CN113281504B

CN113281504B - Novel immunochromatography detection device

Info

Publication number: CN113281504B
Application number: CN202011254054.2A
Authority: CN
Inventors: 张敏; 杨蓉; 宋学文; 王韩梦; 郭秀丽; 赵师伊
Original assignee: Hangzhou Weice Biotechnology Co ltd
Current assignee: Hangzhou Weice Biotechnology Co ltd
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2022-03-08
Anticipated expiration: 2040-11-11
Also published as: WO2022099921A1; CN114414800A; CN113281504A

Abstract

The invention provides a novel immunochromatography detection device, which comprises a sample pretreatment part, does not need pretreatment such as dilution or cracking and the like on a sample during use, and can realize one-step detection. The immunochromatography detection device provided by the invention is particularly suitable for rapid detection of novel coronavirus SARS-CoV-2, can be used for collecting samples by directly adopting a saliva sample mode in the oral cavity, is convenient for patients to sample and detect by themselves, can be used for determining the negative and positive of the novel coronavirus by a one-step method, is convenient to use, simple and efficient, reduces the risk of virus propagation to medical workers and health care workers, reduces the utilization of personal protection equipment, and is suitable for rapid initial diagnosis of the infection condition of the novel coronavirus by various medical institutions or individuals.

Description

Novel immunochromatography detection device

Technical Field

The invention belongs to the technical field of biological detection, relates to a novel immunochromatography detection device, and particularly relates to a one-step immunochromatography detection device containing pretreatment for detecting novel coronavirus.

Background

At present, the detection aiming at the novel coronavirus is mainly virus nucleic acid detection based on PCR, the detection principle is that a unique gene sequence of the virus is taken as a detection target, the exponential level of the selected target DNA sequence is increased through PCR amplification, and the more the amplified target genes are, the stronger the accumulated fluorescent signal is. In the sample without the virus, the result of the increase in the fluorescence signal is not detected because the target gene is not amplified. Novel coronavirus has higher specificity and sensitivity through nucleic acid detection, but this detection method is high to technical requirement, and the sample needs special treatment, requires to possess professional instrument and equipment such as PCR amplification appearance and gel electrophoresis, and is long with the detection to novel coronavirus, needs professional technical staff to operate and judge the testing result, can't be applied to the early preliminary screening of basic level such as community hospital, customs, disease control center.

At present, Chinese patent application No. 202010184358.X and the like disclose a fluorescence immunochromatographic device for detecting COVID-19 and a using method thereof, Chinese patent application No. 202010184382 and the like disclose a colloidal gold immunochromatographic device for detecting COVID-19 and a using method thereof, corresponding products can be applied to early primary screening of a substrate, but certain operation defects still exist, particularly, the method for detecting virus antigens needs to dilute or crack the collected sample for the first time to further detect, namely, a two-step method detection is adopted to collect the sample from the oropharynx or the nasopharynx of a patient, the collected sample is processed in other auxiliary devices except the detecting device after sampling, and then the processed sample is added into the detecting device to perform result detection. Such sampling may increase the risk of viral transmission to medical and health care workers who lack sufficient personal protective equipment, and the operating procedures are not the simplest and are prone to operating errors, thereby having a certain impact on the test results. Therefore, a simple and convenient novel immunochromatography detection device for coronavirus, which is more suitable for self-collection and self-detection of patients, is urgently needed.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a novel immunochromatography detection device which comprises a sample pretreatment part, does not need pretreatment such as dilution or cracking and the like on a sample during use, and can realize one-step detection. The one-step immunochromatographic detection device comprising the pretreatment part can be used for novel coronavirus SARS-CoV-2 immunochromatographic detection, realizes one-step diagnosis of the negative and positive of the novel coronavirus to confirm the negative and positive of the novel coronavirus, is convenient to use, is rapid and efficient, and is suitable for initial rapid diagnosis of the infection condition of the novel coronavirus by various medical institutions or individuals.

The immunochromatography detection device provided by the invention can be used for sampling a saliva sample by directly adopting the oral cavity, and compared with sampling of other upper respiratory tract parts, the oral cavity sampling has less invasiveness and less discomfort for patients. Collecting saliva samples is technically less complex and therefore reduces the risk of virus transmission to health care workers and health care workers by reducing the operating time and allowing patients to collect themselves under supervision. The patient collects themselves under supervision, the utilization of personal protective equipment is reduced, and compared with the existing mode, the mode of completing detection in one step is simpler and has more market demands.

The technical scheme provided by the invention is as follows:

in one aspect, the invention provides a novel immunochromatography detection device, which comprises a sample collection part and a test strip, wherein the test strip comprises a sample pretreatment pad, a sample collected in the sample collection part can be transferred to the sample pretreatment pad, and the sample pretreatment pad comprises a pretreatment reagent for cracking a virus sample, which is dried and fixed.

Further, the test strip comprises a bottom plate, a sample pretreatment pad, a sample pad, a combination pad, a reaction membrane and a water absorption pad, wherein the sample pretreatment pad, the sample pad, the combination pad, the reaction membrane and the water absorption pad are sequentially overlapped together; the sample collection part comprises a sample collection block and a sampling groove.

In some embodiments, the sample pre-treatment pad, the sample pad, the conjugate pad, the reaction membrane, and the bibulous pad overlap one another in sequence and are positioned on the base plate.

In some embodiments, the sample pretreatment pad, the sample pad, the conjugate pad, the reaction membrane, and the absorbent pad are sequentially overlapped together, the sample pad, the conjugate pad, the reaction membrane, and the absorbent pad are located on the bottom plate, and the sample pretreatment pad is located outside the bottom plate.

