A new type of immunochromatographic detection device

Abstract

The present invention provides a novel immunochromatographic detection device, which includes a sample pretreatment part. When used, the sample does not need to be diluted or lysed before use, and one-step detection can be achieved. The immunochromatographic detection device provided by the present invention is particularly suitable for rapid detection of the new coronavirus SARS-CoV-2. Samples can be collected by directly taking saliva samples from the mouth, which is convenient for patients to self-sample and self-test. The one-step method confirms the positive and negative nature of the new coronavirus. It is easy to use, simple and efficient, reduces the risk of virus transmission to medical staff and health care workers, and also reduces the use of personal protective equipment. It is suitable for various medical institutions or individuals to quickly diagnose the infection of the new coronavirus in the early stage.

Description

[0001] This application is a divisional application with application number 2020112540542, application date November 11, 2020, and invention name “A new type of immunochromatographic detection device”. Technical Field

[0002] The present invention belongs to the field of biological detection technology and relates to a novel immunochromatographic detection device, and in particular to a one-step immunochromatographic detection device including pretreatment for detecting the new coronavirus. Background Art

[0003] At present, the detection of the new coronavirus is mainly based on PCR-based viral nucleic acid detection. The detection principle is to use the unique gene sequence of the virus as the detection target, and through PCR amplification, the target DNA sequence we choose increases exponentially. The more target genes are amplified, the stronger the accumulated fluorescent signal. In samples without viruses, since there is no target gene amplification, the results of enhanced fluorescent signals cannot be detected. The detection of the new coronavirus through nucleic acid has high specificity and sensitivity, but this detection method has high technical requirements, and the specimens need special processing. It requires professional instruments and equipment such as PCR amplifiers and gel electrophoresis. The detection of the new coronavirus takes a long time and requires professional technicians to operate and judge the test results. It cannot be used for early preliminary screening at the grassroots level such as community hospitals, customs, and disease control centers.

[0004] At present, in response to the above problems, Chinese patent applications 202010184358.X and others have disclosed a fluorescent immunochromatographic device for detecting COVID-19 and a method of using the same, and Chinese patent applications 202010184382 and others have disclosed a colloidal gold immunochromatographic device for detecting COVID-19 and a method of using the same. The corresponding products can be applied to early preliminary screening at the grassroots level, but they still have certain operational defects, especially the method for detecting viral antigens requires the first dilution or lysis of the collected samples before further detection, that is, a two-step detection method is used to collect samples from the patient's oropharynx or nasopharynx, and after sampling, the collected samples are first processed in other auxiliary devices other than the detection device, and then the processed samples are added to the detection device for result detection. This type of sampling may increase the risk of virus transmission to medical staff and health care workers who lack sufficient personal protective equipment. The operation steps are not the most simplified, and it is easy to cause operational errors, which will have a certain impact on the test results. Therefore, there is an urgent need for an immunochromatographic detection device for the new coronavirus that is more suitable for self-collection and self-testing by patients, and is simpler and more convenient. Summary of the invention

[0005] In order to make up for the defects of the existing technology, the present invention provides a new type of immunochromatographic detection device, which includes a sample pretreatment part. When used, it is not necessary to dilute or lyse the sample for pretreatment, and one-step detection can be achieved. The one-step immunochromatographic detection device including the pretreatment part provided by the present invention can be used for the immunochromatographic detection of the new coronavirus SARS-CoV-2, and the positive and negative of the new coronavirus antigen can be diagnosed in one step to confirm the positive and negative of the new coronavirus. It is easy to use, fast and efficient, and suitable for various medical institutions or individuals to quickly diagnose the infection of the new coronavirus in the early stage.

[0006] The immunochromatographic detection device provided by the present invention can collect samples by directly taking saliva samples from the mouth. Compared with sampling in other upper respiratory tract sites, oral sampling is less invasive and less uncomfortable for patients. Collecting saliva samples is technically less complicated, so the risk of virus transmission to medical staff and health care workers can be reduced by shortening the operation time and allowing patients to collect samples by themselves under supervision. Self-collection by patients under supervision also reduces the use of personal protective equipment. Compared with existing methods, the one-step detection method is simpler and more market-demanding.

[0007] The technical solution provided by the present invention is:

[0008] On the one hand, the present invention provides a novel immunochromatographic detection device, which includes a sample collection site and a test strip, wherein the test strip comprises a sample pretreatment pad, and the sample collected in the sample collection site can be transferred to the sample pretreatment pad, wherein the sample pretreatment pad comprises a dry and fixed pretreatment reagent for virus sample lysis.

[0009] Furthermore, the test strip includes a bottom plate, a sample pretreatment pad, a sample pad, a conjugation pad, a reaction membrane and a water absorbent pad, wherein the sample pretreatment pad, the sample pad, the conjugation pad, the reaction membrane and the water absorbent pad are overlapped together in sequence; the sample collection part includes a sample collection block and a sampling slot.

[0010] In some embodiments, the sample pretreatment pad, the sample pad, the conjugate pad, the reaction membrane and the water absorbent pad are overlapped in sequence and are located on the bottom plate.

[0011] In some other embodiments, the sample pretreatment pad, sample pad, conjugation pad, reaction membrane and absorbent pad are overlapped in sequence, the sample pad, conjugation pad, reaction membrane and absorbent pad are located on the bottom plate, and the sample pretreatment pad is located outside the bottom plate.

[0012] Furthermore, the detection device also includes a card shell, the test strip is installed in the card shell, the front end of the card shell is provided with a sampling groove for accommodating the sample collection block, and the connection between the sample collection block and the test strip is provided with a filtering component.

[0013] Furthermore, the sample collection block is a sponge that can absorb samples, and a portion of the sample collection block is exposed from the sampling slot, and the exposed portion is covered with a sampling cover for squeezing the sample collection block so that the sample passes through the filter component to reach the sample pretreatment pad. The sample collection block can be sealed therein by the cooperation of the sample sampling cover and the sampling slot.

[0014] Furthermore, the card case includes a card case cover and a card case bottom, the card case bottom is provided with a test strip slot, and the front end of the test strip slot includes a pre-treatment pad slot. When the test strip is located in the test strip slot, the sample pre-treatment pad is exactly located in the pre-treatment pad slot.

