A respiratory virus rapid detection kit based on ZIF-8 material and a preparation method thereof

Abstract

The application relates to the field of microorganism detection, in particular to a respiratory virus rapid detection kit based on ZIF-8 material and a preparation method, wherein the kit comprises a virus collecting device, an exhalation cylinder and a ZIF-8 membrane, the ZIF-8 membrane is detachably arranged in the exhalation cylinder, the ZIF-8 membrane comprises a chitosan layer, the chitosan layer is uniformly distributed with ZIF-8 material, virus particles in the exhaled gas of a tester through the exhalation cylinder are adsorbed, a sample processing device comprises a processing liquid, the processing liquid elutes and lyses the virus particles adsorbed by the ZIF-8 material distributed on the chitosan layer, and a virus processing liquid is obtained, a detection reagent is used for detecting the virus processing liquid and giving a detection result, sampling is simple and rapid, the detection can be completed without the participation of professionals, sampling efficiency is higher, and the detection sensitivity is effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganism detection, in particular to a respiratory virus rapid detection kit based on ZIF-8 material and a preparation method. BACKGROUND

[0002] Acute respiratory infection is the most common infectious disease, which has a high incidence and mortality rate in the world. Respiratory pathogens have strong infectivity, fast transmission and acute onset, and can cause serious complications in children, the elderly, patients with chronic diseases, malnutrition and low immunity, etc. Therefore, early and accurate judgment of the type of disease infection is the basis for the rational application of antibiotic treatment, the shortening of the course of disease and the improvement of prognosis, and provides a pathogenic reference for clinical diagnosis and treatment, rational drug use and epidemic monitoring.

[0003] At present, the methods for detecting respiratory viruses mainly include virus isolation and culture, immunological methods and nucleic acid detection methods, etc. Among them, the nucleic acid detection method uses a swab to collect patient samples, which requires the participation of professional personnel and is inconvenient to operate. In addition, the enrichment degree of viruses directly affects the accuracy of the detection results, and the sampling efficiency of the swab and oral saliva detection method is low. SUMMARY

[0004] In order to solve the above problems, the present application provides a respiratory virus rapid detection kit based on ZIF-8 material and a preparation method, which can collect respiratory viruses by blowing and rapidly elute and lyse viruses for rapid detection.

[0005] According to one object of the present application, the present application provides a respiratory virus rapid detection kit based on ZIF-8 material, which comprises:

[0006] A virus collection device, comprising an exhalation cylinder and a ZIF-8 membrane, the ZIF-8 membrane being detachably arranged inside the exhalation cylinder, the ZIF-8 membrane comprising a chitosan layer, the chitosan layer being uniformly distributed with ZIF-8 material to adsorb virus particles in the exhaled gas of a testee through the exhalation cylinder;

[0007] A sample processing device, comprising a processing liquid, the processing liquid eluting and lysing virus particles adsorbed by the ZIF-8 material distributed on the chitosan layer to obtain a virus processing liquid;

[0008] A detection reagent, the detection reagent being used for detecting the virus processing liquid and giving a detection result.

[0009] As a preferred embodiment, the exhalation cylinder comprises an exhalation port, a cylinder and a membrane holder, the exhalation port and the cylinder being in communication, the membrane holder being used for clamping the ZIF-8 membrane and being detachably arranged inside the cylinder.

[0010] As a preferred embodiment, the membrane frame comprises two openable and closable clamps, and the ZIF-8 membrane is fixed between the two clamps.

[0011] As a preferred embodiment, the ZIF-8 membrane further comprises a filter membrane layer, and the filter membrane layer is tearably attached to the chitosan layer.

[0012] As a preferred embodiment, the detection reagent is one of colloidal gold immunochromatography reagent, immunofluorescence chromatography reagent, chemiluminescence reagent and electrochemical reagent.

[0013] As a preferred embodiment, the treatment liquid comprises acetate buffer with pH value of 5.0-5.5, sodium thiocyanate, Triton X-100 and BSA, the content of the sodium thiocyanate is 0.01%-1%, the content of the Triton X-100 is 0.01%-1%, and the content of the BSA is 0.1%-10%.

