Influenza a / b virus, respiratory syncytial virus antigen test strip and kit

Abstract

The application provides an influenza A virus, influenza B virus and respiratory syncytial virus antigen detection test strip and kit, the test strip comprises a bottom plate, a sample pad, a combination pad, a chromatography matrix and a water absorption pad which are sequentially overlapped and are bonded on the bottom plate; wherein the sample pad is added with a biological blocking agent, the biological blocking agent comprises the following components with terminal concentrations: 10-20 mg / mL bovine serum albumin, 1-5 mg / mL sheep IgG and 0.1-3 mg / mL chicken IgY; the combination pad is added with a chemical blocking agent, the chemical blocking agent comprises the following components with terminal concentrations: 0.1%-1% polyvinylpyrrolidone, 0.1%-5% PEG4000, 0.1%-2% Triton X-100 and 0.1%-2% Tween-20. The influenza A virus, the influenza B virus and the respiratory syncytial virus are simultaneously detected, and the specificity is high and the sensitivity is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical diagnosis, and relates to an influenza A / B virus and respiratory syncytial virus antigen detection test strip and kit. BACKGROUND

[0002] Respiratory viruses are a group of viruses that can cause respiratory system and other system diseases through respiratory infection. Common respiratory viruses include influenza virus of orthomyxovirus family, parainfluenza virus, measles virus, mumps virus, respiratory syncytial virus of paramyxovirus family, coronavirus of coronavirus family, rubella virus of togavirus family and adenovirus of adenovirus family, etc. For respiratory viruses, the same virus can cause various clinical symptoms, and different viruses can cause the same clinical symptoms, which causes great difficulty in clinical diagnosis and treatment. Therefore, the detection and diagnosis of such viruses are extremely important for taking effective prevention and treatment measures.

[0003] Influenza viruses are divided into three types, i.e. A, B and C, according to the antigenic properties of viral nucleoprotein (NP). Influenza viruses of A, B and C types all belong to orthomyxovirus family, and influenza A virus is the most likely to cause an epidemic, followed by influenza B virus, and influenza C virus rarely causes an epidemic. Influenza A virus is further divided into various subtypes according to the different antigenic properties of hemagglutinin (HA) and neuraminidase (NA) proteins. Influenza viruses are mainly transmitted through air droplets, and often cause fever, fatigue, muscle soreness and mild to moderate respiratory symptoms. Severe cases can cause pneumonia, myocarditis and heart failure.

[0004] Respiratory syncytial virus (RSV) is an enveloped single-stranded negative-strand RNA virus belonging to paramyxovirus family and pneumovirus genus. RSV nucleotide encodes at least 10 specific proteins, which can divide RSV into two subtypes, i.e. A (RSVA) and B (RSVB). The nucleotide variation within the two subtypes is as high as 20%. Respiratory syncytial virus is widely prevalent in the world, and mainly breaks out in winter and spring in temperate regions; however, in cold regions, it can be detected almost all year round. The two subtypes A and B often coexist, and the relative proportion varies each year, and generally, the season with dominant A subtype epidemic is more. RSV is not only the most common viral pathogen in lower respiratory tract infection of infants and young children, but also has a high incidence in the elderly and immunocompromised high-risk groups, and can cause severe complications, leading to prolonged hospitalization and high mortality.

[0005] In recent years, the incidence of respiratory virus infection has increased significantly, which poses a great threat to global public health and human health. Therefore, timely and accurate identification of the type of respiratory virus infection and its epidemic characteristics is crucial for clinical treatment. Currently, virus isolation and culture method, immunofluorescence, and molecular biology methods such as PCR technology are mainly used for clinical detection of respiratory viruses. The above-mentioned methods need to be performed by specific equipment and professional operators, and need to be operated in the laboratory. Therefore, they do not have the characteristics of simple operation and rapidness. Pathogen culture method is the gold standard for detection, but the culture time is generally 7 days, which is time-consuming and is not conducive to timely clinical treatment.