[0015] The cartridge cover and the cartridge bottom are assembled together to accommodate the sample collection block and the test strip; and after the cartridge cover and the cartridge bottom are assembled together, a sampling slot is formed at the front end.

[0016] In some embodiments, the sample pretreatment pad can be placed separately from the test strip, that is, the sample pretreatment pad is placed alone in the pretreatment pad slot, and the test strip is placed in the test strip slot, and the starting end of the test strip sample pad overlaps with the end of the pretreatment pad. This embodiment also falls within the scope of protection of the present invention.

[0017] Furthermore, the filtering component is a filter screen, and the filter screen is located on the card cover and / or the card bottom, and the width of the filter screen is not greater than the width of the sample pre-treatment pad.

[0018] The cartridge cover is provided with a cartridge cover filter screen, the cartridge bottom is provided with a cartridge bottom filter screen, or only one of the two is provided. When the sampling cover is used to squeeze the sample sampling block, the sample flows through the filter screen to the pre-treatment tank area. The width of the filter screen is equivalent to the width of the sample pre-treatment pad, so that the sample can be just received by the pre-treatment pad after passing through the filter screen without causing leakage and waste.

[0019] Furthermore, a groove is provided on the sample collection block so that it can be fixed in the sampling slot at the front end of the card shell, and the distance between the sample collection block and the filter screen is 1 to 3 mm.

[0020] Furthermore, the distance between the sample collection block and the filter screen is 2 to 3 mm.

[0021] Furthermore, the sample is saliva, and the pretreatment liquid reagent is a lysis reagent for viruses in the saliva sample.

[0022] Furthermore, the lysis reagent comprises: 0.01 mol / L phosphate buffer, 3% casein by mass, 0.5-1.5% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass, wherein the pH of the phosphate buffer is 7.5-8.5.

[0023] Furthermore, the lysis reagent comprises: 0.01 mol / L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass, wherein the pH of the phosphate buffer is 7.5-8.5.

[0024] Furthermore, the marking pad includes colloidal gold or fluorescent marking.

[0025] Furthermore, the preparation method of the pre-treatment pad is:

[0026] 1) Prepare phosphate buffer and adjust the pH to 7.5-8.5;

[0027] 2) preparing a sample pretreatment solution by mixing a certain mass fraction of the above-mentioned phosphate buffer (e.g., 0.01-0.05 mol / L), a certain mass fraction of casein (e.g., 3%-5.5%), a certain mass fraction of Triton X-100 (e.g., 0.01%-1%), and a certain mass fraction of proclin 300 (e.g., 0.01%-0.1%); 3) evenly dispersing the treatment solution on the glass fiber, drying it in a drying oven at 37°C overnight, taking it out and placing it in an aluminum foil bag, sealing it and setting it aside.

[0028] Furthermore, the sample pad is treated with a sample pad treatment solution, and the sample pad treatment solution includes 0.05M pH 8.0 boric acid buffer, casein, surfactant S17, bile acid, and EDTA.

[0029] Furthermore, the conjugate pad is coated with a monoclonal antibody against the novel coronavirus NP protein labeled with colloidal gold particles (or fluorescent microspheres).

[0030] Furthermore, the reaction membrane is a nitrocellulose membrane, and a detection line and a quality control line are sequentially arranged on the reaction pad along the sample flow direction, the detection line is coated with a monoclonal antibody against the NP protein of the new coronavirus, and the quality control line is coated with a goat anti-mouse polyclonal antibody.

[0031] Furthermore, the novel coronavirus NP protein monoclonal antibody is a mouse anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody.

[0032] On the other hand, the present invention provides a novel coronavirus SARS-CoV-2 antigen immunochromatographic saliva detection kit and detection method, comprising the following steps:

[0033] 1) Return the test kit to room temperature, take out a test device from the sealed aluminum foil bag, and gently pull open the sampling cover to expose the front end of the sample sampling block.

[0034] 2) Place the front end of the sample collection block into the oral cavity and hold it there for 3 to 5 minutes.

[0035] 3) Put on the collection cover and press the cover horizontally 1 to 2 times.

[0036] 4) The saliva sample enters the pretreatment pad, the analyte in the saliva reacts with the reagent on the pretreatment pad, and then enters the sample pad by capillary action, and then enters the reaction membrane through the binding pad by capillary action. The reaction complex diffuses forward along the reaction membrane and is captured by the coated antibody fixed on the detection line, thereby displaying a color signal visible to the naked eye at the coated antibody, or in other embodiments, displaying a signal change measurable by an instrument at the coated antibody, and the waste liquid of the chromatography reaction is collected by absorbent paper, pulling the chromatography reaction in that direction.

[0037] 5) Read the test results through the inspection window for 15 to 20 minutes. If both the quality control line C and the detection line T are purple-red, the result is positive; if only the quality control line C is purple-red and the detection line T is not colored, the result is negative; if both the quality control line C and the detection line T are not purple-red, the result is invalid; if the quality control line C does not show purple-red, even if the detection line T shows purple-red, the test result is invalid. Or in other embodiments, the instrument detects the signals of the detection line T and the quality control line C, and when the conversion of the two reaches a certain value, it is determined to be positive.

[0038] On the other hand, the present invention provides the use of stearic acid monoethanolamide in preparing a virus lysis reagent, characterized in that the lysis reagent comprises: 0.01 mol / L phosphate buffer, 3% casein by mass, 0.5-1.5% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass, wherein the pH of the phosphate buffer is 7.5-8.5.

[0039] Furthermore, the lysis reagent comprises: 0.01 mol / L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass, wherein the pH of the phosphate buffer is 7.5-8.5.

[0040] Furthermore, the virus is a new coronavirus.

