Application of high-concentration colloidal gold in preparation of influenza virus detection test strip
By using high-concentration colloidal gold-labeled antibodies and an optimized processing solution, an influenza virus test strip was prepared, solving the problems of low detection sensitivity and cross-reactivity in existing test strips, and achieving highly sensitive and accurate influenza virus detection.
Patent Information
- Application Number
- CN202511484633.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2026-01-23
AI Technical Summary
The existing colloidal gold test strips for influenza A and B have low sensitivity and are prone to cross-reaction, which affects the reliability of the test results and limits their widespread use in clinical diagnosis.
Influenza virus test strips were prepared using high-concentration colloidal gold-labeled monoclonal antibodies against influenza A and B viruses and chicken IgY antibodies with an OD value of 100, combined with optimized sample pad and conjugate pad processing solution formulations.
The test strips have improved their ability to detect samples with low viral loads, reduced the risk of false negatives, minimized non-specific adsorption and cross-reaction, and yielded more accurate and reliable results, making them suitable for primary healthcare institutions and rapid on-site screening.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of immunodetection technology, in particular to the application of high-concentration colloidal gold in the preparation of an influenza virus detection test strip. BACKGROUND
[0002] Influenza virus is a common respiratory pathogen, mainly divided into two types: Influenza A and Influenza B. Influenza virus is the main pathogen causing seasonal influenza and periodic pandemic, posing a persistent and serious threat to global public health security. Therefore, early and rapid detection of influenza virus is of key significance for timely clinical diagnosis, guiding rational drug use and effectively controlling the spread of the epidemic.
[0003] With the progress of medical diagnostic technology, the detection methods of influenza virus have developed from traditional virus isolation culture and serological detection to molecular biology methods such as polymerase chain reaction (PCR), immunological methods such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. In the prior art, PCR method as the gold standard for influenza virus detection has high sensitivity and specificity, but has the disadvantages of complex operation, long time (usually several hours), expensive equipment and need for professional laboratory environment, etc., which is not suitable for primary medical institutions or rapid screening on site. Although ELISA method has high sensitivity, it also has problems such as long detection time and need for special instruments, which limits its popularization and use in rapid screening.
[0004] Colloidal gold immunochromatography test strip as a rapid detection technology has been widely used for qualitative detection of Influenza A and B virus antigens. This method is simple to operate, does not require instruments, and has a short detection time (10-15 minutes), which is suitable for community and outpatient screening. However, the current market Influenza A and B colloidal gold test strip still has certain limitations, such as relatively low detection sensitivity, easy cross-reaction, limited antibody labeling efficiency, etc. These factors affect the reliability of the detection results and restrict the further popularization and application of the technology in clinical diagnosis. SUMMARY
[0005] To solve the above technical problems, the present application uses high-concentration colloidal gold with an OD value of 100 to label Influenza A virus monoclonal antibody 1, Influenza B virus monoclonal antibody 1 and chicken IgY antibody, respectively. The obtained colloidal gold labeling reconstitution solution is mixed and sprayed on the conjugate pad. At the same time, the corresponding capture antibody and goat anti-chicken IgY antibody are sprayed on the nitrocellulose membrane to form the detection line and the quality control line, thereby preparing an influenza virus detection test strip based on high-concentration colloidal gold.
[0006] The first object of the present application is to provide the application of high concentration colloidal gold in the preparation of an influenza virus detection test strip, wherein the test strip uses high concentration colloidal gold labeled influenza virus monoclonal antibody with an OD value of 100.
[0007] The second object of the present application is to provide an influenza virus detection test strip, wherein the influenza virus includes influenza A virus and influenza B virus, and the preparation method of the test strip comprises the following steps:
[0008] (1) preparing a colloidal gold labeling reconstituted solution;
[0009] (2) labeling influenza A virus monoclonal antibody 1 with high concentration colloidal gold, and reconstituting to obtain an influenza A virus monoclonal antibody 1 colloidal gold labeling reconstituted solution, wherein the high concentration colloidal gold has an OD value of 100;
[0010] (3) labeling influenza B virus monoclonal antibody 1 with high concentration colloidal gold, and reconstituting to obtain an influenza B virus monoclonal antibody 1 colloidal gold labeling reconstituted solution, wherein the high concentration colloidal gold has an OD value of 100;
[0011] (4) labeling chicken IgY antibody with high concentration colloidal gold, and reconstituting to obtain a chicken IgY antibody colloidal gold labeling reconstituted solution;
[0012] (5) respectively preparing coating solutions of influenza A virus monoclonal antibody 2, influenza B virus monoclonal antibody 2 and goat anti-chicken IgY antibody;
[0013] (6) mixing the influenza A virus monoclonal antibody 1 colloidal gold labeling reconstituted solution, the influenza B virus monoclonal antibody 1 colloidal gold labeling reconstituted solution and the chicken IgY antibody colloidal gold labeling reconstituted solution, and then spraying them on a conjugate pad, and spraying the coating solutions of the influenza A virus monoclonal antibody 2, the influenza B virus monoclonal antibody 2 and the goat anti-chicken IgY antibody on a nitrocellulose membrane to form an influenza A virus detection line, an influenza B virus detection line and a quality control line;
[0014] (7) sequentially stacking and adhering a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad on a bottom plate, and cutting to obtain the test strip.