[0014] According to another object of the present application, the present application further provides a preparation method of a ZIF-8 material-based respiratory virus rapid detection kit, the ZIF-8 material-based respiratory virus rapid detection kit comprising a virus collection device, a sample treatment device and a detection reagent, the virus collection device comprising an exhalation cylinder and a ZIF-8 membrane, the ZIF-8 membrane being detachably arranged inside the exhalation cylinder, the ZIF-8 membrane comprising a chitosan layer, and the chitosan layer uniformly distributing ZIF-8 material, the method comprising the following steps:

[0015] a. preparing a ZIF-8 membrane;

[0016] b. configuring a treatment liquid of the sample treatment device;

[0017] c. preparing the detection reagent.

[0018] As a preferred embodiment, the ZIF-8 membrane further comprises a filter membrane layer, and the step a comprises:

[0019] a1. providing a chitosan layer;

[0020] a2. attaching the chitosan layer and the filter membrane layer to obtain the ZIF-8 membrane.

[0021] As a preferred embodiment, the step a1 comprises:

[0022] a11. preparing ZIF-8 particles;

[0023] a12, dissolve the ZIF-8 particles with PBS with a pH value of 7.4, and obtain a dissolved solution by ultrasonic oscillation, add glycerol to the dissolved solution and stir;

[0024] a13, add chitosan to the product of step a12 and stir to obtain a film-forming solution;

[0025] a14, dry the film-forming solution to form a film to obtain the chitosan layer.

[0026] As a preferred embodiment, the step b comprises:

[0027] b1, mix anhydrous sodium acetate and water to obtain a mixed solution, and add glacial acetic acid to the mixed solution and mix;

[0028] b2, sequentially add sodium thiocyanate, Tween-20, Triton X-100, BSA, sodium caseinate and Proclin 300 to the product of step b1, and stir uniformly to prepare a treatment solution.

[0029] Compared with the prior art, the technical scheme has the following advantages:

[0030] The virus collection device provides the exhalation cylinder to collect the exhaled gas of the tester, and uses the ZIF-8 membrane provided inside the exhalation cylinder to adsorb the virus particles in the exhaled gas, then elutes and lyses the virus particles by the treatment solution to obtain a virus treatment solution, and finally uses the detection reagent to detect the virus treatment solution to obtain the detection result, which is simple and fast, and can be completed without the participation of professionals.

[0031] The exhalation cylinder collects the exhaled gas, and the tester can perform multiple repeated exhalations to enrich more virus particles, so that the sampling efficiency is higher and the detection sensitivity is effectively improved.

[0032] The ZIF-8 material has adjustable pore size, high stable structure and catalytic activity, etc., which can quickly collect respiratory viruses, and can release the viruses under the acidic conditions of the treatment solution, thereby realizing rapid collection, elution and detection of respiratory viruses.

[0033] The present application will be further described below in conjunction with the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a structural schematic diagram of the exhalation cylinder of the present application;

[0035] Figure 2 It is a front view of the membrane holder in an open state;

[0036] Figure 3Fig. 1 is a perspective view of the membrane frame in an open state according to the present application;

[0037] Figure 4 Fig. 4 is a structural schematic diagram of the ZIF-8 membrane according to the present application;

[0038] Figure 5 Fig. 5 is an assembly schematic diagram of the ZIF-8 membrane and the membrane frame according to the present application;

[0039] Figure 6 Fig. 6 is a structural schematic diagram of the detection reagent according to the present application.

[0040] In the figure: 100, virus collection device; 110, exhalation cylinder; 111, exhalation port; 112, gas cylinder; 113, membrane frame; 113a, clamping body; 113b, taking part; 120, ZIF-8 membrane; 121, chitosan layer; 122, filter membrane layer; 300, detection reagent; 310, binding pad; 320, nitrocellulose membrane; 321, detection line; 322, quality control line; 330, sample pad; 340, absorbent pad; 350, backing. DETAILED DESCRIPTION

[0041] The following description is provided to enable any person skilled in the art to practice the present application. The preferred embodiments in the following description are only examples of the present application and other obvious modifications are possible to those skilled in the art. The basic principles defined in the following description can be applied to other embodiments, modifications, improvements, equivalents and other technical solutions without departing from the spirit and scope of the present application.

