nonstructural proteins by two viral proteases Mpro and PLpro, pp1a and pp1ab forming nonstructural proteins nsp1-11 and nsp1-16, respectively. PLpro (encoded by nsp 3) is responsible for cleavage of nsp1/2, nsp2/3 and nsp3/4 boundaries, while Mpro (encoded by nsp 5) is responsible for the remaining 11 cleavage events, mpro is conserved in coronaviruses, and the substrates of Mpro in different coronaviruses have some common features: the amino acids from n-terminal to c-terminal are numbered in paired form (p 4-p3-p2-p1 ∈ -p1 '), with cleavage sites between p1 and p 1'. In particular, mpro has unique substrate selectivity for glutamine at the P1 site (Leu-Gln +.sup.th (Ser, ala, gly)), a function not possessed by closely related host proteases. The conserved sequence of PLpro is Leu-Xaa-Gly-Gly ∈Xaa (amino acid residue number of substrate is P4-P3-P2-P1 ∈P1', downward arrow indicates cleavage site).
Heme, also known as heme, is chemically named ferriprotoporphyrin ix, which is a prosthetic group of hemoglobin and has a molecular weight of 614D. Heme is composed of protoporphyrin IX and ferrous atom and is a chromogenic group of hemoglobin. Hemoglobin is formed by combining heme and globin, and consists of four peptide chains, two alpha chains and two beta chains, each chain having a cyclic heme containing an iron atom.
Prothrombin (II), one of the blood clotting factors. Is present in plasma, also known as factor II. Is zymogen containing 582 amino acid residue, is cut by factor Xa at Arg-Thr and Arg-Ile, cuts 274 amino acid residues at N end, and the rest 308 amino acid residues are divided into A, B two peptide chains, and are connected by a disulfide bond, namely thrombin (thrombin). Thrombin acts directly on the last step of the blood coagulation process to promote the conversion of soluble fibrinogen in the blood plasma into insoluble fibrin, thereby achieving the purpose of quick-acting hemostasis.
Fibrinogen is also known as the first factor, fibrinogen. Fibrinogen molecules are formed by pairs of three polypeptide chains called alpha (A), beta (B) and gamma chains, each of which is bound as a dimer by S-S bonds. 1 molecule fibrinogen is represented by [ alpha (A) beta (B) gamma ]2, and when blood coagulates, the alpha (A) chain and the Arg-Gly of beta (B) are respectively cut by thrombin, and the fibrin peptide A and the fibrin peptide B are dissociated to form fibrin monomers.
CRISPR-Cas9 targeted gene modification (knockout and knock-in) technology is a powerful molecular biology tool for genome editing, which is developed recently and widely applied to genetic modification of various animal and plant individuals or cell genomes. Multiple genes can be knocked in and/or knocked out simultaneously using CRISPR techniques. The CRISPR technology mainly comprises Cas9 protein and single-stranded guide RNA (sgRNA), wherein the Cas9 protein plays a role in cutting a DNA double strand, the sgRNA plays a role in guiding, and under the guiding of the sgRNA, the Cas9 protein can cut different target positions through a base complementary pairing principle, so that the DNA double strand is broken. In general, when DNA breaks occur in cells, a broken DNA double strand is repaired by a highly efficient non-homologous end joining method (NHEJ), and in the repair process, a mismatch phenomenon of insertion or deletion of a base usually occurs, resulting in frame shift mutation, so that a gene originally encoding a certain peptide chain is changed into a gene encoding another completely different peptide chain sequence, thereby achieving gene knockout.
After DNA double strand breaks, if DNA repair templates enter the cell, the genome break will undergo homologous recombination repair (HDR) according to the repair templates, thereby achieving gene knock-in. The repair template consists of a target gene to be introduced and a homologous sequence (homology arm) at the upstream and downstream of the target sequence, and the length and the position of the homology arm are determined by the size of the editing sequence. HDR repair patterns occur less frequently in cells, typically less than 10%. In order to increase the success rate of gene knock-in, many scientists currently strive to increase HDR efficiency, synchronize edited cells to the most active cell division phase of HDR, and promote repair modes with HDR; or the chemical method is used for inhibiting the gene to carry out NHEJ, so that the HDR efficiency is improved.
Disclosure of Invention
The purpose of the invention is that: provides a novel coronavirus detection method, a detection card, a detection kit and a protective mask, so as to solve the problems in the prior art.
The invention is realized by the following technical scheme:
a novel coronavirus detection method, which is characterized in that: the substrate protein synthesized by the indicator cells is used for developing color or forming gel after being contacted with the enzyme protein of the novel coronavirus to achieve the detection purpose;
wherein the indicator cell is prepared by the following method:
selecting cells containing ACE2 receptors on cell membranes as parent cells for preparing indicator cells;
modifying the genomic sequence of the parent cell by gene editing to express in the cell a substrate protein of the structure: Z-X1-X2-Y;
wherein:
z-is an indicator moiety: the indicator moiety itself or reacts with an external intervening substance, developing or forming a gel;
X1-X2 are linking moieties: a polypeptide chain fragment containing a substrate cleavage site, wherein the substrate cleavage site can react with and break an enzyme protein in the novel coronavirus;
-Y is the control part: has the effect of covering Z or inhibiting the activity of Z, and in the connected state, Z is not indicated, even if Z does not develop color and gel is not formed.