Furthermore, the detection device also comprises a clamping shell, the test strip is arranged in the clamping shell, the front end of the clamping shell is provided with a sampling groove for accommodating the sample collection block, and the joint of the sample collection block and the test strip is provided with a filter component.

Furthermore, the sample collection block is a sponge capable of adsorbing a sample, part of the sample collection block is exposed out of the sampling groove, and the exposed part is sleeved with the sampling cover and used for extruding the sample collection block to enable the sample to reach the sample pretreatment pad through the filtering component. The sample sampling block can be sealed therein by the cooperation of the sample sampling cover and the sampling slot.

Furthermore, the card shell includes card shell cover and card shell bottom, and the card shell bottom is equipped with the test strip groove, and test strip groove front end includes the pretreatment pad groove. When the test strip is positioned in the test strip groove, the sample pretreatment pad is just positioned in the pretreatment pad groove.

The card shell cover and the card shell bottom are assembled together and used for accommodating the sample collecting block and the test strip; and after the card shell cover and the card shell bottom are assembled together, a sampling groove is formed at the front end.

In some embodiments, the sample pretreatment pad can be placed separately from the strip, i.e., the sample pretreatment pad is placed separately in the pretreatment pad slot, the strip is placed in the strip slot, and the starting end of the strip sample pad overlaps with the end of the pretreatment pad, which is also within the scope of the present invention.

Furthermore, the filter part is a filter screen, the filter screen is positioned on the card shell cover and/or the card shell bottom, and the width of the filter screen is not more than that of the sample pretreatment pad.

The card shell is covered and is equipped with card shell lid filter screen, is equipped with card shell end filter screen on the card shell end, perhaps only establishes one of them on the two, when using sampling lid extrusion sample sampling piece, the sample flows to the pretreatment groove region through the filter screen, and the width of filter screen is equivalent with the width of sample pretreatment pad, makes the sample can just be received by pretreatment pad after passing through the filter screen and do not cause to reveal and extravagant.

Furthermore, the sample collecting block is provided with a groove, so that the sample collecting block is fixed in a sampling groove at the front end of the clamping shell, and the distance between the sample collecting block and the filter screen is 1-3 mm.

Further, the distance between the sample collection block and the filter screen is 2-3 mm.

Further, the sample is saliva, and the pretreatment liquid reagent is a lysis reagent for viruses in the saliva sample.

Further, the lysis reagent comprises: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 0.5 to 1.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass, wherein the pH value of the phosphate buffer solution is 7.5 to 8.5.

Further, the lysis reagent comprises: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 1 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass, wherein the PH of the phosphate buffer solution is 7.5-8.5.

Further, the labeling pad comprises colloidal gold or a fluorescent label.

Further, the preparation method of the pretreatment mat is as follows:

1) preparing a phosphate buffer solution, and adjusting the pH value to 7.5-8.5;

2) preparing a sample pretreatment solution from a certain mass fraction of the phosphate buffer (for example, 0.01-0.05mol/L), a certain mass fraction of casein (for example, 3% -5.5%), a certain mass fraction of Triton X-100 (for example, 0.01% -1%), and a certain mass fraction of proclin300 (for example, 0.01% -0.1%); 3) uniformly dispersing the treatment solution on glass fiber, drying overnight in a drying oven at 37 ℃, taking out, placing in an aluminum foil bag, and sealing for later use.

Further, the sample pad is treated with a sample pad treatment solution comprising, 0.05M pH 8.0 boric acid buffer, casein, surfactant S17, cholic acid, EDTA.

Further, the combination pad is coated with a novel coronavirus NP protein monoclonal antibody marked by colloidal gold particles (or fluorescent microspheres).

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

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

On the other hand, the invention provides a novel coronavirus SARS-CoV-2 antigen immunochromatography saliva assay detection kit and a detection method, comprising the following steps:

1) and (4) restoring the detection kit to room temperature, taking out a detection device from the sealed aluminum foil bag, slightly pulling out the sampling cover, and exposing the front end of the sample sampling block.

2) And putting the front end of the sample collection block into the oral cavity for 3-5 min.

3) Cover and gather the lid, press the lid 1 ~ 2 times in the horizontal direction.

4) The saliva sample enters the pretreatment pad, the substance to be detected in the saliva reacts with the reagent on the pretreatment pad, then enters the sample pad through capillary action, then enters the reaction membrane through the combination pad through capillary action, the reaction compound diffuses forwards along the reaction membrane and is captured by the coating antibody fixed on the detection line, so that a macroscopic color signal is displayed at the coating antibody, or in other embodiments, a measurable signal change of an instrument is displayed at the coating antibody, the waste liquid of the chromatography reaction is collected by absorbent paper, and the chromatography reaction is pulled to be carried out in the direction.

5) And judging and reading the test result through the inspection window for 15-20 min. If the quality control line C and the detection line T are purple red, the result is positive; if only the quality control line C shows mauve and the detection line T does not show color, the result is negative; if the quality control line C and the detection line T do not show mauve, the result is invalid; if the quality control line C does not display the mauve color, the test result is invalid even if the detection line T displays the mauve color. Or in other embodiments, the instrument detects signals of the detection line T and the quality control line C, and the signals are converted to reach a certain value, and then the result is determined to be positive.

In a further aspect, the present invention provides the use of stearic acid monoethanolamide for the preparation of a viral lytic reagent, wherein the lytic reagent comprises: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 0.5 to 1.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass, wherein the pH value of the phosphate buffer solution is 7.5 to 8.5.