[0042] First embodiment

[0043] As Figures 1 to 6 described, the respiratory tract virus rapid detection reagent kit based on ZIF-8 material comprises:

[0044] The virus collection device 100 comprises an exhalation cylinder 110 and a ZIF-8 membrane 120, the ZIF-8 membrane 120 is detachably arranged inside the exhalation cylinder 110, the ZIF-8 membrane 120 comprises a chitosan layer 121, and the chitosan layer 121 is uniformly distributed with ZIF-8 material to adsorb virus particles in the exhaled gas of the tester through the exhalation cylinder 110;

[0045] The sample processing device comprises a processing liquid, the processing liquid elutes and lyses the virus particles adsorbed by the ZIF-8 material distributed on the chitosan layer 121 to obtain a virus processing liquid;

[0046] The detection reagent 300 is used for detecting the virus processing liquid and giving a detection result.

[0047] The virus collection device 100 provides the exhalation cylinder 110 to collect the exhaled gas of the tester, and uses the ZIF-8 membrane 120 arranged inside the exhalation cylinder 110 to adsorb the virus particles in the exhaled gas, and then elutes and lyses the virus particles by the treatment liquid to obtain a virus treatment liquid, and finally uses the detection reagent 300 to detect the virus treatment liquid to obtain the detection result. Compared with the traditional swab method, the sampling is simple and fast, and the detection can be completed without the participation of professional personnel. In addition, the exhalation cylinder 110 collects exhaled gas, and the tester can perform multiple repeated exhalation to enrich more virus particles. Compared with the saliva detection card (such as the new crown virus antigen detection card), the sampling efficiency is higher, and the detection sensitivity is effectively improved. In addition, the ZIF-8 material has adjustable pore size, high stability structure and catalytic activity, which can quickly collect respiratory viruses, and release the viruses under the acidic conditions of the treatment liquid, thereby realizing the rapid collection, elution and detection of respiratory viruses.

[0048] As shown in Figure 1 , the exhalation cylinder 110 includes an exhalation port 111, a cylinder 112 and a membrane holder 113. The exhalation port 111 and the cylinder 112 are in communication, and the membrane holder 113 is used to hold the ZIF-8 membrane 120 and is detachably arranged inside the cylinder 112.

[0049] The exhalation port 111 is connected to the left side of the cylinder 112, and the cross-sectional size of the exhalation port 111 gradually decreases from left to right, so that the exhalation port 111 has a ring-shaped horn structure. The cylinder 112 is cylindrical, and the side surface in the middle of the cylinder 112 can be an open structure for guiding gas. The membrane holder 113 is detachably arranged inside the cylinder 112, so as to facilitate the taking and placing of the ZIF-8 membrane 120 held by the membrane holder 113. During detection, the tester contacts the exhalation port 111 with the mouth and blows, and since the exhalation port 111 and the cylinder 112 are in communication, the ZIF-8 membrane 120 located in the cylinder 112 can adsorb the virus particles in the exhaled gas of the tester.

[0050] An opening for placing the membrane holder 113 is formed in the middle side surface of the cylinder 112, so that the membrane holder 113 is taken and placed through the opening. Referring to Figure 2 and Figure 3 , the membrane holder 113 includes two clamping bodies 113a capable of being opened and closed, and the ZIF-8 membrane 120 is fixed between the two clamping bodies 113a. The clamping body 113a is annular to fit the annular cylinder 112, and at this time the ZIF-8 membrane 120 is circular, but is not limited thereto and can be adjusted as a whole according to design needs.

[0051] With reference to the foregoing Figure 3 and Figure 5 , the film holder 113 further comprises a taking part 113b, and the two clamping bodies 113a are connected to the taking part 113b respectively, and the two clamping bodies 113a can clamp and fix the ZIF-8 film 120 between the two clamping bodies 113a by buckling or the like. At this time, the taking part 113b can be acted on to take and place the film holder 113 relative to the air cylinder 112 as a whole. Then, by releasing the buckling of the two clamping bodies 113a, the ZIF-8 film 120 can be taken off relative to the film holder 113.