Further: the enzyme protein of the novel coronavirus reacts with the cleavage site of the X1-X2 part on the substrate protein to break Y in the substrate protein, lose the effect of covering Z or inhibiting the activity of Z, and thus lead Z to react with itself or an external intervention substance, develop color or form gel.
Further: the novel coronavirus enzyme protein is Mpro hydrolase or PLPro protease.
Further: when the enzyme protein of the novel coronavirus is Mpro hydrolase, the X1-X2 part on the substrate protein is Leu-Gln- (Ser, ala, gly), and the substrate cleavage site of the Mpro hydrolase is Leu-Gln ∈ (Ser, ala, gly);
when the enzyme protein of the novel coronavirus is PLPro protease, the X1-X2 part on the substrate protein is Leu-Xaa-Gly-Gly-Xaa, and the substrate cleavage site of the PLpro protease is Leu-Xaa-Gly-Gly ∈Xaa.
The other technical scheme of the invention is as follows: a novel coronavirus detection card, characterized in that: the detection card comprises a detection card body, wherein a sample hole is formed in the detection card body, and an indication cell is arranged on the inner wall of the sample hole.
Further: the detection card body is made of an optical transparent polystyrene material.
The other technical scheme of the invention is as follows: the utility model provides a novel coronavirus detection kit, includes sampling tube and detection card, its characterized in that: the detection card is provided with a sample hole, the inner wall of the sample hole is provided with cultured cells, and the preservation solution of the sampling tube contains the substrate protein as set forth in claim 1.
Further: the cultured cells are Vero cells.
The other technical scheme of the invention is as follows: the utility model provides a protective mask, includes the cover body, the cover body is non-woven fabrics millipore filtration membrane inlayer, melt-blown cloth layer and non-woven fabrics millipore filtration membrane skin from interior to exterior in proper order, its characterized in that: a layer of indicator cells is disposed within the meltblown layer.
Further: the pore size of the micropores on the non-woven fabric microporous filter membrane layer is between viruses and cells.
The invention has the advantages that: in the method, the content of the viruses is increased by culturing the live viruses, so that the detection sensitivity is improved, and only one live virus is needed to be detected; because the method has higher selection specificity on the special enzyme protein of the novel coronavirus, the detection accuracy is higher than that of the antigen; besides high sensitivity and high accuracy, the detection kit has the advantage of self-testing, cross infection during aggregation sampling is avoided, the protective mask has real-time monitoring capability, and can rapidly identify an infected person in a detection period after the infected person has virus transmission capability, and can rapidly identify partial virus contactors, so that virus transmission can be avoided to a certain extent, and epidemic prevention effect is effectively achieved.
Detailed Description
The following is a further description of the present invention by way of methods of use and principles.
The invention discloses a novel coronavirus detection method, which comprises the following steps: preparing targeted indicator cells, and using substrate proteins synthesized by the indicator cells to be in contact with enzyme proteins of the novel coronavirus to develop color or form gel so as to achieve the purpose of detection; wherein the indicator cell is prepared by the following method:
selecting cells containing ACE2 receptor on the cell membrane as parent cells for preparing indicator cells,
modifying the genomic sequence of the parent cell by gene editing to express in the cell a substrate protein of the structure: Z-X1-X2-Y; wherein:
z-is an indicator moiety: the indicator moiety itself or reacts with an external intervening substance, developing or forming a gel;
X1-X2 are linking moieties: a polypeptide chain fragment containing a substrate cleavage site, wherein the substrate cleavage site can react with and break an enzyme protein in the novel coronavirus;
-Y is the control part: has the effect of covering Z or inhibiting the activity of Z, and in the connected state, Z is not indicated, even if Z does not develop color and gel is not formed.
According to the differences of Z-and-Y, the substrate proteins share two framework forms, namely:
architecture 1: z-is an indicator moiety: color development or gel formation;
X1-X2 are linking moieties: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
-Y is the control part: in the connected state, Z is not indicated.
Architecture 2: z-is an indicator moiety: reacting with external intervention substances, developing or forming gel;
X1-X2 are linking moieties: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
-Y is the control part: in the connected state, Z is not indicated.
When cultured cells of the virus are indicator cells, the substrate protein is not required to be added manually, and the detection process of the virus by the method is as follows:
step 1, after the virus contacts with the indicator cell, the virus enters the indicator cell through ACE2 receptor on the indicator cell, and the indicator cell is utilized to synthesize self proteins, wherein the proteins comprise structural proteins, enzyme proteins and auxiliary proteins;
step 2, the synthesized enzyme protein catalyzes the substrate protein on the indicator cell to perform chemical reaction, breaks Y and releases Z;
step 3, when the substrate protein is the framework 1, Z is developed or gel is formed; when the substrate protein is framework 2, Z reacts with external intervention substances to develop color or form gel;
step 4, the color development or gel forming substance is released from the indication cells, and the indication area is enlarged, so that the recognition degree is increased;
and 5, after the cells are lysed, a large amount of viruses and enzyme proteins of the viruses are released from the indicator cells to catalyze chemical reactions, color development or gel formation of substrate proteins on more indicator cells, so that the recognition degree is further increased.