Further, the virus is a novel coronavirus.

The invention has the beneficial effects that: the invention provides a novel immunochromatography detection device, which comprises a sample pretreatment part, does not need pretreatment such as dilution or cracking and the like on a sample during use, and can realize one-step detection. The immunochromatography detection device provided by the invention is particularly suitable for novel coronavirus SARS-CoV-2 immunochromatography detection, can be used for collecting samples by directly adopting a saliva sample mode in the oral cavity, is convenient for patients to sample and detect by themselves, can be used for determining the negative and positive of the novel coronavirus by a one-step method, is convenient to use, is rapid and efficient, reduces the risk of virus propagation to medical workers and health care workers, reduces the utilization of personal protection equipment, and is suitable for various medical institutions or individuals to rapidly diagnose the infection condition of the novel coronavirus at the initial stage.

Drawings

FIG. 1 is a schematic view showing the structure of a novel immunochromatographic assay device in example 1;

FIG. 2 is a view showing an exploded structure of each layer of the novel immunochromatographic assay device in example 1;

FIG. 3 is an exploded view of a sample collection site of the novel immunochromatographic assay device of example 1;

FIG. 4 is a standard color chart of colloidal gold according to example 4;

fig. 5 is a graph showing the correlation between the average values of the signals of the detection lines T without pre-treatment pad and with pre-treatment pad in example 5.

Detailed Description

The present invention is further described in terms of structures or techniques, which are understood and interpreted, if not otherwise indicated, in accordance with the general terminology used in the art. The description is given for the sake of example only, to illustrate how the invention may be implemented, and not to limit it in any way, the scope of which is defined and expressed by the claims.

Detection of

Detection means assaying or testing for the presence or absence of a substance or material. Such as, but not limited to, chemicals, organic compounds, inorganic compounds, metabolic products, drugs or drug metabolites, organic tissues or metabolites of organic tissues, nucleic acids, proteins or polymers. In addition, detection may also indicate the amount of the test substance or material. Assays also refer to immunodetection, chemical detection, enzymatic detection, and the like.

Sample (I)

In the present invention, the sample used in the detection apparatus includes a biological fluid. The initial state of the sample may be liquid, solid or semi-solid, and the solid or semi-solid sample may be converted to a liquid sample by any suitable method, such as mixing, triturating, macerating, incubating, dissolving, enzymatically digesting, etc., and then poured into the collection chamber, and the sample may be tested for the presence of the analyte by the test element. The sample can be obtained from human body, animal, plant, nature, etc. The sample taken from human body can be, for example, blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, gastric juice, etc.; solid or semi-solid samples of feces, hair, cutin, tartar, nails, and the like. Samples taken from plants, which may be, for example, solid samples of roots, stems, leaves, etc.; liquid or semisolid samples such as tissue fluid and cell fluid prepared from root, stem and leaf. The sample taken from nature may be, for example, a liquid sample such as rainwater, river water, seawater, or groundwater; solid or semi-solid samples of soil, rock, ore, petroleum, etc.

In some embodiments, the sample of the present invention is saliva, which facilitates self-sampling.

Testing device

The test device generally includes a test element, which is a component capable of detecting an analyte in a 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, physics, etc. The test element of the present invention may be one type or a combination of two or more types. The test element is provided with a detection area for displaying a detection result, and the detection area displays the detection result after the detection is carried out.

Various test elements may be combined for use in the present invention. One form is a test strip. Test strips for the analysis of analytes, such as drugs or metabolites indicative of a physical condition, in a sample may be in various forms, such as immunoassay or chemical assay forms. The test strip may be used in a non-competitive or competitive assay format. The test strip includes a bibulous material having a sample application area, a reagent area, and a test area. The sample is added to the sample application zone and flows by capillary action to the reagent zone. In the reagent zone, the sample binds to the reagent if the analyte is present. The sample then continues to flow to the detection zone. Other reagents, such as molecules that specifically bind to the analyte, are immobilized at the detection zone. These reagents react with the analyte (if present) in the sample and bind the analyte to the zone, or to one of the reagents of the reagent zone. The label for indicating the detection signal is present in the reagent zone or in a separate label zone.

A typical non-competitive assay format is one in which a signal is generated if the sample contains the analyte and no signal is generated if the analyte is not present. In a competition method, a signal is generated if the analyte is not present in the sample and no signal is generated if the analyte is present.

The test element can be a test paper, and can be made of water-absorbing or non-water-absorbing materials. The test strip may include a variety of materials for liquid sample delivery. One of the test strips may be coated with another material, such as a nitrocellulose membrane coated with filter paper. One region of the test strip may be selected from one or more materials and another region may be selected from a different one or more materials. The test strip may be adhered to some support or hard surface for improved strength when the test strip is held in place.

The analyte is detected by a signal producing system, such as one or more enzymes that specifically react with the analyte, and one or more compositions of the signal producing system are immobilized on the analyte detection zone of the test strip by a method such as that described above for the immobilization of a specific binding substance on the test strip. The signal-producing substance can be on the sample addition zone, reagent zone, or detection zone, or the entire test strip, and the substance can be impregnated on one or more materials of the test strip. A solution containing the signal is applied to the surface of the strip or one or more materials of the strip are immersed in the solution containing the signal. The strip to which the solution containing the signal substance was added was dried.