[0015] Further, the method further comprises the step of pretreating the sample pad and the conjugate pad.
[0016] Further, the pretreatment is to respectively immerse the sample pad and the conjugate pad in a sample pad pretreatment liquid and a conjugate pad pretreatment liquid, wherein:
[0017] The sample pad pre-treatment solution comprises 45-60 mM of Tris-HCl, 0.4-0.6 wt% of casein, 1-2 wt% of polyvinylpyrrolidone K30, 1-2 wt% of surfactant S9, 0.05-0.08 wt% of Tween-20, 0.02-0.1 wt% of preservative, and 0.002-0.01 wt% of blocking agent, and has a pH value of 8.0-9.0.
[0018] The binding pad pre-treatment solution comprises 40-55 mM of Tris-HCl, 0.4-0.6 wt% of BSA or casein, 0.8-1.2 wt% of polyvinylpyrrolidone K30, 0.04-0.08 wt% of Tween-20, 0.02-0.1 wt% of preservative, and has a pH value of 8.0-9.0.
[0019] Further, in step (1), the colloidal gold labeling reconstitution solution comprises 50 mM of Tris-HCl, 0.5 wt% of casein, 20 wt% of sucrose, 5 wt% of trehalose, and 0.02-0.1 wt% of preservative, and has a pH value of 7.5-8.5.
[0020] Further, in step (5), the concentration of the influenza A virus monoclonal antibody 2 coating solution is diluted to 0.5 mg / mL, the concentration of the influenza B virus monoclonal antibody 2 coating solution is diluted to 0.8 mg / mL, and the concentration of the goat anti-chicken IgY antibody coating solution is diluted to 1 mg / mL.
[0021] Further, in step (6), the OD value of the chicken IgY antibody colloidal gold labeling after mixing is 25.
[0022] Further, the detection test strip further comprises a sample diluent.
[0023] Further, the sample diluent comprises 40-45 g / L of NaCl, 6-6.3 g / L of Tris, 0.4-0.6 g / L of anhydrous disodium hydrogen phosphate, 10 g / L of BSA, 0.4-0.6 g / L of anhydrous potassium hydrogen phosphate, 0.8-1.2 mL / L of S22, 0.2 mL / L of ProClin300, and has a pH value of 7-8.
[0024] A third object of the present application is to provide the use of the above-mentioned detection test strip in the detection of influenza viruses.
[0025] The present application has the following beneficial effects:
[0026] The application uses high-concentration colloidal gold with an OD value of 100 to label the antibody, so that the detection capability of the test strip for low-virus-load samples is greatly enhanced, the detection limit for influenza A virus can reach 0.05 ng / mL, and the detection limit for influenza B can reach 0.25 ng / mL, effectively reducing the risk of false negatives in clinical practice. The optimized sample pad and conjugate pad treatment liquid formula effectively reduces non-specific adsorption and reduces background interference; the antibody pairing and labeling process improves the specificity of detection and reduces cross-reactions, making the result interpretation more accurate and reliable. The application overcomes the bottleneck of low sensitivity while maintaining the advantages of colloidal gold method, such as simple operation, rapidity (10-15 minutes for results), and no need for professional equipment, and is very suitable for primary medical institutions, community clinics and on-site emergency screening, meeting the public health needs of high-throughput and rapid screening. BRIEF DESCRIPTION OF DRAWINGS
[0027] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, in which:
[0028] Figure 1 The application provided herein, wherein 1 is a sample pad, 2 is a conjugate pad, 3 is a nitrocellulose membrane, 31 is an influenza B virus detection line, 32 is an influenza A virus detection line, 33 is a quality control line, 4 is a water absorption pad, and 5 is a base plate. DETAILED DESCRIPTION
[0029] The application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting to the application.
[0030] Example 1: Preparation of a test strip
[0031] I. Pretreatment of sample pad and conjugate pad
[0032] A sample pad treatment liquid is prepared, and the components include 50 mM Tris-HCl buffer, 0.5 wt% casein, 1 wt% polyvinylpyrrolidone K30, 1 wt% surfactant S9, 0.05 wt% Tween-20, 0.02 wt% ProClin300 preservative, and 0.003 wt% blocking agent. The pH value of the sample pad treatment liquid is accurately adjusted to 8.5.