[0006] Therefore, it is particularly important to establish a rapid and simple method for detecting respiratory viruses for timely diagnosis and treatment of viral infections. SUMMARY

[0007] To solve the technical problem, the application provides an influenza A / B virus and respiratory syncytial virus antigen detection test strip and kit, which has strong specificity and no non-specificity of the same test strip detection, and high sensitivity.

[0008] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0009] In the first aspect of the application, an influenza A / B virus and respiratory syncytial virus antigen detection test strip is provided, comprising:

[0010] A sample pad, a binding pad, a chromatography matrix and a water absorption pad are adhered to the bottom plate in sequence; wherein,

[0011] The biological blocking agent added in the sample pad comprises the following components at a final concentration: 10-20 mg / mL bovine serum albumin, 1-5 mg / mL sheep IgG and 0.1-3 mg / mL chicken IgY;

[0012] The binding pad is coated with an influenza A virus monoclonal antibody A2-coated colloidal gold complex, an influenza B virus monoclonal antibody B2-coated colloidal gold complex and a respiratory syncytial virus monoclonal antibody R2-coated colloidal gold complex; and the binding pad is added with a chemical blocking agent, which comprises the following components at a final concentration: 0.1%-1% polyvinylpyrrolidone, 0.1%-5% PEG4000, 0.1%-2% Triton X-100, 0.1%-2% Tween-20;

[0013] The chromatography matrix is provided with a quality control line C on the side close to the water absorption pad, and the quality control line C is coated with an anti-mouse IgG secondary antibody;

[0014] A detection line T is provided on the side of the chromatography matrix near the binding pad; the detection line T includes: a detection line A coated with influenza A virus monoclonal antibody A1, a detection line B coated with influenza B virus monoclonal antibody B1, and a detection line R coated with respiratory syncytial virus monoclonal antibody R1 (mouse-derived).

[0015] Furthermore, the colloidal gold complex coated with the influenza A virus monoclonal antibody A2 has a pH of 7.0–7.5 and a labeling amount of 15 μg / mL colloidal gold solution.

[0016] Furthermore, the pH of the colloidal gold complex coated with the influenza B virus monoclonal antibody B2 is 7.0–7.5, and the labeling amount is 15 μg / mL colloidal gold solution.

[0017] Furthermore, the pH of the colloidal gold complex coated with the respiratory syncytial virus monoclonal antibody R2 is 7.0–7.5, and the labeling amount is 18 μg / mL colloidal gold solution.

[0018] Furthermore, the concentration of the influenza A virus monoclonal antibody A1 is 1 mg / mL, and the coating amount is 0.7–1.5 μL / cm.

[0019] Furthermore, the concentration of the influenza B virus monoclonal antibody B1 is 1 mg / mL, and the coating amount is 0.7–1.5 μL / cm.

[0020] Furthermore, the concentration of the respiratory syncytial virus monoclonal antibody R1 (mouse-derived) is 1 mg / mL, and the coating amount is 0.7–1.5 μL / cm.

[0021] Furthermore, the concentration of the anti-mouse IgG secondary antibody is 1 mg / mL, and the coating amount is 0.7–1.5 μL / cm.

[0022] In a second aspect, the present invention provides a method for preparing the influenza A / B virus and respiratory syncytial virus antigen test strip, the method comprising:

[0023] The biological blocking agent is applied to the sample pad to obtain the treated sample pad;

[0024] The colloidal gold complex coated with influenza A virus monoclonal antibody A2, the colloidal gold complex coated with influenza B virus monoclonal antibody B2, and the colloidal gold complex coated with respiratory syncytial virus monoclonal antibody R2 were treated with a stabilizer and then sprayed onto the conjugate pad. The chemical blocking agent was then sprayed onto the conjugate pad to obtain the treated conjugate pad.