The various regions of the test strip may be arranged as follows: the device comprises a sample adding area, a reagent area, a detection area, a control area, a sample adulteration area and a liquid sample absorption area. The control zone is located behind the detection zone. All zones may be arranged on a strip of test paper using only one material. It is also possible to use different materials for the different zones. The zones may be in direct contact with the liquid sample, or different zones may be arranged according to the direction of flow of the liquid sample, with the ends of each zone being contiguous with and overlapping the ends of the other zone. The material used can be a material with good water absorption such as filter paper, glass fiber or nitrocellulose membrane. The test strip may take other forms.

The test strip used in the present invention may be a so-called Lateral flow test strip (Lateral flow test strip), and the specific structure and detection principle of these test strips are well known to those skilled in the art. A typical test strip comprises a sample collection area comprising a sample receiving pad, a labeling area comprising a labeling pad, a detection area comprising a bibulous pad, and a bibulous area comprising a chemical reagent, such as an immunological reagent or an enzymatic reagent, necessary to detect the presence of the analyte. A commonly used detection reagent strip is a nitrocellulose membrane reagent strip, that is, a detection area comprises a nitrocellulose membrane, and a specific binding molecule is fixed on the nitrocellulose membrane to display a detection result; it may be a cellulose acetate film, a nylon film, etc., and it may also include a detection result control region downstream of the detection region, and usually, the control region and the detection region are in the form of a transverse line, which is a detection line or a control line. Such test strips are conventional, but other types of test strips that utilize capillary action for testing are also contemplated. In addition, typically, the test strip has a dry chemical reagent component, such as an immobilized antibody or other reagent, which when exposed to a liquid, flows along the test strip by capillary action, and as it flows, the dry reagent component is dissolved in the liquid, and the next zone is processed to react the dry reagent in that zone, thereby performing the necessary test. The liquid flow is mainly by capillary action. These test elements are described and documented in the following documents: lefukang 'research on regeneration treatment of nitrocellulose membranes and protein adsorption capacity thereof'; malanhuang, Liqiang et al analysis of performance of chromatography material in colloidal gold diagnostic kit; wangyong, Wanglauca et al, a novel colloidal gold immunochromatographic test strip. The detection device can be used in the detection device of the present invention, or can be disposed in the detection chamber to contact the liquid sample, or can be used to detect the presence or quantity of an analyte in the liquid sample entering the detection chamber.

Detailed Description

The present invention is described in further detail below with reference to examples, which are intended to facilitate the understanding of the present invention without limiting it in any way. The reagents not specifically mentioned in this example were all known products and were obtained by purchasing commercially available products.

Embodiment 1 the present invention provides a novel immunochromatography detection apparatus

The novel immunochromatography detection device provided by the embodiment is shown in fig. 1-3, wherein fig. 1 is a schematic structural diagram of the novel immunochromatography detection device, fig. 2 is a split structural diagram of each layer of the novel immunochromatography detection device, and fig. 3 is a split structural diagram of a sample collection part of the novel immunochromatography detection device.

Referring to fig. 1, the present invention provides a novel immunochromatography detection device, which comprises a sample collection part 1 and a test strip 2, wherein the test strip 2 comprises a sample pretreatment pad 3, a sample collected in the sample collection part 1 can be transferred to the sample pretreatment pad 3, and the sample pretreatment pad 3 is provided with a dry and fixed sample pretreatment solution.

Preferably, the test strip 1 comprises a base plate 4, a sample pretreatment pad 3, a sample pad 5, a conjugate pad 6, a reaction membrane 7 and a water absorption pad 8, wherein the sample pretreatment pad 3, the sample pad 5, the conjugate pad 6, the reaction membrane 7 and the water absorption pad 8 are sequentially overlapped together and positioned on the base plate 4. In some embodiments, only the sample pad 5, the conjugate pad 6, the reaction membrane 7, and the 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 site 1 comprises a sample collection block 9 and a sampling slot 10.

Preferably, the detection device further comprises a card shell 11, the test strip 2 is arranged in the card shell 11, a sampling groove 10 for accommodating the sample collection block 8 is arranged at the front end of the card shell 11, and a filter component 12 is arranged at the joint of the sample collection block 9 and the test strip 2.

Preferably, the sample collection block 9 is a sponge capable of adsorbing the sample, a part of the sample collection block 9 is exposed out of the sampling slot 10, and a part of the exposed part is sleeved with a sampling cover 13 for pressing the sample collection block 9 to make the sample reach the sample pretreatment pad 3 through the filtering component. The sample sampling block 9 is sealed therein by the cooperation of the sampling lid 13 with the sampling slot 10.

Preferably, the card housing 11 includes a card housing cover 14 and a card housing bottom 15, the card housing bottom 15 is provided with a test strip groove 16, the front end of the test strip groove 16 includes a pretreatment pad groove 17, and when the test strip 2 is located in the test strip groove 16, the sample pretreatment pad 3 is just located in the pretreatment pad groove 17. The housing cover 14 and the housing bottom 15 are assembled together and used for accommodating the sample collection block 9 and the test strip 2; and the front end forms the sampling slot 10 when the card housing cover 14 and the card housing bottom 15 are assembled together. In some embodiments, the sample pre-treatment pad 3 can be placed separately from the strip 2, i.e., the sample pre-treatment pad 3 is placed in the pre-treatment pad slot 17 alone, the strip 2 is placed in the strip slot 16, and the beginning of the sample pad 5 overlaps the end of the pre-treatment pad 3.

Preferably, the filter component 12 is a filter 18, the filter 18 is located on the card housing cover 14 and/or the card housing bottom 15, and the width of the filter 18 is not greater than the width of the sample pretreatment pad 3.