[0041] The beneficial effects of the present invention are as follows: the present invention provides a novel immunochromatographic detection device, which includes a sample pretreatment part. When used, the sample does not need to be diluted or lysed, and pretreatment such as lysing can be performed, and one-step detection can be achieved. The immunochromatographic detection device provided by the present invention is particularly suitable for immunochromatographic detection of the new coronavirus SARS-CoV-2. Samples can be collected by directly taking saliva samples from the mouth, which is convenient for patients to self-sample and self-test. The positive and negative of the new coronavirus can be confirmed in one step. It is easy to use, fast and efficient, and reduces the risk of virus transmission to medical staff and health care workers. It also reduces the use of personal protective equipment, and is suitable for various medical institutions or individuals to quickly diagnose the infection of the new coronavirus in the early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the structure of the novel immunochromatographic detection device in Example 1;

[0043] Figure 2 This is a diagram showing the structural breakdown of each layer of the novel immunochromatographic detection device in Example 1;

[0044] Figure 3 This is a structural breakdown diagram of the sample collection part of the novel immunochromatographic detection device in Example 1;

[0045] Figure 4 The colloidal gold standard colorimetric card in Example 4;

[0046] Figure 5 This is a correlation diagram of the signal average values ​​of the T line of the detection line without a pre-treatment pad and with a pre-treatment pad in Example 5.

[0047] Detailed description

[0048] The following further describes the structures or technical terms involved in the present invention. If not otherwise specified, they are understood and interpreted according to the general terms commonly used in the art. These descriptions are merely examples of how the present invention is implemented and do not constitute any limitation to the present invention. The scope of the present invention is defined and expressed by the claims.

[0049] Detection

[0050] Detection means to test or examine a substance or material for its presence or absence. The substance or material may be, for example, but not limited to, a chemical substance, an organic compound, an inorganic compound, a metabolite, a drug or a drug metabolite, an organic tissue or a metabolite of an organic tissue, a nucleic acid, a protein or a polymer. In addition, detection may also mean the amount of the substance or material tested. Assays may also refer to immunoassays, chemical assays, enzyme assays, etc.

[0051] sample

[0052] In the present invention, the sample used in the detection device includes biological liquid. The initial state of the sample can be liquid, solid or semi-solid. The solid or semi-solid sample can be converted into a liquid sample by any appropriate method, such as mixing, crushing, macerating, incubating, dissolving, enzymolysis, etc., and then poured into the collection chamber, and the sample is detected by the test element to determine whether it contains the analyte. The sample can be taken from the human body, animals, plants, nature, etc. Samples taken from the human body can be, for example, liquid samples such as blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, gastric juice, etc.; solid or semi-solid samples such as feces, hair, keratin, tartar, finger / toe nails, etc. Samples taken from plants can be, for example, solid samples such as roots, stems, leaves, etc.; liquid or semi-solid samples such as tissue fluid and cell fluid prepared from roots, stems, and leaves. Samples taken from nature can be, for example, liquid samples such as rainwater, river water, seawater, groundwater, etc.; solid or semi-solid samples such as soil, rocks, ores, and petroleum.

[0053] In some embodiments, the sample described in the present invention is saliva, which is more convenient for self-sampling and testing.

[0054] Test equipment

[0055] The test device generally includes a test element, and the so-called test element refers to a component that can detect the analyte in the sample to be tested. The detection of the analyte by the test element can be based on any technical principle, such as immunology, chemistry, electricity, optics, molecular science, physics, etc. The test element of the present invention can be one or a combination of two or more test elements. The test element has a detection area for displaying the test result, and after the test is performed, the test area displays the test result.

[0056] Various test elements can be combined and applied to the present invention. One form is a test strip. The test strip used to analyze the analyte in the sample (such as drugs or metabolites indicating physical conditions) can be in various forms, such as immunoassays or chemical analysis. The test strip can adopt a non-competitive or competitive analysis mode. The test strip comprises a water-absorbing material with a sample application area, a reagent area and a test area. The sample is added to the sample application area and flows to the reagent area through capillary action. In the reagent area, if the analyte is present, the sample binds to the reagent. The sample then continues to flow to the detection area. Other reagents, such as molecules that specifically bind to the analyte, are fixed in the detection area. These reagents react with the analyte in the sample (if present) and bind the analyte to the area, or bind to a reagent in the reagent area. The marker used to display the detection signal is present in the reagent area or a separate labeling area.

[0057] The typical non-competitive assay format is that if the sample contains the analyte, a signal is generated, and if it does not contain the analyte, no signal is generated. In a competitive assay, if the analyte is not present in the sample, a signal is generated, and if the analyte is present, no signal is generated.

[0058] The test element can be a test paper, which can be made of absorbent or non-absorbent materials. The test paper can include multiple materials for liquid sample delivery. One material of the test paper can be covered on another material, such as filter paper covered on a nitrocellulose membrane. One area of ​​the test paper can be made of one or more materials, while another area can be made of one or more different materials. The test paper can be adhered to a certain support or hard surface to increase the strength of holding the test paper.

[0059] The analyte is detected through a signal generating system, such as by using one or more enzymes that react specifically with the analyte, and by using the method of fixing the specific binding substance on the test paper as described above, to fix the composition of one or more signal generating systems on the analyte detection area of ​​the test paper. The substance that generates the signal can be in the sample addition area, the reagent area, or the detection area, or on the entire test paper, and the substance can be filled with one or more materials of the test paper. A solution containing the signal substance is added to the surface of the test paper or one or more materials of the test paper are immersed in a solution containing the signal substance. The test paper to which the solution containing the signal substance is added is dried.

[0060] The various zones of the test strip can be arranged in the following manner: sample addition zone, reagent zone, test zone, control zone, zone for determining whether the sample is adulterated, and liquid sample absorption zone. The control zone is located after the test zone. All zones can be arranged on a test strip using only one material. Different materials can also be used in different zones. Each zone can be directly in contact with the liquid sample, or different zones can be arranged according to the direction of liquid sample flow, with the end of each zone connected to the front end of another zone and overlapping. The material used can be a material with good water absorption such as filter paper, glass fiber or nitrocellulose membrane. The test strip can also be in other forms.