[0025] Spray the anti-mouse IgG secondary antibody on the control line C on the chromatography substrate, spray the influenza A virus monoclonal antibody A1 on the detection line A on the chromatography substrate, spray the influenza B virus monoclonal antibody B1 on the detection line B on the chromatography substrate, and spray the respiratory syncytial virus monoclonal antibody R1 (mouse origin) on the detection line R on the chromatography substrate to obtain the chromatography substrate containing the detection line T and the control line C;

[0026] The treated sample pad, the treated conjugate pad, the chromatography substrate containing the detection line T and the control line C, and the water absorption pad are sequentially and mutually overlapped and pasted on the base plate to obtain the influenza A / B virus and respiratory syncytial virus antigen detection test strip.

[0027] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

[0028] 1. The influenza A / B virus and respiratory syncytial virus antigen detection test strip provided by the present application has no cross-reaction between the kit and other pathogenic microorganisms, and has strong specificity. And high sensitivity: the minimum detection limit of H1N is 1.58x10 3 TCID 50 / mL; the minimum detection limit of H3N2 is 4.2x10 2 TCID 50 / mL; the minimum detection limit of FluB is 6.3x10 2 TCID 50 / mL; the minimum detection limit of RSV A is 7.2x10 2 TCID 50 / mL; the minimum detection limit of RSV B is 7.9x10 2 TCID 50 / mL.

[0029] 2. The influenza A / B virus and respiratory syncytial virus antigen detection test strip provided by the present application is simple to operate, and can detect virus infection after symptoms appear, and the result can be obtained in 15 minutes, so that the disease can be diagnosed quickly. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1 The appearance of the influenza A / B virus and respiratory syncytial virus antigen detection test strip of the present application is shown in the figure.

[0032] Figure 2 The figure is a structural schematic diagram of the influenza A / B virus and respiratory syncytial virus antigen detection test strip of the present application; the reference signs are as follows: 1: water absorption pad; 2: chromatography matrix; 3: binding pad; 4: sample pad; 5: bottom plate; the transverse arrow indicates the chromatography direction;

[0033] Figure 3 The figure is a structural schematic diagram of the influenza A / B virus and respiratory syncytial virus antigen detection test strip of the present application; the reference signs are as follows: 1: water absorption pad; 2: chromatography matrix; 3: binding pad; 4: sample pad; 5: bottom plate; the transverse arrow indicates the chromatography direction; DETAILED DESCRIPTION

[0034] The advantages and various effects of the present application will be more clearly presented hereinafter in combination with specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, rather than limit the present application.

[0035] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the present application belongs. If there is a conflict, the present specification takes precedence.

[0036] Unless otherwise specifically indicated, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0037] The influenza A virus monoclonal antibody, influenza B virus monoclonal antibody, respiratory syncytial virus monoclonal antibody, anti-mouse IgG secondary antibody, goat IgG and chicken IgY used in the embodiments of the present application are all commercially available products.

[0038] The technical solution provided by the embodiments of the present application is to solve the above technical problems, and the general idea is as follows:

[0039] According to a typical embodiment of the present application, an influenza A / B virus and respiratory syncytial virus antigen detection test strip is provided, comprising:

[0040] A sample pad, a binding pad, a chromatography matrix and a water absorption pad are sequentially adhered to the bottom plate; wherein,

[0041] The biological blocking agent added in the sample pad comprises the following components with final concentrations: 10-20 mg / mL bovine serum albumin, 1-5 mg / mL goat IgG and 0.1-3 mg / mL chicken IgY;

[0042] The binding pad is coated with a colloidal gold complex coated with an influenza A virus monoclonal antibody A2, a colloidal gold complex coated with an influenza B virus monoclonal antibody B2 and a colloidal gold complex coated with a respiratory syncytial virus monoclonal antibody R2; and a chemical blocking agent is added to the binding pad, the chemical blocking agent including the following components at the final concentrations: 0.1%-1% polyvinylpyrrolidone, 0.1%-5% PEG4000, 0.1%-2% Triton X-100, 0.1%-2% Tween-20;

[0043] A quality control line C is provided on one side of the chromatography matrix close to the water absorption pad, and the quality control line C is coated with an anti-mouse IgG secondary antibody;

[0044] A detection line T is provided on one side of the chromatography matrix close to the binding pad; the detection line T includes: a detection line A coated with an influenza A virus monoclonal antibody A1, a detection line B coated with an influenza B virus monoclonal antibody B1 and a detection line R coated with a respiratory syncytial virus monoclonal antibody R1 (mouse origin).