Preferably, the sample collection block 9 is provided with a groove 19 so as to be fixed in the sampling groove 10, and the distance between the sample collection block 9 and the filter screen 18 is 1-3 mm.

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

Preferably, the sample is saliva.

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

Preferably, the virus is a novel coronavirus.

EXAMPLE 2 preparation of an immunochromatographic assay device (colloidal gold method) for detecting novel coronavirus

The preparation procedure of the immunochromatographic assay test device (colloidal gold method) for detecting the novel coronavirus SARS-CoV-2 provided in this example includes:

the method comprises the following steps: preparation sample pretreatment pad

1) Phosphate buffer was prepared and the pH was adjusted to 7.5.

2) Taking the phosphate buffer solution with a certain mass fraction: 0.01mol/L, certain mass fraction of casein: 3%, Triton X-100 with a certain mass fraction: 1%, proclin300 in a certain mass fraction: 0.01% of the solution was prepared as a pretreatment liquid.

3) Uniformly dispersing the pretreatment liquid on glass fiber, drying overnight in a drying oven at 37 ℃, taking out, placing in an aluminum foil bag, and sealing for later use.

Step two: sample pad treatment

1) Prepare boric acid buffer and adjust PH to 7.5.

2) Taking a certain mass fraction of boric acid buffer solution: 0.01mol/L), a certain mass fraction of casein: 0.1%, certain mass fraction of surfactant S17: 1 percent; cholic acid with a certain mass fraction: 0.1 percent, certain mass fraction of EDTA: 0.6% was configured as sample pad treatment fluid.

3) Uniformly dispersing the sample pad treatment solution on glass fiber, drying in a drying oven at 37 ℃ overnight, taking out, and placing in an aluminum foil bag for later use.

Step three: bond pad treatment

1) Adjusting the pH value of the colloidal gold solution to 9.0, uniformly mixing the colloidal gold solution with the novel coronavirus NP protein monoclonal antibody for reaction to obtain a labeled antibody, and redissolving the labeled antibody by using a redissolution.

2) The labeled antibody compound solution of 1) is uniformly sprayed on untreated glass fibers by a film-scribing metal spraying instrument in a certain mass fraction (for example, 2 uL/cm).

3) And (3) placing the sprayed glass fiber in a drying oven at 37 ℃ for overnight drying, taking out the glass fiber, placing the glass fiber in an aluminum foil bag, and sealing the aluminum foil bag for later use.

Step four: nitrocellulose membrane scribing

1) Phosphate buffer was prepared and the pH was adjusted to 8.

2) A certain mass fraction of phosphate buffer (for example, 0.1mol/L) is taken to dilute the novel coronavirus NP protein monoclonal antibody to 1.0mg/ml to be used as a T line solution.

3) The goat anti-mouse polyclonal antibody is diluted to 0.5mg/ml by taking a certain mass fraction of phosphate buffer (e.g. 0.1mol/L) as a C line solution.

4) And (4) scribing the film by using a scribing goldprojector at a certain mass fraction (such as 0.5 uL/cm).

5) And (4) drying the scratched film in a drying oven at 37 ℃ overnight, taking out, placing in an aluminum foil bag, and sealing for later use.

Step five: assembled cutting and bagging

1) Cutting the pre-treatment pad obtained in the step one, the sample pad treated in the step two, the combination pad obtained in the step three and the nitrocellulose membrane treated in the step four into proper widths, adhering the nitrocellulose membrane and the absorbent paper to a bottom plate in sequence, and firmly lapping 1-2mm between every two layers, wherein the width of the pre-treatment pad is larger than or equal to that of the sample pad.

2) And cutting the stuck large card into test cards with the width of 3-6mm by using a cutting machine, and putting the test cards into a card shell.

3) And fixing the sample collecting block at the front end of the clamping shell.

4) The sample collection cap is closed.

5) Packaging into aluminum foil bags, and sealing for storage.

Preferably, the pretreatment pad in the fifth step can be also independently adhered to the bottom plate for cutting, or the bottom plate is not required to support, and the width of the pretreatment pad is larger than or equal to that of the sample pad.

Example 3: preparation of another immunochromatography detection device (colloidal gold method) for detecting novel coronavirus

The same as in example 2, except that stearic acid monoethanolamide was used instead of Triton X-100 in the preparation of the pre-treated pad, and the remaining ingredients were the same as in example 2, and were divided into three formulations for preparing the pre-treated pad according to the mass fraction of stearic acid monoethanolamide.

Formula 1: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 0.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass.

And (2) formula: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 1 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass.

And (3) formula: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 1.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass.

Example 4: preparation of immunochromatography detection device (fluorescence method) for detecting novel coronavirus

The preparation steps of the immunochromatographic assay test device (fluorescence method) for detecting the novel coronavirus SARS-CoV-2 provided in this example include:

the method comprises the following steps: preparation pretreatment pad

1) Phosphate buffer was prepared and the pH was adjusted to 8.5.

Step two: sample pad treatment

1) Phosphate buffer was prepared and the pH was adjusted to 8.5.

2) Taking phosphate buffer solution with certain mass fraction: 0.01mol/L), a certain mass fraction of bovine serum albumin: 0.1 percent, surfactant Triton X-100 with certain mass fraction: 1% and povidone K30 with a certain mass fraction: 0.05% was configured as sample pad treatment solution.

Step three: bond pad treatment

1) Adjusting the pH value of the fluorescent microsphere solution to 8.5, uniformly mixing the fluorescent microsphere solution with the novel coronavirus NP protein monoclonal antibody for reaction to obtain an antibody marker, and redissolving the antibody marker by using a redissolution.