[0052] As shown in Figure 4 , the ZIF-8 film 120 is a double-layer film, comprising a chitosan layer 121 and a filter membrane layer 122, the filter membrane layer 122 is closely attached to the chitosan layer 121, and the two can be torn open, the filter membrane layer 122 is used to intercept larger particulate matter such as bacteria in air, and the chitosan layer 121 is uniformly distributed with ZIF-8 material, which is used to adsorb virus particles in the gas filtered by the filter membrane layer 122, when the virus particles are collected, the two can be torn open, and the filter membrane layer 122 is discarded, and the chitosan layer 121 is retained for the next detection.

[0053] The filter membrane layer 122 can be a 0.45 μm microporous filter membrane, which can use common mixed fiber microporous filter membrane, polypropylene filter membrane, polyether sulfone filter membrane, polyvinylidene fluoride filter membrane, polytetrafluoroethylene filter membrane, nylon filter membrane, etc. on the market. The chitosan layer 121 serves as a support structure, and is uniformly distributed with ZIF-8 material, ZIF-8 material is a porous material containing many cage structures, and the pore diameter is 20 nm to 250 nm, which is comparable to the diameter of virus particles. Similarly, the filter membrane layer 122 is also a porous structure, wherein the pore diameter of the filter membrane layer 122 is greater than the pore diameter of the cage structure, and when the ZIF-8 film 120 is arranged in the air cylinder 110, the filter membrane layer 122 is close to the air outlet 111 of the air cylinder 110 relative to the chitosan layer 121. In this way, when air passes through the ZIF-8 film 120, the filter membrane layer 122 first intercepts larger particulate matter such as bacteria in the air, and virus particles with a pore diameter comparable to the cage structure are adsorbed inside the cage structure, and smaller particles pass through the cage structure, thereby achieving the purpose of separating viruses.

[0054] Further illustrate, ZIF-8 is a porous crystalline material formed by Zn(II) and 2-methylimidazole coordination self-assembly, with adjustable pore size, high stability structure and catalytic activity, etc. ZIF-8 as a permeable porous material, its specific surface area is large and porosity is high, and it has adsorption capacity for various viruses. And ZIF-8 material has the characteristics of stability under alkaline conditions and dissolution under certain acidic conditions. Based on this, using the different characteristics of ZIF-8 in different pH conditions, using ZIF-8 material to quickly collect respiratory viruses, and releasing viruses and detecting viruses under acidic conditions, the device can realize the rapid collection, elution and detection of respiratory viruses.

[0055] The treatment liquid includes acetate buffer with pH value of 5.0-5.5, sodium thiocyanate, Triton X-100 and BSA, the content of the sodium thiocyanate is 0.01%-1%, the content of the Triton X-100 is 0.01%-1%, and the content of the BSA is 0.1%-10%.

[0056] The Triton X-100 (polyoxyethylene octylphenyl ether) is a non-ionic surfactant which can dissolve lipids to increase the permeability of cell membranes to antibodies. The BSA (bovine serum albumin) is used as a stabilizer in the process of virus protein preservation and immune response. The acetate buffer with pH value of 5.0-5.5 can dissolve ZIF-8 material to release the adsorbed viruses, and the sodium thiocyanate and the Triton X-100 can lyse viruses to release target proteins.

[0057] The detection reagent 300 is one of colloidal gold immunochromatography reagent, immunofluorescence chromatography reagent, chemiluminescence reagent and electrochemical reagent.

[0058] The colloidal gold immunochromatography reagent includes new coronavirus detection reagent, epidemic influenza detection reagent, parainfluenza virus detection reagent and chlamydia pneumoniae detection reagent, etc., and its basic principle is double antibody sandwich method, one strain of virus characteristic protein antibody labeled with colloidal gold is placed on the gold label pad, and another strain of antibody is coated on the nitrocellulose membrane, when detecting, the complex of antibody colloidal gold and virus characteristic protein is combined with another strain of antibody on the nitrocellulose membrane, forming an obvious color band.