[0045] The principle of the test strip is as follows: the detection lines (A / B / R) on the nitrocellulose membrane are coated with an influenza A virus monoclonal antibody A1, an influenza B virus monoclonal antibody B1 and a respiratory syncytial virus monoclonal antibody R1 (mouse origin), respectively; and colloidal gold is used to label the influenza A virus monoclonal antibody A2, the influenza B virus monoclonal antibody B2 and the respiratory syncytial virus monoclonal antibody R2 as tracers and fixed on the glass fiber membrane at the sample addition hole. During detection, the sample to be detected is added to the sample addition hole, the substances to be detected in the sample can first react with the pre-coated colloidal gold labeled antibodies on the glass fiber membrane to form antigen-antibody complexes, then the complexes are chromatographed under the capillary effect, and finally the complexes are combined with the antibodies pre-coated on the detection lines (A / B / R) on the nitrocellulose membrane to form purple-red bands. According to the color development of the bands, the sample can be determined to be positive for the influenza A virus, the influenza B virus, the respiratory syncytial virus or a combination thereof. If there is no substance to be detected in the sample, no purple-red band will appear on the detection line, and the sample is negative. No matter whether the sample contains the substance to be detected or not, a purple-red band should appear on the quality control line C, which serves as an internal control standard for determining whether the chromatography process is normal and whether the reagent is invalid.

[0046] The chromatography matrix can be a nitrocellulose membrane, the binding pad is a glass fiber membrane containing gold-labeled antibodies, and the bottom plate can be a PVC bottom plate.

[0047] The present application only needs one test strip, which can minimize the consumption of consumables and achieve the purpose of simultaneously detecting the influenza A virus, the influenza B virus and the respiratory syncytial virus.

[0048] In the experimental exploration process, the present inventors found that when the sample pad and the conjugate pad were only treated with phosphate and the like, the three index antibodies mixed together would produce false positive results when detected on the same test strip. In order to eliminate the non-specificity of the detection on the same test strip, the present application adds a blocking agent to the conjugate pad and the sample pad. The blocking agent added to the conjugate pad is a chemical blocking agent, including 0.1%-1% polyvinylpyrrolidone, 0.1%-5% PEG4000, 0.1%-2% Triton X-100, and 0.1%-2% Tween-20. The biological blocking agent added to the sample pad includes bovine serum albumin (10-20 mg / mL), sheep IgG (1-5 mg / mL), and chicken IgY (0.1-3 mg / mL). Therefore, the antigen detection kit of the present application has greater advantages over other kits as a whole.

[0049] The influenza A / B virus and respiratory syncytial virus antigen test strip of the present application will be described in detail below in combination with examples and experimental data.

[0050] Example 1, Preparation of the influenza A / B virus and respiratory syncytial virus antigen test kit

[0051] I. Influenza A / B virus and respiratory syncytial virus antigen test kit

[0052] 1. Sample pad treatment

[0053] Prepare the sample pad treatment solution: weigh 0.296 g of sodium dihydrogen phosphate, 2.901 g of disodium hydrogen phosphate, 10 g of bovine serum albumin, and 2.14 g of Triton X-100 into a 1 L ultrapure water solution, and add sheep IgG and chicken IgY to make the final concentrations 2 mg / mL and 0.5 mg / mL, respectively. Take a 200 mm*300 mm sample pad and place it in a stainless steel tray. Uniformly bubble the sample pad treatment solution on the sample pad, turn it over every 10 min, and after 20 min, take it out and dry it in a 37°C drying oven (about 12-16 hours). Cut it into 15 mm*300 mm and store it.