[0061] The test strip applied to the present invention can be a so-called lateral flow test strip, and the specific structure and detection principle of these test strips are well-known technologies for those skilled in the art in the prior art. Common test strips include a sample collection area, a labeling area, a test area and a water absorption area, wherein the sample collection area includes a sample receiving pad, the labeling area includes a labeling pad, and the water absorption area can include a water absorption pad, wherein the test area includes necessary chemical substances that can detect whether the analyte is contained, such as an immunoreagent or an enzyme chemical reagent. The commonly used test strip is a nitrocellulose membrane test strip, that is, the test area includes a nitrocellulose membrane, and specific binding molecules are fixed on the nitrocellulose membrane to display the test result; it can also be a cellulose acetate membrane or a nylon membrane, etc., and of course, the test result control area can also be included downstream of the test area, and usually, the control area and the test area appear in the form of horizontal lines, which are test lines or control lines. Such a test strip is a traditional test strip, and of course, it can also be other types of test strips that utilize capillary action to detect. In addition, the general detection reagent strip carries dry chemical reagent components, such as fixed antibodies or other reagents. When encountering liquid, the liquid flows along the reagent strip with capillary action. As it flows, the dry reagent components are dissolved in the liquid, and then processed in the next area to react with the dry reagent in the area, thereby performing necessary tests. The flow of liquid is mainly carried out by capillary action. These test elements are described and recorded in the following documents: "Regeneration of Nitrocellulose Membrane and Study on Its Protein Adsorption Ability" by Li Fugang; "Analysis of Chromatographic Membrane Material Performance in Colloidal Gold Diagnostic Kit" by Ma Hongyan, Li Qiang, etc.; "A New Colloidal Gold Immunochromatographic Test Strip" by Wang Yong, Wang Luhai, etc. They can all be used in the detection device of the present invention, or be arranged in the detection chamber to contact the liquid sample, or be used to detect the presence or amount of the analyzed substance in the liquid sample entering the detection chamber. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the examples. It should be noted that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. The reagents not particularly specified in the present examples are all known products and were obtained by purchasing commercially available products.

[0063] Example 1 A novel immunochromatographic detection device provided by the present invention

[0064] The novel immunochromatographic detection device provided in this embodiment is as follows Figure 1-3 As shown, Figure 1 This is a schematic diagram of the structure of a new immunochromatographic detection device. Figure 2 This is a diagram showing the structural breakdown of each layer of the new immunochromatographic detection device. Figure 3This is a structural breakdown diagram of the sample collection site of the new immunochromatographic detection device.

[0065] like Figure 1 The present invention provides a novel immunochromatographic detection device, which includes a sample collection part 1 and a test strip 2, wherein the test strip 2 includes a sample pretreatment pad 3, and the sample collected in the sample collection part 1 can be transferred to the sample pretreatment pad 3, and the sample pretreatment pad 3 has a dry and fixed sample pretreatment liquid.

[0066] Preferably, the test strip 1 comprises a bottom plate 4, a sample pretreatment pad 3, a sample pad 5, a conjugation pad 6, a reaction membrane 7 and a water absorbent pad 8, which are overlapped in sequence and located on the bottom plate 4. In some embodiments, only the sample pad 5, the conjugation pad 6, the reaction membrane 7 and the water absorbent pad 8 may be located on the bottom plate, and the sample pretreatment pad 3 may be located outside the bottom plate 4. The sample collection portion 1 comprises a sample collection block 9 and a sampling slot 10.

[0067] Preferably, the detection device further comprises a cartridge 11 , in which the test strip 2 is installed, a sampling slot 10 for accommodating the sample collection block 8 is provided at the front end of the cartridge 11 , and a filter component 12 is provided at the connection between the sample collection block 9 and the test strip 2 .

[0068] Preferably, the sample collection block 9 is a sponge that can absorb samples, and a portion of the sample collection block 9 is exposed from the sampling slot 10, and the exposed portion is covered with a sampling cover 13, which is used to squeeze the sample collection block 9 so that the sample passes through the filter component to reach the sample pre-processing pad 3. The sample collection block 9 can be sealed therein by the cooperation of the sampling cover 13 and the sampling slot 10.

[0069] Preferably, the cartridge 11 includes a cartridge cover 14 and a cartridge bottom 15, the cartridge bottom 15 is provided with a test strip slot 16, the front end of the test strip slot 16 includes a pre-treatment pad slot 17, when the test strip 2 is located in the test strip slot 16, the sample pre-treatment pad 3 is just located in the pre-treatment pad slot 17. The cartridge cover 14 and the cartridge bottom 15 are assembled together to accommodate the sample collection block 9 and the test strip 2; and after the cartridge cover 14 and the cartridge bottom 15 are assembled together, the front end forms a sampling slot 10. In some embodiments, the sample pre-treatment pad 3 can be placed separately from the test strip 2, that is, the sample pre-treatment pad 3 is placed separately in the pre-treatment pad slot 17, the test strip 2 is placed in the test strip slot 16, and the starting end of the sample pad 5 overlaps with the end of the pre-treatment pad 3.

[0070] Preferably, the filter component 12 is a filter screen 18 , and the filter screen 18 is located on the housing cover 14 and / or the housing bottom 15 . The width of the filter screen 18 is not greater than the width of the sample pre-treatment pad 3 .

[0071] Preferably, a groove 19 is provided on the sample collection block 9 so that it is fixed in the sampling slot 10 , and the distance between the sample collection block 9 and the filter screen 18 is 1 to 3 mm.

[0072] Preferably, the distance between the sample collection block 9 and the filter screen 18 is 2-3 mm.

[0073] Preferably, the sample is saliva.

[0074] Preferably, the sample pretreatment solution is a lysis solution for lysing viruses.

[0075] Preferably, the virus is a new coronavirus.

[0076] Example 2 Preparation of an Immunochromatographic Detection Device (Colloidal Gold Method) for Detecting Novel Coronavirus

[0077] The preparation steps of the immunochromatographic test device (colloidal gold method) for detecting the new coronavirus SARS-CoV-2 provided in this embodiment include:

[0078] Step 1: Prepare the sample pretreatment pad

[0079] 1) Prepare phosphate buffer and adjust the pH to 7.5.

[0080] 2) A certain mass fraction of the above phosphate buffer: 0.01 mol / L, a certain mass fraction of casein: 3%, a certain mass fraction of Triton X-100: 1%, and a certain mass fraction of proclin 300: 0.01% are prepared into a pretreatment solution.