2) Phosphate buffer was prepared and the pH was adjusted to 8.5.

3) A certain mass fraction of phosphate buffer solution (for example, 0.01mol/L), a certain mass fraction of bovine serum albumin (for example, 0.1%), a certain mass fraction of sucrose (for example, 0.1%), and a certain mass fraction of proclin300 (for example, 0.01%) are taken to prepare the treatment solution for the bonding pad.

4) Uniformly dispersing the treatment solution on glass fiber, drying overnight in a drying oven at 37 ℃, taking out, and placing in an aluminum foil bag for later use.

5) Spraying the antibody marker compound solution in the step 1) on the glass fiber of the step 4) in a certain mass fraction (for example, 1uL/cm) by using a film-scratching gold spraying instrument, placing the glass fiber in a drying oven at 37 ℃ for overnight drying, taking out the glass fiber, and placing the glass fiber in an aluminum foil bag for sealing for later use.

Step four: drawing nitrocellulose membrane (reaction membrane)

1) Phosphate buffer was prepared and the pH was adjusted to 8.

2) A certain mass fraction of phosphate buffer (for example, 0.1mol/L) is taken, and the novel coronavirus NP protein monoclonal antibody is diluted to 1.0mg/ml to be used as a T line solution.

3) A certain mass fraction of phosphate buffer (e.g., 0.1mol/L) is taken, and the goat anti-mouse polyclonal antibody is diluted to 0.5mg/ml to be used as a C line solution.

4) The film is scribed at a certain mass fraction (e.g. 0.5uL/cm) with a scribing goldplexer.

Step five: assembled cutting and bagging

1) Cutting the pre-treatment pad obtained in the step one, the sample pad treated in the step two, the combination pad obtained in the step three and the nitrocellulose membrane treated in the step four into proper widths, sequentially adhering the nitrocellulose membrane and the absorbent paper to a bottom plate, and firmly lapping 1-2mm between every two layers.

4) The sample collection cap is closed.

5) Packaging into aluminum foil bags, and sealing for storage.

Furthermore, the pretreatment pad in the fifth step can be separately adhered to the bottom plate for cutting, or the bottom plate is not required to support, and the width of the pretreatment pad is larger than or equal to that of the sample pad.

EXAMPLE 5 preparation of an alternative immunochromatographic assay device (fluorescence method) for detection of novel coronaviruses

The same as example 4, except that stearic acid monoethanolamide was used instead of Triton X-100 in the preparation of the pre-pad, the following three formulations were prepared according to the differences in the mass fractions, and the other ingredients were the same as in example 4:

the formula A is as follows: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 0.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass.

And the formula B is as follows: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 1 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass.

And a formula C: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 1.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass.

Example 6: example 2 verification of an immunochromatographic assay device for detecting a novel coronavirus, which was prepared by colloidal gold method

The virus culture 1.51 multiplied by 106TCID50/mL is respectively diluted into 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times, 6400 times and 9600 times as a sample to be detected, and negative results appear when a signal of a detection line T line is interpreted after the dilution times are more than 6400 times in the existing system. The results were interpreted using a standard colorimetric card (see FIG. 4).

As shown in FIG. 4, if the detection line T is greater than or equal to G3, the detection result is positive; if the detection line T is less than G3, the detection result is negative, and the quality control line C is always greater than or equal to G8; if the quality control line C is less than G8 or no color is developed, the test result will be invalid even if the detection line T is developed.

The test was carried out in three groups, wherein the first group was carried out by directly using the saliva test kit without pretreatment pad (without sample lysis) (see table 1 for specific results), the second group was carried out by carrying out sample lysis treatment before using the saliva test kit without pretreatment pad (see table 2 for specific results), and the third group was carried out by using the saliva test kit with pretreatment pad provided in example 2 (see table 3 for specific results), each treatment was repeated 20 times, and the test results were interpreted by using a standard colorimetric card (G represents the shade of color to interpret the test results). The first, second and third sets of test strips are identical in structure and reagents except for the following differences: the first group is not subjected to pretreatment; the second group has a pretreatment, but the test strips are also conventional test strips without a pretreatment pad, and the third group is test strips that include a pretreatment pad.

The specific experimental process is as follows:

1) the detection device is rewarmed at 15-30 ℃.

2) The virus cultures were diluted 100-fold, 200-fold, 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, 9600-fold, respectively.

3) After the saliva sample was collected, the diluted virus cultures were added to the saliva sample in the gradient of 2) to obtain 8 samples, and 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, and in addition, 1 sample was not added with the virus culture, and the culture concentration was 0.

4) Taking out the existing detection device without the pretreatment pad and the matched lysate from the sealed aluminum foil bag; the test device with the pre-pad provided in example 2 was removed from the sealed aluminum foil pouch and placed on a clean and horizontal surface.

5) Dividing each sample in 2) into three portions, wherein the second portion is added to the lysis solution of the existing detection device without the pre-treatment pad, and the first portion and the third portion are not added to the lysis solution.

6) 60 microliter of sample without added lysate is dripped on the detection device of the first group; 60 microliters of the sample with the lysate was added dropwise to the second set of detection devices; and opening a sample sampling cover of the third group of detection devices with the pretreatment pads, placing the exposed sample sampling blocks into samples which are not added into the lysis solution, standing for a certain time (for example, 1 to 2 minutes), covering the sample sampling cover, and horizontally pressing for 1 to 2 times.

7) And (5) after the test piece is placed on a horizontal desktop at room temperature for 15-20 min, the test result is interpreted through an inspection window.