When the cultured cells of the virus are not indicator cells, the substrate protein is manually added, and the virus detection process of the method is as follows:
step 1, after the virus contacts with cultured cells, the virus enters the cells through ACE2 receptors on cell membranes, and the substances of the cells are utilized to synthesize self proteins, wherein the proteins comprise structural proteins, enzyme proteins and auxiliary proteins;
step 2, after the synthesized enzyme protein contacts with the substrate protein, chemical reaction can occur, Y is disconnected, and Z is released;
step 3, when the substrate protein is the framework 1, Z is developed or gel is formed; when the substrate protein is architecture 2, Z reacts with external intervening substances, developing color or forming a gel.
Preferably: the Mpro hydrolase in the novel coronavirus reacts with a substrate cleavage site on X1-X2, so that Y in a substrate protein is disconnected, and when the substrate protein is a framework 1, the Y loses the effect of covering Z, and the Z is exposed and developed or forms gel; when the substrate protein is the framework 2, the Y loses the effect of inhibiting the activity of Z, and the Z reacts with external intervention substances to develop color or form gel.
Preferably: the X1-X2 part of the substrate protein is Leu-Gln- (Ser, ala, gly), and the cleavage site of the substrate is Leu-Gln ∈ (Ser, ala, gly);
the invention also discloses a protective mask, which comprises a mask body, wherein the mask body comprises a non-woven fabric microporous filter membrane inner layer, a melt-blown cloth layer and a non-woven fabric microporous filter membrane outer layer from inside to outside, in sequence, wherein: an indicator cell is arranged inside the melt-blown cloth layer, and the pore size of the micropores on the non-woven microporous filter membrane layer is between the viruses and the cells.
Preferably; the two layers of melt-blown cloth are arranged, the inner layer is melt-blown cloth with one layer of indication cells, and the outer layer is melt-blown cloth which is resident and does not contain the indication cells, so that the melt-blown cloth is suitable for workers in a virus environment;
preferably: the protective mask should be stored at 2-8deg.C, the service time should not exceed 24h, and should be used at ambient temperature below 40deg.C;
preferably; the chip is arranged on the protective mask and used for reporting unqualified results to the disease control center or providing a two-dimensional code of the disease control center so as to facilitate the reporting of the results by an infected person;
preferably; the outside of the protective mask is added with a heat preservation layer, so that the use temperature of the protective mask is wider.
The invention also discloses a novel coronavirus detection card, wherein the detection card is provided with a sample hole, and the inner wall of the sample hole is provided with indicating cells.
Preferably: the detection card is prepared from an optical transparent polystyrene material;
preferably: the detection card is stored at the temperature of 2-8 ℃;
preferably; and a chip is arranged on the detection card and used for reporting unqualified results to the disease control center or providing a two-dimensional code of the disease control center so as to facilitate the reporting of the results by an infected person.
The invention also discloses a virus detection kit, which comprises a sampling tube and a detection card, wherein 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 cultured cells.
Preferably: the detection card is prepared from an optical transparent polystyrene material;
preferably: the cultured cells are Vero cells;
preferably: the virus detection kit is stored at the temperature of 2-8 ℃;
the present solution is further described below by the detection principle.
The invention discloses a novel coronavirus detection method, which comprises the steps of preparing targeted indicator cells, and utilizing substrate proteins synthesized by the indicator cells to be in contact with enzyme proteins of the novel coronavirus for color development or forming gel to achieve the purpose of detection; the detailed preparation method of the indicator cell comprises the following steps:
1, selection of mother cells: taking a cell containing an ACE2 receptor on a cell membrane as a parent cell for preparing an indicator cell;
2, designing a substrate: the structure of the substrate protein is Z-X1-X2-Y,
architecture 1: z-is an indicator moiety: color development or gel formation;
X1-X2 are linking moieties: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
-Y is the control part: in the connected state, Z is not indicated.
The substrate protein can be catalyzed and decomposed by enzyme proteins of novel coronaviruses to generate Z-X1 and X2-Y, and the Y loses the effect of covering Z, so that the Z-X1 is colored or forms gel.
Architecture 2: z-is an indicator moiety: reacting with external intervention substances, developing or forming gel;
X1-X2 are linking moieties: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
-Y is the control part: in the connected state, Z is not indicated.
The substrate protein can be catalyzed and decomposed by enzyme proteins of novel coronaviruses to generate Z-X1 and X2-Y, wherein Y loses the effect of inhibiting the activity of Z, and the Z-X1 and an external intervention substance are subjected to chemical reaction, color development or gel formation;
3, modifying the genome sequence of the parent cell by a gene editing technique, so that the substrate protein can be fully expressed in the cell.
Preferably: the mother cell is a Vero cell;
preferably: the substrate protein is a framework 1, the substrate protein is formed by connecting a haemoglobin alpha chain and a novel coronavirus polyprotein nsp5-11 together in a covalent bond form through a polypeptide chain (hereinafter referred to as haemoglobin alpha chain-nsp 5-11),
preferably: the substrate protein is a framework 2, and is formed by connecting a thrombin double chain and a novel coronavirus polyprotein nsp5-11 together in a covalent bond form through a polypeptide chain (hereinafter referred to as thrombin-nsp 5-11);
preferably, the gene editing technique is a CRISPR-Cas9 gene editing technique.