[0081] 3) Disperse the pretreatment solution evenly on the glass fiber, dry it in a drying oven at 37°C overnight, take it out and place it in an aluminum foil bag and seal it for later use.

[0082] Step 2: Sample pad processing

[0083] 1) Prepare boric acid buffer and adjust the pH to 7.5.

[0084] 2) Take a certain mass fraction of boric acid buffer: 0.01 mol / L), a certain mass fraction of casein: 0.1%, a certain mass fraction of surfactant S17: 1%, a certain mass fraction of bile acid: 0.1%, and a certain mass fraction of EDTA: 0.6% to prepare a sample pad treatment solution.

[0085] 3) Disperse the sample pad treatment solution evenly on the glass fiber, dry it in a drying oven at 37°C overnight, take it out, and then seal it in an aluminum foil bag for later use.

[0086] Step 3: Bonding pad treatment

[0087] 1) Adjust the pH of the colloidal gold solution to 9.0, mix and react with the novel coronavirus NP protein monoclonal antibody to obtain a labeled antibody, and re-dissolve it with a re-solution.

[0088] 2) The labeled antibody reconstituted solution prepared in 1) is uniformly sprayed onto the untreated glass fiber at a certain mass fraction (eg, 2 uL / cm) using a film spraying apparatus.

[0089] 3) Place the sprayed glass fiber in a 37°C drying oven to dry overnight, take it out and seal it in an aluminum foil bag for later use.

[0090] Step 4: Scratching the nitrocellulose membrane

[0091] 1) Prepare phosphate buffer and adjust the pH to 8.

[0092] 2) Take a certain mass fraction of phosphate buffer (e.g. 0.1 mol / L) to dilute the novel coronavirus NP protein monoclonal antibody to 1.0 mg / ml as the T-line solution.

[0093] 3) Take a certain mass fraction of phosphate buffer (e.g. 0.1 mol / L) to dilute the goat anti-mouse polyclonal antibody to 0.5 mg / ml as the C-line solution.

[0094] 4) Use a film-scratching gold spraying instrument to scrape the film at a certain mass fraction (such as 0.5uL / cm).

[0095] 5) Place the scribed membrane in a 37°C drying oven to dry overnight, then take it out and seal it in an aluminum foil bag for later use.

[0096] Step 5: Assembling, cutting and bagging

[0097] 1) Cut the pretreatment pad obtained in step 1, the sample pad treated in step 2, the binding pad obtained in step 3, the nitrocellulose membrane treated in step 4, and the absorbent paper into appropriate widths and stick them to the base plate in sequence, with each layer being 1-2 mm apart. The width of the pretreatment pad is greater than or equal to the width of the sample pad.

[0098] 2) Use a chopper to cut the glued large card into 3-6mm wide test cards and put them into the card shell.

[0099] 3) Fix the sample collection block at the front end of the cartridge.

[0100] 4) Put on the sample collection cover.

[0101] 5) Store in an aluminum foil bag and seal.

[0102] Preferably, the pre-treatment pad in step 5 can also be glued to the bottom plate and cut separately, or without the support of the bottom plate, and the width of the pre-treatment pad is greater than or equal to the width of the sample pad.

[0103] Example 3: Preparation of another immunochromatographic detection device (colloidal gold method) for detecting novel coronavirus

[0104] The same as Example 2, the difference is that stearic acid monoethanolamide is used instead of TritonX-100 in the preparation of the pretreatment pad, and the other ingredients are the same as the other contents in Example 2, and are divided into three formulations for preparing the pretreatment pad according to the different mass fractions of stearic acid monoethanolamide.

[0105] Formula 1: 0.01 mol / L phosphate buffer, 3% casein by mass, 0.5% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass.

[0106] Formula 2: 0.01 mol / L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass.

[0107] Formulation 3: 0.01 mol / L phosphate buffer, 3% casein by mass, 1.5% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass.

[0108] Example 4: Preparation of an immunochromatographic detection device (fluorescence method) for detecting novel coronavirus

[0109] The preparation steps of the immunochromatographic test device (fluorescence method) for detecting the new coronavirus SARS-CoV-2 provided in this embodiment include:

[0110] Step 1: Prepare the pre-treatment pad

[0111] 1) Prepare phosphate buffer and adjust the pH to 8.5.

[0112] 2) A certain mass fraction of the above phosphate buffer: 0.01 mol / L, a certain mass fraction of casein: 3%, a certain mass fraction of Triton X-100: 1%, and a certain mass fraction of proclin 300: 0.01% are prepared into a pretreatment solution.

[0113] 3) Disperse the pretreatment solution evenly on the glass fiber, dry it in a drying oven at 37°C overnight, take it out and place it in an aluminum foil bag and seal it for later use.

[0114] Step 2: Sample pad processing

[0115] 1) Prepare phosphate buffer and adjust the pH to 8.5.

[0116] 2) A certain mass fraction of phosphate buffer: 0.01 mol / L), a certain mass fraction of bovine serum albumin: 0.1%, a certain mass fraction of surfactant Triton X-100: 1%, and a certain mass fraction of povidone K30: 0.05% are prepared into a sample pad treatment solution.

[0117] 3) Disperse the sample pad treatment solution evenly on the glass fiber, dry it in a drying oven at 37°C overnight, take it out, and then seal it in an aluminum foil bag for later use.

[0118] Step 3: Bonding pad treatment

[0119] 1) Adjust the pH of the fluorescent microsphere solution to 8.5, mix and react with the novel coronavirus NP protein monoclonal antibody to obtain an antibody marker, and re-dissolve it with a re-solution.

[0120] 2) Prepare phosphate buffer and adjust the pH to 8.5.

[0121] 3) A certain mass fraction of phosphate buffer (eg, 0.01 mol / L), a certain mass fraction of bovine serum albumin (eg, 0.1%), a certain mass fraction of sucrose (eg, 0.1%), and a certain mass fraction of proclin300 (eg, 0.01%) are prepared into a binding pad treatment solution.

[0122] 4) Disperse the treatment solution evenly on the glass fiber, dry it in a drying oven at 37°C overnight, take it out, and then seal it in an aluminum foil bag for later use.