[0059] Figure 6A colloidal gold immunochromatography reagent is shown, which includes a conjugate pad (colloidal gold pad) 310, a nitrocellulose membrane 320 on which a detection line 321 (T zone) and a quality control line 322 (C zone) are formed, a sample pad 330, an absorbent pad 340, and a backing 350, wherein the sample pad 330, the conjugate pad 310, the nitrocellulose membrane 320, and the absorbent pad 340 are sequentially connected and arranged on the backing 350. In use, a drop of virus treatment liquid is added to the sample pad 330, and the detection result is displayed by the reaction of the T zone and the C zone, i.e. a negative or positive result is given.

[0060] The use method of the respiratory virus rapid detection kit based on ZIF-8 material is as follows:

[0061] Step 1, take out the ZIF-8 film piece 120 and place it on the film piece holder 113 and clamp it.

[0062] Step 2, place the film piece holder 113 with the fixed ZIF-8 film piece 120 into the opening on the middle side of the air cylinder 112.

[0063] Step 3, the tester blows the air outlet 111 of the air cylinder 110 10 times.

[0064] Step 4, remove the film piece holder 113 from the air cylinder 112, and take the ZIF-8 film piece 120 from the film piece holder 113, then spread the chitosan layer 121 and the filter membrane layer 122 of the ZIF-8 film piece 120, and discard the filter membrane layer 122.

[0065] Step 5, place the chitosan layer 121 into the treatment liquid and shake for 1 min to dissolve it to obtain a virus treatment liquid.

[0066] Step 6, add the virus treatment liquid to the detection reagent 300, and the detection result is given by the detection reagent 300.

[0067] In summary, the virus collection device 100 provides an exhalation cylinder 110 to collect the exhaled gas from the test subject. The ZIF-8 membrane 120 disposed inside the exhalation cylinder 110 adsorbs virus particles in the exhaled gas. The virus particles are then eluted and lysed using a processing solution to obtain a virus processing solution. Finally, the virus processing solution is detected using the detection reagent 300 to obtain the test result. Furthermore, sampling is simple and rapid, and testing can be completed without the intervention of professional personnel. In addition, the method of collecting exhaled gas using the exhalation cylinder 110 allows the test subject to exhale repeatedly to enrich more virus particles, resulting in higher sampling efficiency and effectively improving detection sensitivity. Moreover, utilizing the adjustable pore size, highly stable structure, and catalytic activity of the ZIF-8 material, respiratory viruses can be collected rapidly, and the viruses can be released under acidic conditions of the processing solution, thereby achieving rapid collection, elution, and detection of respiratory viruses. Furthermore, the ZIF-8 membrane 120 can be clamped onto the membrane holder 113, and the membrane holder 113 is detachably mounted on the exhalation pump 110 to facilitate the replacement and use of the ZIF-8 membrane 120.

[0068] Second Embodiment

[0069] like Figures 1 to 6 As shown, the preparation method of the rapid respiratory virus detection kit based on ZIF-8 material is described. The rapid respiratory virus detection kit based on ZIF-8 material includes a virus collection device 100, a sample processing device, and a detection reagent 300. The virus collection device 100 includes an exhalation cylinder 110 and a ZIF-8 membrane 120. The ZIF-8 membrane 120 is detachably disposed inside the exhalation cylinder 110. The ZIF-8 membrane 120 includes a chitosan layer 121 on which ZIF-8 material is uniformly distributed. The method includes the following steps:

[0070] a. Prepare ZIF-8 membrane 120.

[0071] The ZIF-8 membrane 120 includes a chitosan layer 121 on which ZIF-8 material is uniformly distributed. The ZIF-8 material has the characteristics of adjustable pore size, high stability structure and catalytic activity, which can rapidly collect respiratory viruses and release viruses under the acidic conditions of the treatment solution, thereby realizing rapid collection, elution and detection of respiratory viruses.

[0072] like Figure 4 As shown, the ZIF-8 membrane 120 further includes a filter membrane layer 122. Step a includes: a1, providing a chitosan layer 121; a2, bonding the chitosan layer 121 and the filter membrane layer 122 to obtain the ZIF-8 membrane 120.