The detection results are shown in table 1, table 2 and table 3, when the dilution times are the same, the first group shows that the results are obviously weaker, and the real results are difficult to detect (without cracking); the second group and the third group have consistent color development results and have no difference in chromaticity, so that the detection device is feasible for virus detection. The detection device does not need an additional pretreatment step, and is directly dripped on the test strip, so that the steps are reduced, and possible secondary pollution to operators caused by the pretreatment step is reduced.

TABLE 1 results of samples directly added to the existing detection device without lysis

Table 2 results of adding the existing testing device to test after the sample is cracked

TABLE 3 results of the direct addition of the sample to the test device of the present invention

EXAMPLE 7 validation of an alternative immunochromatographic assay device for detection of novel coronaviruses prepared by colloidal gold method as provided in example 3

The test devices for preparing the

pre-pad using formulations

1, 2, and 3 were divided into three groups, wherein formulation 1 corresponds to the fourth group (0.5%), formulation 2 corresponds to the fifth group (1%), and formulation 3 corresponds to the sixth group (1.5%). Saliva samples were collected for testing as in example 6, each set was repeated 20 times and the test results were interpreted using a standard colorimetric card.

The specific experimental procedures 1) to 3) were the same as in example 6, followed by the procedures of:

4) the test devices for pre-treatment pads prepared according to

formulations

1, 2 and 3, respectively, were removed from the sealed aluminum foil pouch and placed on a clean and horizontal plane.

5) Dividing each sample in the step 3) into three parts, opening sample sampling covers of the detection devices with the pretreatment pads in the fourth group, the fifth group and the sixth group in sequence, exposing sample sampling blocks, placing the sample sampling blocks into the samples, covering the sample sampling covers after one group corresponds to one sample, standing for the same time as in the embodiment 6, and pressing for 1-2 times in the horizontal direction.

As shown in tables 4, 5, and 6, when Triton X-100 was replaced with stearic acid monoethanolamide, the lowest detection threshold was still 95% positive when diluted 9600 times, and the lowest detection threshold was greatly increased as compared with table 3. When the current treatment pad is prepared by adopting the formula 2, the positive rate of the detection result is the highest when the content of the obtained stearic acid monoethanolamide is 1%, and the virus is more completely released. In addition, it was found that when the stearic acid monoethanolamide content was 1% at the time of 9600-fold dilution, the positive detection rate was 100%. This fully indicates that the addition of stearic acid monoethanolamide to the lysis reagent treated on the sample pretreatment pad is critical to the lysis effect, and the content of stearic acid monoethanolamide in mass fraction is preferably 1%.

TABLE 4 results of the samples directly added to the apparatus of formulation 1 of example 3 for testing

TABLE 5 results of the samples directly added to the detection apparatus of formulation 2 of example 3 for detection

TABLE 6 results of the samples directly added to the formulation 3 testing apparatus of example 3 for testing

EXAMPLE 8 verification of the immunochromatographic assay device for detecting novel coronaviruses prepared by the fluorescence method provided in example 4

Diluting the virus culture 1.51 × 106TCID50/mL into 500 times, 1000 times, 2000 times, 4000 times, 5000 times, 7500 times, 10000 times and 12000 times respectively to be used as samples to be detected, and after the dilution times are more than 10000 times in the existing system, the T line signal value is similar to the background value, and the sample is judged to be negative.

The test is carried out in two groups, wherein the first group is a saliva test kit which is firstly subjected to sample lysis treatment and then used without a pretreatment pad, the second group is a saliva test kit which is provided by the embodiment 4 and used with a pretreatment pad for detection, the test is repeated for 3 times and is carried out by using a fluorescence immunoassay analyzer, and the specific experimental process is as follows:

1) the detection device is rewarmed at 15-30 ℃.

2) The virus culture was diluted 500-fold, 1000-fold, 2000-fold, 4000-fold, 5000-fold, 7500-fold, 10000-fold, and 12000-fold respectively to obtain 8 samples, 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 respectively to obtain saliva samples, and 1 sample was not added with the virus culture, and the culture concentration was 0.

3) After the saliva sample was collected, the diluted virus culture was added according to the gradient in 2), respectively.

4) The existing detection device without the pretreatment pad and the matched lysate are taken out of the sealed aluminum foil bag, and the detection device with the pretreatment pad is taken out of the sealed aluminum foil bag and placed on a clean and horizontal plane.

5) Dividing the sample in 3) into two parts, wherein the first part is added into the lysis solution of the existing detection device without the pretreatment pad, and the second part is not added into the lysis solution.

6) A certain amount of sample (e.g. 60 microliters) with lysate is added dropwise to the first set of test devices; and opening a sample sampling cover of the second group of detection devices with the pretreatment pads, placing the exposed sample sampling blocks into samples which are not added into the lysis solution, standing for a certain time (for example, 1 to 2 minutes), covering the sample sampling cover, and horizontally pressing for 1 to 2 times.

7) The test pieces were placed on a horizontal table at room temperature, and the test results were read by a fluorescence immunoassay analyzer after 15 minutes.

The detection results are shown in tables 7, 8 and 9, the C-line signals of the quality control lines of the two groups of detection results are basically stable, when the dilution multiples are the same, the T-line signals of the detection lines are basically similar, the average value (Mean T) of the T-line signals of the two groups of data is taken to make linear correlation, and as shown in table 9 and fig. 5, the correlation R2 of the two groups of data is 0.9998, which is highly correlated data, so that the detection device is feasible for detecting the fluorescent immunochromatographic virus, and the virus can be directly detected by a one-step method without pretreatment such as cracking of a sample. Therefore, an additional pretreatment step is not needed, and possible secondary pollution to operators caused by the pretreatment step is reduced. The pretreatment pad arranged in front of the test strip sample pad comprises a lysis reagent, so that the same effect of independent lysis can be obtained.