[0123] 5) The antibody marker complex solution in 1) is sprayed onto the glass fiber in 4) at a certain mass fraction (e.g., 1 uL / cm) using a film spray gold apparatus, placed in a 37° C. drying oven overnight, taken out, and sealed in an aluminum foil bag for later use.

[0124] Step 4: Scratching nitrocellulose membrane (reaction membrane)

[0125] 1) Prepare phosphate buffer and adjust the pH to 8.

[0126] 2) Take a certain mass fraction of phosphate buffer (e.g. 0.1 mol / L) and dilute the novel coronavirus NP protein monoclonal antibody to 1.0 mg / ml as the T-line solution.

[0127] 3) Take a certain mass fraction of phosphate buffer (eg, 0.1 mol / L) and dilute the goat anti-mouse polyclonal antibody to 0.5 mg / ml as the C-line solution.

[0128] 4) Use a film-scratching gold spraying instrument to perform film-scratching at a certain mass fraction (e.g., 0.5uL / cm).

[0129] 5) Place the scribed membrane in a 37°C drying oven to dry overnight, then take it out and seal it in an aluminum foil bag for later use.

[0130] Step 5: Assembling, cutting and bagging

[0131] 1) Cut the pre-treatment pad obtained in step 1, the sample pad treated in step 2, the conjugate pad obtained in step 3, the nitrocellulose membrane treated in step 4, and the absorbent paper into appropriate widths and stick them to the base plate in sequence, with each layer firmly overlapped by 1-2 mm.

[0132] 2) Use a chopper to cut the glued large card into 3-6mm wide test cards and put them into the card shell.

[0133] 3) Fix the sample collection block at the front end of the cartridge.

[0134] 4) Put on the sample collection cover.

[0135] 5) Store in an aluminum foil bag and seal.

[0136] Furthermore, the pre-treatment pad in step 5 can also be glued to the base plate and cut separately, or without the support of the base plate, and the width of the pre-treatment pad is greater than or equal to the width of the sample pad.

[0137] Example 5 Preparation of another immunochromatographic detection device (fluorescence method) for detecting novel coronavirus

[0138] The same as Example 4, the difference is that stearic acid monoethanolamide is used to replace TritonX-100 in the preparation of the pretreatment pad, and the following three formulas are prepared according to different mass fractions, and the other ingredients are the same as Example 4:

[0139] Formula A: 0.01 mol / L phosphate buffer, 3% casein by mass, 0.5% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass.

[0140] Formula B: 0.01 mol / L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass.

[0141] Formula C: 0.01 mol / L phosphate buffer, 3% casein by mass, 1.5% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass.

[0142] Example 6: Validation of the immunochromatographic detection device for detecting novel coronavirus prepared by the colloidal gold method provided in Example 2

[0143] The virus culture was diluted 100-fold, 200-fold, 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, and 9600-fold to 1.51×106 TCID50 / mL respectively as the test samples. After the dilution factor was greater than 6400-fold in the existing system, a negative result would appear when interpreting the signal of the test line T line. When interpreting the test results, a standard colorimetric card (see Figure 4 ) was used for comparison.

[0144] As Figure 4 shown, if the test line T≥G3, the test result is positive; if the test line T<G3, the test result is negative, and the quality control line is always C≥G8; if the quality control line C<G8 or does not develop color, even if the test line T develops color, the test result will be invalid.

[0145] It was carried out in three groups. The first group directly used a saliva detection kit without a pretreatment pad (without sample lysis either) (specific results are shown in Table 1). The second group first performed sample lysis treatment and then used a saliva detection kit without a pretreatment pad (specific results are shown in Table 2). The third group used the saliva detection kit with a pretreatment pad provided in Example 2 for detection (specific results are shown in Table 3). Each treatment was repeated 20 times, and a standard colorimetric card was used to interpret the test results (G represents the depth of color to interpret the test results). The test strip structures and reagents of the first group, the second group, and the third group were the same, except for the following differences: the first group did not undergo pretreatment; the second group had pretreatment, but the test strip was still a traditional test strip without a pretreatment pad, and the third group was a test strip including a pretreatment pad.

[0146] The specific experimental process is as follows:

[0147] 1) The detection device was rewarmed at 15-30°C.

[0148] 2) The virus culture was diluted 100-fold, 200-fold, 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, and 9600-fold respectively.

[0149] 3) After collecting the saliva samples, the diluted virus culture was added to the saliva samples according to the gradients in 2) respectively to obtain 8 samples. The virus culture concentrations of the samples were 1 / 9600, 1 / 6400, 1 / 3200, 1 / 1600, 1 / 800, 1 / 400, 1 / 200, and 1 / 100 respectively. In addition, there was another sample without adding the virus culture, and its culture concentration was 0.

[0150] 4) The existing detection device without a pretreatment pad and the supporting lysis solution were taken out from the sealed aluminum foil bag; the detection device with a pretreatment pad provided in Example 2 was taken out from the sealed aluminum foil bag and placed on a clean and flat surface.

[0151] 5) Each sample in 2) is divided into three parts, wherein the second part is added to the lysis solution of the existing detection device without the pre-treatment pad, and the first part and the third part are not added to the lysis solution.

[0152] 6) Take 60 microliters of the sample without lysis solution and drip it onto the detection device of the first group; take 60 microliters of the sample with lysis solution and drip it onto the detection device of the second group; open the sample sampling cover of the detection device of the third group with the pretreatment pad, expose the sample sampling block, place it in the sample without lysis solution and let it stand for a certain time (e.g., 1-2 minutes), then cover the sample sampling cover and press it horizontally 1-2 times.

[0153] 7) Place the container on a horizontal table for 15 to 20 minutes at room temperature and read the test results through the inspection window.

[0154] The test results are shown in Table 1, Table 2, and Table 3. When the dilution multiple is the same, the first group shows significantly weaker results, and it is difficult to detect the true results (without lysis); the color development results of the second and third groups are consistent, and there is no difference in chromaticity. Therefore, it is feasible to use the detection device of the present invention to detect viruses. A pretreatment pad is set in front of the sample pad on the test strip, which includes a lysis reagent, and can truly realize the direct one-step detection of viruses without pretreatment such as lysis of the sample. Such a detection device does not require additional pretreatment steps, and is directly added to the test strip, which reduces the steps and reduces the possible secondary contamination of the operator caused by the pretreatment steps.