The virus detection method is a living virus culture method, and is used for judging whether a detection sample contains viruses or not through color development or gel formation.
When the cultured cells are indicator cells, the substrate proteins are not required to be added manually, if the detection sample contains viruses, the viruses can enter the indicator cells through receptors on the indicator cells, and the substances of the indicator cells are utilized to synthesize self proteins, including structural proteins, enzyme proteins and auxiliary proteins; the synthesized enzyme protein catalyzes the substrate protein on the indicator cell to perform chemical reaction, breaks Y and releases Z; z develops or forms a gel when the substrate protein is architecture 1; when the substrate protein is framework 2, Z reacts with external intervention substances to develop color or form gel; the color development or gel formation substance is released from the indicator cells, and the indicator area is enlarged, thereby increasing the recognition degree; after the cells are lysed, a large amount of viruses and enzyme proteins of the viruses are released to catalyze more substrate proteins on the indicator cells to undergo chemical reactions, develop colors or form gels, thereby further increasing the recognition degree;
when the cultured cells are not indicator cells, the substrate protein needs to be manually added, if the detected sample contains viruses, the viruses enter the cells through ACE2 receptors on cell membranes after contacting the cultured cells, and the substances of the cells are utilized to synthesize self proteins, wherein the proteins comprise structural proteins, enzyme proteins and auxiliary proteins; after contacting the synthesized enzyme protein with the substrate protein, chemical reaction can occur, Y is disconnected, and Z is released; z develops color or forms gel when the substrate protein is in architecture 1, and Z reacts with external intervening substances to develop color or form gel when the substrate protein is in architecture 2;
if the test sample does not contain virus, no color change or gel formation occurs.
Preferably, the test sample is gas exhaled through the mouth and nose or a sample collected by an inspection swab;
preferably, the cultured cells of the virus are indicator cells;
preferably, the receptor is ACE2;
preferably, the enzyme protein is an Mpro hydrolase;
preferably, when the substrate protein is in the structure 1, the substrate protein is hemoglobin alpha chain-nsp 5-11, heme groups of the substrate protein are blocked by nsp5-11 and do not develop color or develop light red, and the substrate protein can be hydrolyzed by Mpro to release hemoglobin alpha polypeptide chain to develop red;
preferably, when the substrate protein is framework 2, the substrate protein is thrombin-nsp 5-11, and the substrate protein is prothrombin, and can be hydrolyzed by Mpro to release two chains of thrombin, and the two chains are connected by a disulfide bond to generate activated thrombin.
The protective mask is made of melt-blown cloth with a layer of indication cells, and comprises a mask body, a nose clip and a mask belt; the mask body sequentially comprises an inner layer of a non-woven fabric microporous filter membrane, a melt-blown cloth layer and an outer layer of the non-woven fabric microporous filter membrane from inside to outside, wherein the non-woven fabric is non-woven cloth formed by air flow or mechanical net forming, water jet, needle punching or hot rolling reinforcement; the non-woven fabric microporous filter membrane layer is made by using a high molecular chemical material, and a pore-forming additive is smeared on a non-woven fabric after special treatment, wherein the pore size of the pore-forming additive is between viruses and cells, and the viruses are allowed to pass but the cells are forbidden to pass; a layer of indicator cells is attached to the melt-blown fabric; the nose clip is made of a bendable plastic material; the mask belt is knitted by cotton fiber threads and spandex threads.
Preferably, the storage condition of the protective mask is 2-8 ℃;
preferably, the protective mask should not be used for more than 24 hours;
preferably, the two layers of the melt-blown cloth layer of the protective mask used by the staff in the virus environment are all two layers, the inner layer is melt-blown cloth with one layer of indication cells, the outer layer is melt-blown cloth without the indication cells after standing, and the melt-blown cloth layer of the protective mask used by other staff only contains one layer and is melt-blown cloth with one layer of indication cells;
preferably, the protective mask should be used under the environment condition of not higher than 40 ℃, and when the temperature is too low, a heat preservation layer needs to be adhered to the outer side of the protective mask.
The detection card can be used as a part of a virus detection kit, the virus detection kit comprises an inspection swab, a sampling tube, a detection card and a dropper, wherein the inspection swab consists of a plastic rod (such as polystyrene) and a swab head (such as artificial fiber) and is used for collecting a sample; the sampling tube contains preservation liquid and is used for collecting samples collected by the inspection swab; the detection card is provided with a sample hole, and a layer of indicating cells are attached to the sample hole arm; the dropper is used for dropping the sample in the sampling tube into the sample hole of the detection card.
The using method of the virus detection kit comprises the following steps: taking out the inspection swab, inserting the swab head into nostril or pharyngeal portion, rotating left for three circles, and rotating right for three circles; inserting the swab head into the preservation solution of the sampling tube, repeatedly stirring, then extruding the outer wall of the sampling tube by hands, and completely extruding the liquid on the swab head into the preservation solution in the sampling tube; taking out the detection card, dripping the preservation solution in the sampling tube into a sample hole in the detection card, and standing for 4 hours; if the color is changed or a gel is formed, the result is positive, and if the color is not changed or a gel is not formed, the result is negative. The used kit should be put into a medical waste garbage bag and discarded after uniform autoclaving.