[0054] 2. Conjugate pad treatment

[0055] Configuration of the pad processing solution: take 0.296 g of sodium dihydrogen phosphate, 2.901 g of disodium hydrogen phosphate, 10 g of bovine serum albumin, 60 g of sucrose into 1 L of ultrapure water solution, and add Tween-20, PEG4000, polyvinylpyrrolidone and Triton X-100 to make their final concentrations 0.5%, 1%, 0.2%, 0.2% respectively. Take a 240mm*300mm pad and place it in a stainless steel tray, evenly bubble the pad processing solution on the pad, wait for it to be completely soaked, take it out after 10 minutes and place it in a 37°C drying oven for drying treatment (about 12-16 hours), cut it into 6mm*300mm and store it.

[0056] 3. Preparation of colloidal gold solution

[0057] Using the chemical reduction method of trisodium citrate, take 10 mL of chloroauric acid and add it to 990 mL of ultrapure water solution, heat to boiling, then adjust to 100-150°C, then quickly add 15 mL of trisodium citrate to the chloroauric acid aqueous solution, continue to boil for 10 min, wait until it turns purple red, cool to room temperature, and restore the volume to 1 L.

[0058] 4. Preparation of influenza A virus, influenza B virus, and respiratory syncytial virus gold-labeled antibodies

[0059] (1) Use 0.1M potassium carbonate to adjust the pH of the colloidal gold solution to the appropriate value.

[0060] (2) Use 10mM PB to dilute the influenza A virus monoclonal antibody, influenza B virus monoclonal antibody, and respiratory syncytial virus monoclonal antibody stock solution to a concentration of 0.1mg / mL.

[0061] (3) According to the volume of the label, add the corresponding volume of diluted antibody, and shake the reaction at room temperature for 30 min.

[0062] (4) Add a certain volume of 2% PEG20000 / 20% BSA for blocking, and continue to shake the reaction in the shaker for 30 min.

[0063] (5) After centrifugation at 14000 rpm for 25 min, discard the supernatant, add a certain volume of gold-labeled antibody resuspension solution to resuspend the precipitate, adjust each antibody to 4 OD, and store at 4°C for later use.

[0064] 5. Preparation of gold-labeled pad

[0065] Mix the adjusted 4 OD gold-labeled antibody according to a certain proportion, take out the treated pad, use a pipette to evenly drop 1.2 mL of gold-labeled antibody mixed solution on the pad, and place it in a 37°C drying oven for treatment (2-3 hours) and storage.

[0066] 6. NC membrane coating

[0067] The C-line coating antibody, influenza A virus coating antibody, influenza B virus coating antibody, and respiratory syncytial virus coating antibody were diluted to 0.8 mg / mL using a coating solution to obtain a C-line coating solution, an influenza A virus coating solution, an influenza B virus coating solution, and a respiratory syncytial virus coating solution. Each coating solution was uniformly drawn on a nitrocellulose membrane using a membrane drawing instrument at 1 μL / cm to form a quality control line C, an influenza A virus detection line A1, an influenza B virus detection line B1, and a respiratory syncytial virus detection line R1. The C-line was positioned and adjusted at a distance of 5 ± 1 mm from the upper end of the water-absorbing pad, and the R-line was positioned and adjusted at a distance of 6 ± 1 mm from the lower edge of the NC membrane. The distance between the A-line, the B-line, and the R-line was 4 mm.

[0068] 7. Test strip assembly

[0069] (1) The water-absorbing pad was cut into a size of 20 ± 1 mm x 300 ± 1 mm using a cutting machine. The treated sample pad, gold marker pad, NC membrane, and water-absorbing pad were sequentially built on a PVC bottom plate to form a colloidal gold chromatography test strip, wherein the water-absorbing pad and the NC membrane overlapped by 2 mm, the NC membrane and the gold marker pad overlapped by 2 mm, the gold marker pad and the sample pad overlapped by 2 mm, and the remaining visible length of the NC membrane was 21 mm.

[0070] (2) The large card was cut into a test strip of 3 mm ± 0.1 mm.

[0071] (3) The prepared test strip was fixed to the membrane strip area of the detection card shell-bottom, the face card shell was covered, and the detection card was placed in the pressing shell machine to press tightly. After the shell cover was closed, there was no gap.