[0155] Table 1 Results of testing by adding samples directly to existing detection devices without lysis

[0156]

[0157] Table 2 Results of testing after sample lysis and addition to existing detection device

[0158]

[0159] Table 3 Results of testing by adding samples directly into the detection device of the present invention

[0160]

[0161] Example 7 Validation of another immunochromatographic detection device for detecting novel coronavirus prepared by the colloidal gold method provided in Example 3

[0162] The virus culture was diluted 100-fold, 200-fold, 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, and 9600-fold to 1.51×106 TCID50 / mL as the test samples. After the dilution factor was greater than 6400-fold in the existing system, a negative result would appear when reading the signal of the test line T line. When reading the test results, a standard color comparison card (see Figure 4 ) was used for comparison.

[0163] If Figure 4 as shown, if the test line T≥G3, the test result is positive; if the test line T<G3, the test result is negative, and the quality control line is always C≥G8; if the quality control line C<G8 or is not colored, even if the test line T is colored, the test result will be invalid.

[0164] The detection devices using pretreatment pads prepared with Formulation 1, Formulation 2, and Formulation 3 were divided into three groups. Among them, Formulation 1 corresponded to the fourth group (0.5%), Formulation 2 corresponded to the fifth group (1%), and Formulation 3 corresponded to the sixth group (1.5%). The same saliva samples as in Example 6 were collected for testing, and each group was repeated 20 times. A standard color comparison card was used to read the test results.

[0165] The specific experimental procedures 1) to 3) were the same as those in Example 6, and the subsequent procedures were as follows:

[0166] 4) The detection devices using pretreatment pads prepared with Formulation 1, Formulation 2, and Formulation 3 were taken out from the sealed aluminum foil bags and placed on a clean and flat surface.

[0167] 5) Each sample in 3) was divided into three parts. The sample sampling covers of the detection devices with pretreatment pads in the fourth group, fifth group, and sixth group were opened in sequence, the sample sampling blocks were exposed and placed into the samples. One group corresponded to one sample. After standing for the same time as in Example 6, the sample sampling covers were covered, and pressed horizontally 1-2 times.

[0168] 7) After being placed on a horizontal table at room temperature for 15-20 min, the test results were read through the viewing window.

[0169] The test results are shown in Table 4, Table 5, and Table 6. When Triton X-100 is replaced with stearic acid monoethanolamide, the minimum detection threshold still has a positive detection rate of 95% when diluted 9600 times. Compared with Table 3, the minimum detection threshold is greatly improved. When the current treatment pad is prepared using Formula 2, that is, when the content of stearic acid monoethanolamide is 1%, the positive rate of the test result is the highest, and the virus is released more completely. In addition, it was found that when diluted 9600 times, when the content of stearic acid monoethanolamide is 1%, the positive detection rate is 100%. This fully demonstrates that the addition of stearic acid monoethanolamide in the lysis reagent treated on the sample pre-treatment pad is critical to the lysis effect, and its mass fraction content is preferably 1%.

[0170] Table 4 Results of testing by adding samples directly to the test device of Formula 1 of Example 3

[0171]

[0172] Table 5 Results of testing by adding samples directly to the test device of formula 2 of Example 3

[0173]

[0174] Table 6 Results of testing by adding samples directly to the test device of formula 3 of Example 3

[0175]

[0176] Example 8 Validation of the Immunochromatographic Detection Device for Detecting Novel Coronavirus Prepared by the Fluorescence Method Provided in Example 4

[0177] The virus culture 1.51×106TCID50 / mL was diluted 500 times, 1000 times, 2000 times, 4000 times, 5000 times, 7500 times, 10000 times, and 12000 times as the test samples. In the existing system, when the dilution multiple was greater than 10000 times, the T line signal value was close to the background value and was judged to be negative.

[0178] Two groups were divided into two groups, wherein the first group was subjected to sample lysis treatment before using the saliva detection kit without a pre-treatment pad, and the second group was tested using the saliva detection kit with a pre-treatment pad provided in Example 4, which was repeated 3 times and tested using a fluorescent immunoassay analyzer. The specific experimental process is as follows:

[0179] 1) The detection device is rewarmed at 15-30℃.

[0180] 2) The virus culture was diluted to 500 times, 1000 times, 2000 times, 4000 times, 5000 times, 7500 times, 10000 times, and 12000 times, respectively, to obtain 8 samples in total. The virus culture concentrations of the samples were 1 / 500, 1 / 1000, 1 / 2000, 1 / 4000, 1 / 5000, 1 / 7500, 1 / 10000, and 1 / 12000 for saliva samples, respectively. In addition, there was another sample to which no virus culture was added, and its culture concentration was 0.

[0181] 3) After collecting the saliva samples, the diluted virus culture was added according to the gradient in 2).

[0182] 4) Take out the existing detection device without the pre-treatment pad and the matching lysis solution from the sealed aluminum foil bag, take out the detection device with the pre-treatment pad from the sealed aluminum foil bag, and place them on a clean and horizontal surface.

[0183] 5) The sample in 3) is divided into two parts, wherein the first part is added to the lysis solution of the existing detection device without the pre-treatment pad, and the second part is not added to the lysis solution.

[0184] 6) Take a certain amount of sample with lysis solution (e.g., 60 μl) and drip it onto the detection device of the first group; open the sample sampling cover of the detection device of the second group with the pretreatment pad, expose the sample sampling block and place it in the sample that has not been added to the lysis solution and let it stand for a certain period of time (e.g., 1-2 minutes), then cover the sample sampling cover and press it horizontally 1-2 times.

[0185] 7) Place on a horizontal table at room temperature and read the test results using a fluorescent immunoassay analyzer after 15 minutes.