Preferably: the inspection swab is a cotton swab;
preferably: when the substrate protein is hemoglobin alpha chain-nsp 5-11, the preservation solution in the sampling tube is a DMEM liquid culture medium;
preferably: when the substrate protein is thrombin-nsp 5-11, the preservation liquid level in the sampling tube is a DMEM liquid medium containing calcium ions and fibrinogen;
preferably, the sampling tube has the function of a dropper and replaces a common dropper;
preferably, the virus detection kit should be stored at 2-8 ℃.
The detection kit comprises an inspection swab, a sampling tube, a detection card, a lysate tube and a dropper, wherein the inspection swab consists of a plastic rod (such as polystyrene) and a swab head (such as artificial fiber) and is used for collecting samples; the sampling tube contains preservation liquid and is used for collecting samples collected by the inspection swab; the detection card is provided with a sample hole, and a layer of cultured cells is attached to the sample hole arm; the dropper is used for dropping the sample in the sampling tube into the sample hole of the detection card; the lysate tube contains lysate for lysing the cultured cells, and releasing viruses and viral proteins from the cultured cells.
The using method of the virus detection kit comprises the following steps: taking out the inspection swab, inserting the swab head into nostril or pharyngeal portion, rotating left for three circles, and rotating right for three circles; inserting the swab head into the preservation solution of the sampling tube, repeatedly stirring, then extruding the outer wall of the sampling tube by hands, and completely extruding the liquid on the swab head into the preservation solution in the sampling tube; taking out the detection card, dripping the preservation solution in the sampling tube into a sample hole in the detection card, and standing for 2h; dropwise adding the lysate into the sample hole, and standing for 0.5h; if the color is changed or a gel is formed, the result is positive, and if the color is not changed or a gel is not formed, the result is negative. The used kit should be put into a medical waste garbage bag and discarded after uniform autoclaving.
Preferably: the cultured cells are Vero cells;
preferably: the inspection swab is a cotton swab;
preferably: when the substrate protein is the framework 1, the preservation solution in the sampling tube is a DMEM liquid culture medium containing hemoglobin alpha chain-nsp 5-11;
preferably: when the substrate protein is the framework 2, the preservation liquid level in the sampling tube is a DMEM liquid medium containing thrombin-nsp-5-11 and fibrinogen;
preferably: the sampling tube has the function of a dropper and replaces a common dropper;
preferably: the lysate is a modified version of RIPA lysate without SDS.
Preferably, the virus detection kit should be stored at 2-8 ℃.
Example 1
1. Preparation of indicator cells
1, selection of mother cells: cells containing ACE2 receptors on cell membranes are taken as parent cells for preparing indicator cells, and Vero cells are taken as parent cells in the example;
2, designing a substrate: the structure of the substrate protein is Z-X1-X2-Y,
wherein: z-is an indicator moiety: a colored protein or polypeptide;
X1-X2 are linking moieties: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
-Y is the control part: in the connected state, Z is not indicated.
The substrate protein can be catalyzed and decomposed by enzyme proteins of a novel coronavirus to generate Z-X1 and X2-Y, colored proteins are released to change color, the Z chain of the example is a hemoglobin alpha chain, the X chain is "-Trp-Ala-Leu-Gln-Gly-", and the Y chain is a novel coronavirus polyprotein nsp5-11;
3, modifying the genome sequence of the parent cell by a gene editing technology, so that the substrate protein is fully expressed in the cell, modifying the gene sequence of a promoter synthesizing the genome of the hemoglobin alpha chain by a CRISPR-Cas9 gene editing technology, sequentially adding the gene sequence of a polypeptide chain fragment synthesized into Trp-Ala-Leu-Gln-Gly-, the gene sequence of a novel coronavirus polyprotein nsp5-11 and a poly (A) tail at the tail end of the genome, thereby inducing Vero cells to synthesize the hemoglobin alpha chain-nsp 5-11, wherein the nsp5-11 of the protein blocks the heme group of the hemoglobin alpha chain to make the heme group not appear or appear light red, ensuring that the Mpro hydrolase can rapidly hydrolyze the hemoglobin alpha chain-nsp 5-11 to release the hemoglobin alpha polypeptide chain to appear red, modifying the gene sequence of the synthesized X1-X2 chain if necessary, and improving the capacity of the Mpro hydrolase to hydrolyze the hemoglobin alpha chain-nsp 5-11, and indicating that the cell is a novel cell after the gene editing. Among them, CRISPR-Cas9 gene editing technology is the prior art and will not be described in detail.