TABLE 7 detection results of a detection device without a pretreatment pad

TABLE 8 detection results of the detection device with the pre-treatment pad of the present invention

TABLE 9 comparison of signal averages for detection lines T without pre-treatment pad and with pre-treatment pad

EXAMPLE 9 validation of an alternative immunochromatographic assay device for detection of novel coronaviruses prepared by fluorescence as provided in example 5

The detection device for preparing the pre-treatment pad by using the formula A, the formula B and the formula C is divided into three groups, wherein the formula A corresponds to the group I, the formula B corresponds to the group II, and the formula C corresponds to the group III. The same saliva sample as in example 8 was collected and tested, and repeated 3 times, using a fluorescence immunoassay analyzer.

The specific experimental procedures 1) to 3) were the same as in example 8, followed by the procedures of:

4) the test device for the pre-treated pads prepared according to formulas a, B, and C was taken out of the sealed aluminum foil pouch and placed on a clean and horizontal plane.

5) Dividing the sample in the step 3) into three parts, opening the sample sampling covers of the detection devices in the first group, the second group and the third group, exposing the sample sampling blocks, putting the sample sampling blocks into the sample, covering the sample sampling covers after one group corresponds to one sample and standing for the same time as in the embodiment 8, and pressing for 1-2 times in the horizontal direction.

As shown in tables 10, 11, and 12, when Triton X-100 was replaced with stearic acid monoethanolamide, a signal was detected even at a dilute concentration of 1/12000, which is more sensitive than that in example 8, and when the current treatment pad was prepared according to formulation B, in which the content of stearic acid monoethanolamide was 1%, the positive rate was the highest and the virus release was more complete. This fully indicates that the addition of stearic acid monoethanolamide to the lysis reagent treated on the sample pretreatment pad is critical to the lysis effect, and the content of stearic acid monoethanolamide in mass fraction is preferably 1%.

TABLE 10 test results of group I test devices

TABLE 11 test results of group II test devices

TABLE 13 test results of group III test devices

All patents and publications mentioned in the specification of the invention are indicative of the techniques disclosed in the art to which this invention pertains and are intended to be applicable. All patents and publications cited herein are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The invention described herein may be practiced in the absence of any element or elements, limitation or limitations, which limitation or limitations is not specifically disclosed herein. For example, the terms "comprising", "consisting essentially of … …" and "consisting of … …" in each instance herein may be substituted for the remaining 2 terms of either. The word "a" or "an" herein means only "one", and does not exclude only one, but may mean 2 or more. The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described, but it is recognized that various modifications and changes may be made within the scope of the invention and the claims which follow. It is to be understood that the embodiments described herein are preferred embodiments and features and that modifications and variations may be made by one skilled in the art in light of the teachings of this disclosure, and are to be considered within the purview and scope of this invention and the scope of the appended claims and their equivalents.

Claims

1. The immunochromatography detection device is characterized by comprising a sample collection part and a test strip, wherein the test strip comprises a sample pretreatment pad, a sample collected in the sample collection part can be transferred to the sample pretreatment pad, and the sample pretreatment pad comprises a pretreatment reagent which is dried and fixed and is used for splitting a virus sample; the pretreatment liquid reagent is a cracking reagent for the novel coronavirus in the sample; the lysis reagent comprises: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 0.5 to 1.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass, wherein the pH value of the phosphate buffer solution is 7.5 to 8.5.

2. The detection device of claim 1, wherein the test strip further comprises a bottom plate, a sample pad, a label pad, a reaction membrane and a water absorption pad, wherein the sample pretreatment pad, the sample pad, the label pad, the reaction membrane and the water absorption pad are sequentially overlapped together; the sample collection part comprises a sample collection block and a sampling groove.

3. The test device of claim 2, further comprising a housing, wherein the test strip is disposed in the housing, a sampling slot for accommodating the sample collection block is disposed at the front end of the housing, and a filter member is disposed at the connection between the sample collection block and the test strip.

4. The test device of claim 3, wherein the sample collection block is a sponge capable of adsorbing the sample, the sample collection block partially exposes out of the sampling slot, and the exposed portion is covered with a sampling cover for pressing the sample collection block to allow the sample to pass through the filter member to the sample pretreatment pad.

5. The detecting device for detecting the rotation of the motor rotor according to the claim 4, wherein the card shell comprises a card shell cover and a card shell bottom, the card shell bottom is provided with a test strip groove, and the front end of the test strip groove comprises a pretreatment pad groove; the filter component is a filter screen, the filter screen is positioned on the card shell cover and/or the card shell bottom, and the width of the filter screen is not more than that of the sample pretreatment pad.

6. The detection device according to claim 5, wherein the sample collection block is provided with a groove so as to be fixed in the sampling groove at the front end of the card shell, and the distance between the sample collection block and the filter screen is 1-3 mm.

7. The test device of claim 2, wherein the label pad comprises colloidal gold or a fluorescent label.

8. Use of stearic acid monoethanolamide for the preparation of a novel coronavirus splitting reagent, wherein said splitting reagent comprises: 0.01mol/L phosphate buffer solution, 3 percent of casein by mass, 0.5 to 1.5 percent of stearic acid monoethanolamide by mass and 0.01 percent of proclin300 by mass, wherein the pH value of the phosphate buffer solution is 7.5 to 8.5.