[0186] The test results are shown in Tables 7, 8 and 9. The C line signals of the quality control line of the two groups of test results are basically stable. When the dilution multiples are the same, the T line signals of the test line are basically similar. The average value (Mean T) of the T line signals of the two groups of data is taken for linear correlation, as shown in Tables 9 and Figure 5 As shown, the correlation R2 of the two sets of data is 0.9998, which is highly correlated data. Therefore, it is feasible to use the detection device of the present invention to perform fluorescent immunochromatographic virus detection, which can truly realize the one-step detection of viruses without pre-treatment such as lysis of the sample. In this way, no additional pre-treatment steps are required, which reduces the possible secondary contamination of the operator caused by the pre-treatment steps. The pre-treatment pad set in front of the test strip sample pad includes a lysis reagent, which can achieve the same effect as independent lysis.

[0187] Table 7 Test results of the test device without pre-treatment pad

[0188]

[0189] Table 8 Test results of the pre-treatment pad test device of the present invention

[0190]

[0191] Table 9 Comparison of the average signal values ​​of the T line of the detection line without pre-treatment pad and with pre-treatment pad

[0192]

[0193] Example 9 Validation of another immunochromatographic detection device for detecting novel coronavirus prepared by the fluorescence method provided in Example 5

[0194] The virus culture 1.51×106TCID50 / mL was diluted 500 times, 1000 times, 2000 times, 4000 times, 5000 times, 7500 times, 10000 times, and 12000 times as the test samples. In the existing system, when the dilution multiple was greater than 10000 times, the T line signal value was close to the background value and was judged to be negative.

[0195] The detection devices using the pretreatment pads prepared using formula A, formula B, and formula C were divided into three groups, formula A corresponding to group I, formula B corresponding to group II, and formula C corresponding to group III. The same saliva samples as in Example 8 were collected for testing, repeated 3 times, and tested using a fluorescent immunoassay analyzer.

[0196] The specific experimental process 1) to 3) is the same as that of Example 8, and the subsequent process is:

[0197] 4) Take out the detection device of the pre-treatment pad prepared according to Formula A, Formula B, and Formula C from the sealed aluminum foil bag and place it on a clean and level surface.

[0198] 5) Divide the samples in 3) into three parts, open the sample sampling covers of the detection devices of Group I, Group II, and Group III, expose the sample sampling blocks and place them in the samples, one group corresponds to one sample, and let them stand for the same time as in Example 8, then cover the sample sampling covers and press horizontally 1-2 times.

[0199] 7) Place on a horizontal table at room temperature and read the test results using a fluorescent immunoassay analyzer after 15 minutes.

[0200] The test results are shown in Tables 10, 11 and 12. When Triton X-100 is replaced with stearic acid monoethanolamide, the signal can be detected at a dilute concentration of 1 / 12000, which is higher than the detection sensitivity in Example 8. When the current treatment pad is prepared using Formula B, that is, when the content of stearic acid monoethanolamide is 1%, the positive rate of the test result is the highest, and the virus is released more completely. This fully demonstrates that the addition of stearic acid monoethanolamide in the lysis reagent treated on the sample pre-treatment pad is critical to the lysis effect, and its mass fraction content is preferably 1%.

[0201] Table 10 Test results of group I test device

[0202]

[0203] Table 11 Test results of group II test device

[0204]

[0205] Table 13 Test results of group III test device

[0206]

[0207] All patents and publications mentioned in the present specification indicate that these are public technologies in the field and can be used in the present invention. All patents and publications cited here are also listed in the references, just as each publication is specifically cited separately. The present invention described here can be implemented in the absence of any one or more elements, one or more restrictions, and such restrictions are not specifically stated here. For example, in each example here, the terms "comprising", "substantially consisting of..." and "consisting of..." can be replaced by the other two terms of one of the two. The so-called "one" here only means "one", but does not exclude that it only includes one, and can also mean that it includes more than two. The terms and expressions used here are descriptive and not limited by them. There is no intention to indicate that these terms and explanations described in this book exclude any equivalent features, but it can be known that any appropriate changes or modifications can be made within the scope of the present invention and the claims. It can be understood that the embodiments described in the present invention are some preferred embodiments and features, and any person skilled in the art can make some changes and variations based on the essence of the present invention, and these changes and variations are also considered to belong to the scope of the present invention and the scope limited by the independent claims and the appended claims.

Claims

1. An immunochromatographic detection device, characterized in that: The invention comprises a cartridge and a test strip, wherein the test strip is installed in the cartridge, wherein the test strip comprises a sample pretreatment pad, a sample pad, a marking pad, a reaction membrane and a water-absorbing pad which are overlapped in sequence, wherein the sample pretreatment pad comprises a dry and fixed pretreatment reagent for virus sample lysis; the pretreatment reagent comprises: 0.01 mol / L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide by mass, and 0.01% proclin 300 by mass, wherein the pH of the phosphate buffer is 7.5-8.5; and the virus is a novel coronavirus.

2. The detection device according to claim 1, characterized in that It also includes a sample collection part; the sample collection part is provided with a sample collection block and a sampling cover, and the sample collection block is squeezed by the sampling cover to allow the sample to pass through the filtering component in the cartridge to reach the sample pre-processing pad.

3. The detection device according to claim 2, characterized in that: The test strip also includes a bottom plate; the front end of the card shell is provided with a sampling slot for accommodating a sample collection block; and the filter component is located at the connection between the sample collection block and the test strip.

4. The detection device according to claim 3, characterized in that: The sample collection block is a sponge that can absorb samples. A portion of the sample collection block is exposed from the sampling slot, and the exposed portion is covered with a sampling cover.

5. The detection device according to claim 4, characterized in that: The cartridge comprises a cartridge cover and a cartridge bottom, the cartridge bottom is provided with a test strip slot, and the front end of the test strip slot comprises a pre-treatment pad slot; the filter component is a filter screen, the filter screen is located on the cartridge cover and / or the cartridge bottom, and the width of the filter screen is not greater than the width of the sample pre-treatment pad.

6. The detection device according to claim 5, characterized in that: A groove is arranged on the sample collection block so that it is fixed in the sampling slot at the front end of the card shell, and the distance between the sample collection block and the filter screen is 1 to 3 mm.

7. The detection device according to any one of claims 1 to 6, characterized in that: The sample is saliva, and the pretreatment reagent is a lysis reagent for viruses in the saliva sample.

8. The detection device according to claim 7, characterized in that: The marking pad includes colloidal gold or fluorescent marking.

Images