[0073] Step a1 includes:

[0074] a11. Preparation of ZIF-8 particles. This involves adding 40 mL of a 400 mmol / L 2-methylimidazole methanol solution dropwise to 40 mL of a 100 mmol / L Zn(NO3)2 methanol solution, diluting with methanol to 200 mL, stirring at room temperature for 6 h, obtaining a white precipitate, centrifuging, washing three times with methanol, and finally drying under vacuum at 40 °C for 30 min to obtain the ZIF-8 particles.

[0075] a12. Dissolve the ZIF-8 particles in PBS with a pH of 7.4 and sonicate to obtain a solution. Add glycerol to the solution and stir. Specifically, weigh 0.1 g of ZIF-8 particles and dissolve them in PBS with a pH of 7.4. Then, sonicate for 5 min to obtain a solution. Add 0.6 g of glycerol to the solution and stir in a three-necked flask for about 20 min.

[0076] a13. Add chitosan to the product of step a12 and stir to obtain a film-forming solution. The chitosan is 2g, and while adding chitosan to the product of step a12, stir continuously for about 20 minutes. Then heat and stir at 80°C for about 30 minutes to allow the obtained film-forming solution to stand and remove bubbles generated by stirring.

[0077] a14. The film-forming solution is dried to form a film, resulting in the chitosan layer 121. Specifically, the film-forming solution obtained in step a13 is poured evenly into a prepared petri dish and allowed to dry naturally at room temperature until a film is formed, which is the chitosan layer 121.

[0078] In step a2, the chitosan layer 121 and the filter membrane layer 122 are overlapped and pressed together so that the two thin films are simply bonded together to form the ZIF-8 membrane 120, which can then be cut into the required shape as needed.

[0079] b. Prepare the processing solution for the sample processing device. The processing solution is used to elute and lyse virus particles to obtain a virus processing solution. Step b includes:

[0080] b1. Mix anhydrous sodium acetate and water to obtain a mixed solution, then add glacial acetic acid to the mixed solution and mix well. Specifically, mix 16g of anhydrous sodium acetate and 500mL of water to obtain a mixed solution, then add 29mL of glacial acetic acid to the mixed solution and mix well.

[0081] b2. Add 80 g of sodium thiocyanate, 20 g of Tween-20, 40 g of Triton X-100, 100 g of BSA, 10 g of sodium caseinate, and 100 mL of Proclin 300 to the product of step b1 in sequence, and stir to obtain a treatment solution.

[0082] Tween-20 (polysorbate-20) is a surfactant used as an emulsifier, dispersant, solubilizer, and stabilizer. Triton X-100 (polyethylene glycol octylphenyl ether) is a non-ionic surfactant that can dissolve lipids to increase the permeability of the cell membrane to antibodies. BSA (bovine serum albumin) is used as a stabilizer in the preservation of viral proteins and in the process of immune response. Sodium caseinate is a safe and harmless thickening agent and emulsifier. Proclin 300, as a liquid biological preservative, can dissolve ZIF-8 material to release the adsorbed virus, and the sodium thiocyanate and Triton X-100 can lyse the virus to release the target protein.

[0083] Further comprising the following steps after step b2:

[0084] The treatment solution prepared in step b2 is divided into 1.5 mL cryogenic tubes, and each tube contains 300 ul of the treatment solution. Then the filled treatment solution is labeled with the name and batch number.

[0085] c. Prepare the detection reagent 300. The detection reagent 300 is used to detect the viral treatment solution and give the detection result. The detection reagent 300 is one of colloidal gold immunochromatography reagent, immunofluorescence chromatography reagent, chemiluminescence reagent and electrochemical reagent. Taking the colloidal gold immunochromatography reagent as an example, referring to Figure 6 , it includes a conjugate pad 310 and a nitrocellulose membrane 320, and the step c includes:

[0086] c1. Prepare the conjugate pad. Take red colloidal gold in a centrifuge tube, then add borate buffer with a pH value of 8.5 and mix well by blowing, then add anti-2019-nCoV monoclonal antibody, ultrasonic, block and centrifuge to obtain the supernatant, and finally reconstitute to obtain COVAg marker. COVAg marker is spread on glass fiber and dried, then stored at room temperature.