2. Preparation of protective mask
1, culture of indicator cells: culturing the indicator cells in a culture flask in a culture medium rich in ferric ions, heme, MEM, fetal bovine serum, two antibiotics and the like at 37 ℃ for 72 hours, and carrying out passage 1 time and further culturing for 72 hours;
2, flushing 3 times by using PBS solution, adding pancreatin into the culture flask, removing pancreatin within 3 minutes, putting into a culture box at 37 ℃ for digestion, and gently shaking the culture flask every about 10 seconds until sliding of the quicksand cells is observed;
3, after the indicator cells are digested, transferring the indicator cells into a culture medium rich in iron ions, heme, MEM, fetal calf serum, two antibiotics and the like, soaking the melt-blown cloth in the culture medium, and culturing at 37 ℃ for 24 hours;
4, taking out the melt-blown cloth, cleaning the melt-blown cloth for 3 times by using PBS solution, soaking the melt-blown cloth in protective liquid containing nutrient substances such as bovine serum albumin, sucrose, MEM and the like for 2 hours at 37 ℃, taking out the melt-blown cloth, and air-drying;
5, preparation of protective mask: stacking the inner layer of the sterile non-woven fabric microporous filter membrane, the melt-blown cloth layer and the outer layer of the non-woven fabric microporous filter membrane together, manufacturing a mask main body through high-frequency welding, and then cutting into a single mask on a production line. Installing a nose clip on one side of the mask, sewing by hemming, and putting on an ear rope on the edge of the mask to obtain the protective mask; the melt-blown cloth layer is the melt-blown cloth with a layer of indication cells prepared by the method, and a layer of melt-blown cloth without the indication cells can be added outside the melt-blown cloth for workers in virus environment, and the layer of oil-blown cloth can be used for being resided in a resident electrode device, and the inner side of the melt-blown cloth layer is coated with a layer of glycerol protective agent.
3. Preparation of virus detection kit
1, preparing a detection card carrier by adopting an optical transparent pure polystyrene material, wherein a sample hole is formed;
2, adding the prepared indicator cell solution into a sample hole, and culturing for 24 hours at 37 ℃;
3, drying the liquid in the sample hole, cleaning the sample hole for 3 times by using PBS solution, adding protective solution containing nutrients such as bovine serum albumin, sucrose, MEM and the like, incubating for 2 hours at the constant temperature of 37 ℃, and drying the sample hole by beating to obtain the detection card;
and 4, placing the prepared detection card into a sealing bag for sealing, and placing the sealed detection card, an inspection swab, a sampling tube, a dropper and a use instruction into an external packaging box to obtain the virus detection kit, wherein the preservation solution in the sampling tube is a DMEM liquid culture medium.
The working principle of this embodiment is as follows:
1, working principle of virus detection mask: when an infected person exhales, the virus exhales, part of the virus can be adsorbed on the indication cells of the mask, enter the cells through ACE2 receptors on the indication cells, and synthesize self protein in the cells, wherein Mpro hydrolase synthesized by the virus breaks down substrate protein on the indication cells to release haemoglobin alpha chains, so that the haemoglobin alpha chains are changed in color and are identified. If the color of the inner side of the mask changes, the user is indicated to be an infected person or a potential infected person; if the color of the outer side of the mask changes, the user is indicated to contact the virus environment; a person working in a virus environment for a long time can select a mask with two layers of oil sprayed, and the outer layer does not contain indication cells, so that only the inner side of the mask can change in color.
2, working principle of virus detection kit: according to the instruction, a user can collect a sample by using an inspection swab, the sample is transferred into a preservation solution of a sampling tube to prepare a sample solution, the sample solution of the sampling tube is dripped into a sample hole of a detection card by using a dropper, if the user is an infected person, viruses in the sample solution can be adsorbed on an indicator cell and enter the cell through a receptor on the indicator cell to synthesize self protein in the cell, wherein Mpro hydrolase synthesized by the viruses can decompose substrate protein in the indicator cell to release a hemoglobin alpha chain, so that the color change is generated and the virus is identified; if the user is not an infected person, the sample hole of the detection card will not change color.
Example 2
Preparation of indicator cells
1, selection of mother cells: cells containing ACE2 receptors on cell membranes are taken as parent cells for preparing indicator cells, and Vero cells are taken as parent cells in the example;
2, designing a substrate: the structure of the substrate protein is Z-X1-X2-Y,
wherein: z-is an indicator moiety: react with external intervention substances to form gel;
X1-X2 are linking moieties: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
-Y is the control part: in the connected state, Z is not indicated.
The substrate protein can be catalyzed and decomposed by enzyme proteins of novel coronaviruses to generate Z-X1 and X2-Y, the Z-X1 reacts with external intervention substances to form gel, the Z chain of the example is double chains of thrombin, the X chain is "-Trp-Ala-Leu-Gln-Gly-", the Y chain is novel coronavirus polyprotein nsp5-11, and the external intervention substances are fibrinogen separated from livestock and poultry blood;
3, modifying the genome sequence of the parent cell by a gene editing technology, so that the substrate protein is fully expressed in the cell, modifying the gene sequence of a promoter of a genome of synthetic prothrombin by a CRISPR-Cas9 gene editing technology, modifying the gene sequences corresponding to action sites 'Arg-Thr' and 'Arg-Ile' of factor Xa into the gene sequence of synthetic '-Trp-Ala-Leu-Gln-Gly-', modifying the gene sequence corresponding to 274 amino acid residues cut off by factor Xa into the gene sequence corresponding to novel coronavirus polyprotein nsp5-11, adding a poly (A) tail at the tail of the genome of prothrombin, thereby inducing Vero cells to synthesize thrombin-nsp 5-11, which is prothrombin capable of being activated by Mpro, and ensuring that Mpro hydrolase can rapidly hydrolyze thrombin-nsp 5-11 by verification, inducing synthetase, and if necessary, synthesizing X1-X2, and improving the gene sequence of Mpro hydrolase, namely, indicating that the thrombin is capable of being edited by the cell. Among them, CRISPR-Cas9 gene editing technology is the prior art and will not be described in detail.