[0072] 8. Sample extraction solution

[0073] 0.296 g of sodium dihydrogen phosphate, 2.901 g of disodium hydrogen phosphate, 5 g of sodium caseinate, 0.6 g of NP-40, and 150 uL of preservative were dissolved in 1 L of ultrapure water solution.

[0074] 9. Sampling sample tube: including a tube and a dropper, 500 μL (about 20 drops) of sample extraction solution was added to the sample tube, and a sampling swab was placed.

[0075] 10. Packaging: the final product kit includes a detection card, a sample extraction solution, a sampling sample tube, and an instruction manual.

[0076] 2. Detection method

[0077] (1) Sample collection: The sampler holds the head of the collected person with one hand and holds the swab with the other hand to the nostril, slowly and deeply along the bottom of the lower nasal passage. Since the nasal passage is arc-shaped, it should not be forced too hard to avoid injury and bleeding. When the tip of the swab reaches the back wall of the nasopharynx, rotate it gently for one turn (if reflex cough occurs, stop for a while), then slowly take out the swab. The sample should be immediately treated with sample extraction solution after collection.

[0078] (2) Sample processing: Take out the sample tube and add 500 μL (about 20 drops) of sample extraction solution to the sample tube (the lowermost scale of the sample tube is about 500 μL). Put the collected swab into the sample tube, rotate the swab close to the inner wall of the sample tube about 10 times, so that the swab is completely immersed in the sample extraction solution. Squeeze the swab with your fingers a few times from the outside of the sample tube to make the solution stay in the tube as much as possible, take out the swab and cover the dropper.

[0079] (3) Sample detection: Invert the sample tube above the sample hole of the detection card, gently squeeze the sample tube, and drop 3 drops (about 70 μL) into the sample hole of the detection card. Start timing, observe the results after 15 minutes at room temperature, and make a judgment.

[0080] The judgment method is as follows:

[0081] a. Negative: only the quality control line area (C) appears a band, and no band appears at the detection line area (A / B / R).

[0082] b. Positive: both the quality control line area (C) and the detection line area (A) appear a band, indicating infection with influenza A virus;

[0083] the quality control line area (C) and the detection line area (B) appear a band, indicating infection with influenza B virus;

[0084] the quality control line area (C) and the detection line area (R) appear a band, indicating infection with respiratory syncytial virus;

[0085] the quality control line area (C) and the detection line area (A / B) appear a band, indicating infection with influenza A / B virus;

[0086] the quality control line area (C) and the detection line area (A / R) appear a band, indicating infection with influenza A virus and respiratory syncytial virus;

[0087] the quality control line area (C) and the detection line area (B / R) appear a band, indicating infection with influenza B virus and respiratory syncytial virus;

[0088] the quality control line area (C) and the detection line area (A / B / R) appear a band, indicating infection with all three;

[0089] c. Invalid: the quality control line area (C) does not appear a red band. Specifically, Figure 3as shown.

[0090] Comparative Example 1

[0091] In this comparative example, the binding pad without chemical blocking agent + sample pad without biological blocking agent were used, and the others were the same as in Example 1.

[0092] Comparative Example 2

[0093] In this comparative example, the binding pad with chemical blocking agent + sample pad without biological blocking agent were used, and the others were the same as in Example 1.

[0094] Comparative Example 3

[0095] In this comparative example, the binding pad without chemical blocking agent + sample pad with biological blocking agent were used, and the others were the same as in Example 1.

[0096] Experimental Example 1, Specificity Test

[0097] To verify the effect of adding chemical blocking agent to the binding pad and adding biological blocking agent to the sample pad on the detection effect of the test strip, the kits of Example 1 and Comparative Examples 1-3 were tested, respectively.

[0098] The virus stock solutions of H1N1 (ATCC VR-1469), H3N2 (ATCC VR-1680), FluB (ATCC VR-1735), RSV A (ATCC VR-26), and RSV B (ATCC VR-1580) from ATCC were diluted to 7.9 x 10 4 TCID 50 / mL, 2.1 x 10 4 TCID 50 / mL, 3.15 x 10 4 TCID 50 / mL, 3.6 x 10 4 TCID 50 / mL, 3.95 x 10 4 TCID 50 / mL, and sample extraction solution were tested for specificity, and the detection results were as follows:

[0099] Table 1

[0100]

[0101]

[0102] As can be seen from Table 1, compared with Comparative Examples 1-3, in Example 1 of the present application, the binding pad with chemical blocking agent and the sample pad with biological blocking agent are used in combination, so that the test strip has high specificity.