[0087] c2. Prepare the nitrocellulose membrane. Dilute the anti-2019-nCoV monoclonal antibody and Super C with coating buffer to the appropriate concentration, and use a membrane marker to coat the C and T regions of the nitrocellulose membrane. The coated nitrocellulose membrane is dried and stored at room temperature.

[0088] C3, Preparation of the large card. The absorption pad, the marker pad, and the sample pad were cut to a specified width, pasted on the PVC backing 350 with nitrocellulose membrane, and then stored at room temperature.

[0089] Finally, in the assembly of the finished product, the large card was cut into small strips, assembled in a plastic card shell, and then sealed in an aluminum foil bag together with the desiccant. The aluminum foil bag, the treatment solution, and other accessories such as the virus collection device 100 were packed in a box to assemble the respiratory virus rapid detection kit based on ZIF-8 material.

[0090] Table 1: Consistency analysis with the comparative reagent

[0091]

[0092] Clinical sensitivity = A / (A+C) x 100% = 97.67% (95% confidence interval: 91.91%-99.36%);

[0093] Clinical specificity = D / (B+D) x 100% = 98.21% (95% confidence interval: 93.72%-99.51%);

[0094] Clinical accuracy = (A+D) / (A+B+C+D) x 100% = 97.98% (95% confidence interval: 94.92%-99.21%);

[0095] In addition, the consistency coefficient (Kappa coefficient) of the detection results of the reagent and the clinical reference standard was calculated.

[0096] P0 = (A+D) / (A+B+C+D) = 0.98;

[0097] Pe = [(A+C)(A+B) / (A+B+C+D)+(B+D)(C+D) / (A+B+C+D)] / (A+B+C+D) = 0.5098;

[0098] Kappa coefficient = (P0-Pe) / (1-Pe) = 0.9592.

[0099] In order to verify the application effect of the new coronavirus detection kit based on ZIF-8 material in clinic, we selected 198 patients with suspected symptoms of new crown, and detected them with nucleic acid reagent and the reagent at the same time. The reagent detection and nucleic acid detection results were compared, of which 86 cases were positive in nucleic acid detection, and 112 cases were negative in nucleic acid detection. 86 cases were positive in reagent detection, and 112 cases were negative in reagent detection. Among them, 2 cases were positive in nucleic acid detection and negative in reagent detection, and 2 cases were negative in nucleic acid detection and positive in reagent detection. Compared with the results of nucleic acid detection, the clinical sensitivity of the reagent was 97.67%, the clinical specificity was 98.21%, and the clinical accuracy was 97.98%. The consistency coefficient Kappa value was 0.9592, which was greater than 0.9, indicating that the detection results of the reagent and the contrast reagent had high consistency.

[0100] In summary, by using the characteristics of ZIF-8 that are stable under different pH conditions, the device for rapidly collecting respiratory viruses using ZIF-8 material, releasing viruses under acidic conditions, and detecting viruses can achieve rapid collection, elution, and detection of respiratory viruses, thereby effectively improving the detection sensitivity.

[0101] The above-described embodiments are only used to illustrate the technical ideas and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and cannot be limited to the patent application range of the present application only by the above-described embodiments, that is, any equivalent changes or modifications made according to the spirit disclosed by the present application still fall within the patent application range of the present application.