2. Preparation of virus detection kit
1, preparation of a detection card: the culture medium for culturing the indicator cells was a medium enriched with galactose, mannose, fucose, hexosamine, sialic acid, calcium ions, vitamin K, MEM, fetal bovine serum, two antibiotics, and the like, and the rest was the same as in example 1;
2, placing the prepared detection card into a sealing bag for sealing, and placing the sealed detection card, an inspection swab, a sampling tube, a dropper and a use instruction into an external packaging box to obtain the virus detection kit, wherein preservation liquid in the sampling tube is a DMEM liquid culture medium containing calcium ions and fibrinogen.
The working principle of this embodiment is as follows:
the working principle of the virus detection kit is as follows: according to the instruction, a user can collect a sample by using an inspection swab, the sample is transferred into a preservation solution of a sampling tube to prepare a sample solution, the sample solution of the sampling tube is dripped into a sample hole of a detection card by using a dropper, if the user is an infected person, viruses in the sample solution can be adsorbed on an indicator cell and enter the cell through a receptor on the indicator cell to synthesize own protein in the cell, wherein Mpro hydrolase synthesized by the viruses decomposes substrate protein in the indicator cell to generate and release activated thrombin, and the activated thrombin converts soluble fibrinogen into insoluble fibrin, so that gel is formed and identified; if the user is not an infected person, the sample well of the test card will not form a gel.
Example 3
1. Selection of the Carrier
An animal and plant cell or a microorganism (such as virus, bacteria, etc.) is selected as a carrier for preparing the substrate protein, and Vero cells are used as the carrier for preparing the substrate protein in the example.
2. Preparation of indicator cells
1, designing a substrate: the structure of the substrate protein is Z-X1-X2-Y,
wherein: z-is an indicator moiety: forming a gel;
x is a linking moiety: a fragment of a polypeptide chain comprising a cleavage site for a substrate;
y is a control part: in the connected state, Z is not indicated.
The substrate protein can be catalyzed and decomposed by enzyme proteins of novel coronaviruses to generate Z-X1 and X2-Y, insoluble protein Z is released, so that gel is formed, the Z chain of the example is fibrin, the X1-X2 chain is "-Trp-Ala-Leu-Gln-Gly-", and the Y chain is fibrin peptide A and fibrin peptide B;
2, modifying the genome sequence of Vero cells by gene editing technology to make the above substrate protein fully expressed in the cells, in this example, modifying the gene sequence of the promoter of the genome of synthetic fibrinogen alpha (a), beta (B) and gamma chains by CRISPR-Cas9 gene editing technology, and modifying the gene sequence of the action site of synthetic thrombin into the gene sequence of synthetic "-Trp-Ala-Leu-gin-Gly-", the action site being located at the "Arg-Gly" position of fibrinogen alpha (a) and beta (B) chains, thereby inducing Vero cells to produce another fibrinogen which is soluble protein, can be hydrolyzed by Mpro to produce fibrinogen peptide a, fibrinogen peptide B and insoluble fibrin, and by verification, ensuring that Mpro hydrolase can rapidly hydrolyze the fibrinogen to release insoluble fibrin, thereby forming gel, and if necessary, modifying the gene sequence of synthetic X1-X2 chains, optimizing the proteolytic ability of the Mpro hydrolase, i.e. the new cells are indicated. Among them, CRISPR-Cas9 gene editing technology is the prior art and will not be described in detail.
3. Preparation of substrate proteins
1, culture of indicator cells: culturing the indicator cells in a culture flask in a culture medium rich in MEM, fetal bovine serum, two antibiotics, etc., at 37deg.C for 72 hr, passaging for 1 time, and culturing for 72 hr;
2, collecting the indicator cell culture fluid, and separating and purifying fibrinogen, wherein the method for separating fibrinogen is the prior art and will not be described in detail.
4. Preparation of virus detection kit
1, preparation of a sampling tube: adding the fibrinogen prepared by the method into a DMEM liquid culture medium, subpackaging into sterile plastic tubes, and covering the tube orifice with a cover to obtain a sampling tube;
2, preparation of a detection card: vero cells were selected as the culture cells, and the culture medium of the culture cells was rich in MEM, fetal bovine serum, two antibiotics, and the like, and the rest was the same as in example 1;
and 3, placing the prepared detection card into a sealing bag for sealing, and placing the sealed detection card, an inspection swab, a sampling tube, a dropper, a lysate tube and a use instruction into an external packaging box to obtain the virus detection kit, wherein the lysate in the lysate tube is an improved RIPA lysate without SDS.
The working principle of this embodiment is as follows:
the working principle of the virus detection kit is as follows: according to the instruction, a user can collect a sample by using an inspection swab, the sample is transferred into a preservation solution of a sampling tube to prepare a sample solution, the sample solution of the sampling tube is dripped into a sample hole of a detection card by using a dropper, a lysate is dripped after 2 hours, and the lysate is stood for half an hour to observe a result, if the user is an infected person, after the lysate lyses cells, viruses and proteins synthesized by the viruses are released, and the Mpro hydrolase in the sample solution breaks down fibrinogen to generate insoluble fibrin to form gel; if the user is not an infected person, the sample well of the test card will not form a gel.