[0103] Experimental Example 2, Sensitivity Test

[0104] The minimum detection limit was determined after gradient dilution of virus stock of ATCC-derived H1N1 (ATCC VR-1469), H3N2 (ATCC VR-1680), FluB (ATCC VR-1735), RSV A (ATCC VR-26), RSV B (ATCC VR-1580), and 3-5 replicates were performed for each gradient dilution, and 20 replicates were performed for each, and the virus level with 90-95% positive detection rate was determined as the minimum detection limit. The kit of Example 1 of the present application was used for the detection, and the detection results were as follows:

[0105] 1. Minimum detection limit test for H1N1

[0106] Table 2 - Actual data for detection of different titers of H1N1

[0107]

[0108] Table 3 - Actual data for minimum detection limit of H1N1

[0109]

[0110]

[0111] 2. Minimum detection limit test for H3N2

[0112] Table 4 - Actual data for detection of different titers of H3N2

[0113]

[0114] Table 5 - Actual data for minimum detection limit of H3N2

[0115]

[0116] 3. Minimum detection limit test for FluB

[0117] Table 6 - Actual data for detection of different titers of FluB

[0118]

[0119]

[0120] Table 7 - Actual data for minimum detection limit of FluB

[0121]

[0122] 4. Minimum detection limit test for RSV A

[0123] Table 8 - Actual data for detection of different titers of RSV A

[0124]

[0125] Table 9 - Actual data for minimum detection limit of RSV A

[0126]

[0127]

[0128] 5. Minimum detection limit of RSV B

[0129] Table 10 - Actual data for detection of different titers of RSV B

[0130]

[0131] Table 11 - Actual data for minimum detection limit of RSV B

[0132]

[0133]

[0134] 3. Conclusion

[0135] From the above, it is finally determined that the detection sensitivity of the kit of the present application is as shown in Table 12.

[0136] Table 12

[0137] Detection index Virus strain Lowest detection limit H1N1 ATCC VR-1469 1.58 x 10 3 TCID 50 / mL]]> H3N2 ATCC VR-1680 4.2 x 10 2 TCID 50 / mL]]> FluB ATCC VR-1735 6.3 x 10 2 TCID 50 / mL]]> RSVA ATCC VR-26 7.2 x 10 2 TCID 50 / mL]]> RSVB ATCC VR-1580 7.9 x 10 2 TCID 50 / mL]]>

[0138] Experimental Example 3, Cross-validation

[0139] 1. Test strains

[0140] Different microorganisms in the following table were diluted to the corresponding concentration / titer using a sample extraction solution to verify the specificity of the kit of the present application, and the related pathogens and titers are as shown in Table 13.

[0141] Table 13 - Test information of cross-validation strains

[0142]

[0143]

[0144] 2. Test results

[0145] Table 14 - Test results of cross-validation strains

[0146]

[0147] 3. Conclusion

[0148] From the above data, the results of detecting other pathogenic microorganisms by the kit of the present application are all negative, which proves that the kit of the present application has no cross-reaction with other pathogenic microorganisms and has strong specificity.

[0149] Finally, it should be noted that the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0150] While the preferred embodiments of the application have been described, additional modifications and changes can occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims shall cover all such modifications and changes as fall within the true spirit and scope of the application.