Claims

1. A rapid respiratory virus detection kit based on ZIF-8 material, characterized in that, include: The virus collection device (100) includes an exhalation cylinder (110) and a ZIF-8 membrane (120). The ZIF-8 membrane (120) is detachably disposed inside the exhalation cylinder (110). The ZIF-8 membrane (120) includes a chitosan layer (121) on which ZIF-8 material is uniformly distributed to adsorb virus particles in the gas exhaled by the test subject through the exhalation cylinder (110). A sample processing device, comprising a processing solution, wherein the processing solution elutes and lyses virus particles adsorbed by the ZIF-8 material distributed on the chitosan layer (121) to obtain a virus processing solution. A test reagent (300) is used to test the virus treatment solution and provide test results; The treatment solution comprises an acetate buffer solution with a pH of 5.0–5.5, sodium thiocyanate, Triton X-100, and BSA, wherein the content of sodium thiocyanate is 0.01%–1%, the content of Triton X-100 is 0.01%–1%, and the content of BSA is 0.1%–10%; 40 mL of 400 mmol / L 2-methylimidazole methanol solution is added dropwise to 40 mL of 100 mmol / L... The ZIF-8 particles were prepared by diluting the ZIF-8 particles in methanol solution to 200 mL, stirring at room temperature for 6 h, centrifuging to obtain a white precipitate, washing the precipitate three times with methanol, and finally drying it under vacuum at 40 °C for 30 min. The ZIF-8 particles were dissolved in PBS with a pH of 7.4 and ultrasonically vibrated to obtain a solution. Glycerol was added to the solution and stirred. Chitosan was added to the product and stirred to obtain a film-forming solution. The film-forming solution was dried to form a film, resulting in the chitosan layer (121).

2. The rapid respiratory virus detection kit based on ZIF-8 material as described in claim 1, characterized in that, The exhalation pump (110) includes an exhalation port (111), a pump (112), and a diaphragm holder (113). The exhalation port (111) and the pump (112) are connected. The diaphragm holder (113) is used to hold the ZIF-8 diaphragm (120) and is detachably disposed inside the pump (112).

3. The rapid respiratory virus detection kit based on ZIF-8 material as described in claim 2, characterized in that, The diaphragm holder (113) includes two clamps (113a) that can be opened and closed, and the ZIF-8 diaphragm (120) is fixed between the two clamps (113a).

4. The rapid respiratory virus detection kit based on ZIF-8 material as described in claim 1, characterized in that, The ZIF-8 membrane (120) further includes a filter membrane layer (122), which is closely attached to the chitosan layer (121) and the two can be torn apart.

5. The rapid respiratory virus detection kit based on ZIF-8 material as described in claim 1, characterized in that, The detection reagent (300) is one of the following: colloidal gold immunochromatographic reagent, immunofluorescence chromatography reagent, chemiluminescence reagent, and electrochemical reagent.

6. A method for preparing a rapid respiratory virus detection kit based on ZIF-8 material, characterized in that, The rapid respiratory virus detection kit based on ZIF-8 material includes a virus collection device (100), a sample processing device, and a detection reagent (300). The virus collection device (100) includes an exhalation cylinder (110) and a ZIF-8 membrane (120). The ZIF-8 membrane (120) is detachably disposed inside the exhalation cylinder (110). The ZIF-8 membrane (120) includes a chitosan layer (121) on which ZIF-8 material is uniformly distributed. The method includes the following steps: Step a: Fabricate ZIF-8 membrane (120); Step b: Prepare the processing solution for the sample processing device; The ZIF-8 membrane (120) further includes a filter membrane layer (122), and step a includes: Step a1: Provide a chitosan layer (121); Step a2: The chitosan layer (121) and the filter membrane layer (122) are bonded together to obtain the ZIF-8 membrane (120). Step a1 includes: Step a11: Prepare ZIF-8 particles; Step a12: Dissolve the ZIF-8 particles in PBS with a pH of 7.4 and sonicate to obtain a solution. Add glycerol to the solution and stir. Step a13: Add chitosan to the product of step a12 and stir to obtain a film-forming solution; Step a14: Dry the film-forming solution to form a film, and obtain the chitosan layer (121). Step a11 includes: adding 40 mL of a 400 mmol / L 2-methylimidazole methanol solution dropwise to 40 mL of a 100 mmol / L... The ZIF-8 particles were prepared by diluting the ZIF-8 granules in a methanol solution to 200 mL with methanol, stirring at room temperature for 6 h, centrifuging to separate the precipitate, washing the precipitate three times with methanol, and finally drying it under vacuum at 40 °C for 30 min. Step b includes: Step b1: Mix anhydrous sodium acetate and water to obtain a mixed solution, add glacial acetic acid to the mixed solution and mix well; Step b2: Add sodium thiocyanate, Tween-20, Triton X-100, BSA, sodium caseinate and Proclin 300 to the product of step b1 in sequence, and stir evenly to obtain the treatment solution.

Images