In summary, in the scheme of the invention, the content of viruses is increased by culturing the live viruses, so that the detection sensitivity is improved, and only one live virus is needed to be detected; because the method has higher selection specificity on the special enzyme protein of the novel coronavirus, the detection accuracy is higher than that of the antigen; besides high sensitivity and high accuracy, the detection kit has the advantage of self-testing, cross infection during aggregation sampling is avoided, the protective mask has real-time monitoring capability, and can rapidly identify an infected person in a detection period after the infected person has virus transmission capability, and can rapidly identify partial virus contactors, so that virus transmission can be avoided to a certain extent, and epidemic prevention effect is effectively achieved.
Claims (10)
1. A novel coronavirus detection method, which is characterized in that: the substrate protein synthesized by the indicator cells is used for developing color or forming gel after being contacted with the enzyme protein of the novel coronavirus to achieve the detection purpose;
wherein the indicator cell is prepared by the following method:
selecting cells containing ACE2 receptors on cell membranes as parent cells for preparing indicator cells;
modifying the genomic sequence of the parent cell by gene editing to express in the cell a substrate protein of the structure: Z-X1-X2-Y;
wherein:
z-is an indicator moiety: the indicator moiety itself or reacts with an external intervening substance, developing or forming a gel;
X1-X2 are linking moieties: a polypeptide chain fragment containing a substrate cleavage site, wherein the substrate cleavage site can react with and break an enzyme protein in the novel coronavirus;
-Y is the control part: has the effect of covering Z or inhibiting the activity of Z, and in the connected state, Z is not indicated, even if Z does not develop color and gel is not formed.
2. The method for detecting coronavirus according to claim 1, wherein: the enzyme protein of the novel coronavirus reacts with the cleavage site of the X1-X2 part on the substrate protein to break Y in the substrate protein, lose the effect of covering Z or inhibiting the activity of Z, and thus lead Z to react with itself or an external intervention substance, develop color or form gel.
3. The novel coronavirus detection method according to claim 1 or 2, characterized in that: the novel coronavirus enzyme protein is Mpro hydrolase or PLPro protease.
4. The method for detecting coronavirus according to claim 3, wherein: when the enzyme protein of the novel coronavirus is Mpro hydrolase, the X1-X2 part on the substrate protein is Leu-Gln- (Ser, ala, gly), and the substrate cleavage site of the Mpro hydrolase is Leu-Gln ∈ (Ser, ala, gly);
when the enzyme protein of the novel coronavirus is PLPro protease, the X1-X2 part on the substrate protein is Leu-Xaa-Gly-Gly-Xaa, and the substrate cleavage site of the PLpro protease is Leu-Xaa-Gly-Gly ∈Xaa.
5. A novel coronavirus detection card, characterized in that: the detection card comprises a detection card body, wherein a sample hole is formed in the detection card body, and an indication cell is arranged on the inner wall of the sample hole.
6. The novel coronavirus detection card of claim 5, wherein: the detection card body is made of an optical transparent polystyrene material.
7. The utility model provides a novel coronavirus detection kit, includes sampling tube and detection card, its characterized in that: the detection card is provided with a sample hole, the inner wall of the sample hole is provided with cultured cells, and the preservation solution of the sampling tube contains the substrate protein as set forth in claim 1.
8. The novel coronavirus detection kit of claim 7, wherein: the cultured cells are Vero cells.
9. The utility model provides a protective mask, includes the cover body, the cover body is non-woven fabrics millipore filtration membrane inlayer, melt-blown cloth layer and non-woven fabrics millipore filtration membrane skin from interior to exterior in proper order, its characterized in that: a layer of indicator cells is disposed within the meltblown layer.
10. The protective mask of claim 9 wherein: the pore size of the micropores on the non-woven fabric microporous filter membrane layer is between viruses and cells.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210546332.4A CN117604067A (en) | 2022-05-20 | 2022-05-20 | Novel coronavirus detection method, detection card, detection kit and protective mask |
| PCT/CN2023/087592 WO2023221690A1 (en) | 2022-05-20 | 2023-04-11 | Detection method, detection card and detection kit for sars-cov-2, and protective mask |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210546332.4A CN117604067A (en) | 2022-05-20 | 2022-05-20 | Novel coronavirus detection method, detection card, detection kit and protective mask |
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| CN117604067A true CN117604067A (en) | 2024-02-27 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202210546332.4A Pending CN117604067A (en) | 2022-05-20 | 2022-05-20 | Novel coronavirus detection method, detection card, detection kit and protective mask |
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| WO (1) | WO2023221690A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021173865A2 (en) * | 2020-02-25 | 2021-09-02 | Bambu Vault Llc | Compounds for detection of viral pathogens |
| US20210301319A1 (en) * | 2020-03-26 | 2021-09-30 | Cellex, Inc. | Protease assays and their applications |
| WO2021262734A2 (en) * | 2020-06-22 | 2021-12-30 | Duke University | Fluorescent rapid neutralization assay for viral infections |
| CN215455610U (en) * | 2021-06-10 | 2022-01-11 | 保山中医药高等专科学校 | Mask with respiratory track detects function |
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