[0151] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. An antigen test strip for detecting influenza A / B virus, respiratory syncytial virus, characterized in that, It comprises: a bottom plate, a sample pad, a binding pad, a chromatographic matrix and an absorbent pad which are adhered to the bottom plate in turn; wherein, the sample pad is added with a biological blocking agent, the biological blocking agent comprises the following components at the final concentration: 10-20 mg / mL bovine serum albumin, 1-5 mg / mL goat IgG and 0.1-3 mg / mL chicken IgY; the binding pad is coated with an influenza A virus monoclonal antibody A2 coated colloidal gold complex, an influenza B virus monoclonal antibody B2 coated colloidal gold complex and a respiratory syncytial virus monoclonal antibody R2 coated colloidal gold complex; and the binding pad is added with a chemical blocking agent, the chemical blocking agent comprises the following components at the final concentration: 0.1%-1% polyvinylpyrrolidone, 0.1%-5% PEG4000, 0.1%-2% Triton X-100, 0.1%-2% Tween-20; the chromatographic matrix is provided with a quality control line C on the side close to the absorbent pad, and the quality control line C is coated with an anti-mouse IgG secondary antibody; the chromatographic matrix is provided with a detection line T on the side close to the binding pad; the detection line T comprises: a detection line A coated with an influenza A virus monoclonal antibody A1, a detection line B coated with an influenza B virus monoclonal antibody B1 and a detection line R coated with a respiratory syncytial virus monoclonal antibody R1 of mouse origin; the pH of the influenza A virus monoclonal antibody A2 coated colloidal gold complex is 7.0-7.5, and the labeling amount is 15 μg / mL colloidal gold solution; the pH of the influenza B virus monoclonal antibody B2 coated colloidal gold complex is 7.0-7.5, and the labeling amount is 15 μg / mL colloidal gold solution; the pH of the respiratory syncytial virus monoclonal antibody R2 coated colloidal gold complex is 7.0-7.5, and the labeling amount is 18 μg / mL colloidal gold solution.

2. The test strip for detecting influenza A / B virus and respiratory syncytial virus antigens according to claim 1, characterized by, The concentration of the influenza A virus monoclonal antibody A1 is 1 mg / mL, and the coating amount is 0.7-1.5 μL / cm.

3. The test strip for detecting influenza A / B virus and respiratory syncytial virus antigens according to claim 1, wherein, The concentration of the influenza B virus monoclonal antibody B1 is 1 mg / mL, and the coating amount is 0.7-1.5 μL / cm.

4. The test strip of claim 1, wherein the antigens are selected from the group consisting of: Influenza A virus, Influenza B virus, and Respiratory Syncytial Virus. The concentration of the respiratory syncytial virus monoclonal antibody R1 of mouse origin is 1 mg / mL, and the coating amount is 0.7-1.5 μL / cm.

5. A method for preparing the antigen test strip for detecting influenza A / B virus and respiratory syncytial virus according to any one of claims 1 to 4, characterized in that, The method comprises: coating the biological blocking agent on the sample pad to obtain a treated sample pad; spraying the influenza A virus monoclonal antibody A2 coated colloidal gold complex, the influenza B virus monoclonal antibody B2 coated colloidal gold complex and the respiratory syncytial virus monoclonal antibody R2 coated colloidal gold complex on the binding pad after treatment with a stabilizer, and spraying the chemical blocking agent on the binding pad to obtain a treated binding pad; Spray anti-mouse IgG secondary antibody on the control line C on the chromatography matrix, spray influenza A virus monoclonal antibody A1 on the detection line A on the chromatography matrix, spray influenza B virus monoclonal antibody B1 on the detection line B on the chromatography matrix, and spray respiratory syncytial virus monoclonal antibody R1 of mouse origin on the detection line R on the chromatography matrix to obtain a chromatography matrix containing the detection line T and the control line C; Paste the treated sample pad, the treated conjugate pad, the chromatography matrix containing the detection line T and the control line C, and the water absorption pad on the base plate in sequence to obtain an influenza A / B virus and respiratory syncytial virus antigen detection test strip.

6. An antigen detection kit for influenza A / B virus, respiratory syncytial virus, characterized by, The kit comprises any one of the influenza A / B virus and respiratory syncytial virus antigen detection test strips.

7. The influenza A / B virus, respiratory syncytial virus antigen detection kit according to claim 6, characterized by, The kit further comprises a detection card or / and a sample extraction solution or / and a sampling sample tube.

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