[0033] The sample pad is placed in a soaking container and soaked with the above-mentioned sample pad treatment liquid at an amount of 0.06 mL / cm² for 10 minutes. Subsequently, the sample pad is taken out and spun dry to remove excess liquid, and transferred to a 45°C oven for baking until completely dry. The dried sample pad is sealed and stored in an environment with a humidity of ≤30% for standby use.
[0034] The conjugate pad treatment solution was prepared with components including 50 mM Tris-HCl buffer, 0.5 wt% casein, 1 wt% polyvinylpyrrolidone K30, 0.05 wt% Tween-20 and 0.02 wt% ProClin 300 preservative, and the pH value was adjusted to 8.5. The conjugate pad was added into the treatment solution in an amount of 0.06-0.14 mL / cm2and soaked for 10 minutes. The subsequent spin-drying, 45°C baking drying and low-humidity environment sealing steps were the same as those in the sample pad treatment method.
[0035] II. Preparation of colloidal gold labeling reconstitution solution
[0036] The colloidal gold labeling reconstitution solution was prepared with components including 50 mM Tris-HCl, 0.5 wt% casein, 20 wt% sucrose, 5 wt% trehalose and 0.02 wt% ProClin 300, and the pH value was adjusted to 8.0 using sodium hydroxide solution. The solution was used for subsequent reconstitution of gold-labeled antibodies.
[0037] III. Labeling and reconstitution of influenza A virus monoclonal antibody 1
[0038] Take 40 μL of high-concentration colloidal gold solution with a concentration of 100 OD, add 60 μL of 0.01 M borate solution with a pH value of 7.4, and mix immediately. Add 6 μg of mouse anti-human influenza A virus monoclonal antibody 1 to the mixture, mix gently, and react at room temperature for 1.5 hours. Then add 100 μL of blocking solution (containing 1 wt% casein and 100 mM borate, pH=8.5), continue to mix and react for 20 minutes to complete the blocking. Centrifuge the reaction solution at 10000 rpm at 4°C for 10 minutes. Carefully aspirate the supernatant, add 50 μL of the second-step prepared gold labeling reconstitution solution to the precipitate, and vortex thoroughly to completely resuspend the precipitate, thus obtaining the mouse anti-human influenza A virus monoclonal antibody 1 gold labeling reconstitution solution.
[0039] IV. Labeling and reconstitution of influenza B virus monoclonal antibody 1
[0040] Take 40 μL of high concentration colloidal gold solution with a concentration of 100 OD in a centrifuge tube, quickly add 60 μL of boric acid solution with a concentration of 0.01 M and a pH of 7.4, and immediately vortex mix. Add 6 μg of mouse anti-human influenza B virus monoclonal antibody 1 to the mixture, gently mix, and react at room temperature for 1.5 hours. Then add 100 μL of blocking solution (containing 1 wt% casein and 100 mM boric acid, pH 8.5), continue to mix and react for 20 minutes to complete the blocking. Centrifuge the reaction solution at 10,000 rpm for 10 minutes at 4°C. Carefully aspirate the supernatant, and add 50 μL of the gold-labeled reconstitution solution prepared in the above step to the precipitate, vortex thoroughly to completely resuspend the precipitate, and obtain the mouse anti-human influenza B virus monoclonal antibody 1 colloidal gold-labeled reconstitution solution.
[0041] Five, labeling and reconstitution of chicken IgY antibodies
[0042] Take 40 μL of high concentration colloidal gold solution with a concentration of 100 OD, quickly add 60 μL of boric acid solution with a concentration of 0.01 M and a pH of 7.4, and immediately mix. Add 6 μg of chicken IgY antibody to the mixture, gently mix, and react at room temperature for 1.5 hours. Then add 100 μL of blocking solution (containing 1 wt% casein and 100 mM boric acid, pH 8.5), continue to mix and react for 20 minutes to complete the blocking. Centrifuge the reaction solution at 10,000 rpm for 10 minutes at 4°C. Carefully aspirate the supernatant, and add 50 μL of the gold-labeled reconstitution solution prepared in the above step to the precipitate, vortex thoroughly to completely resuspend the precipitate, and obtain the chicken IgY antibody colloidal gold-labeled reconstitution solution.
[0043] Six, setting of detection lines and quality control lines
[0044] First, prepare the coating diluent, which contains 1x PBS (phosphate buffer) and 2 wt% sucrose solution. Dilute the mouse anti-human influenza A virus monoclonal antibody 2 to 0.5 mg / mL with the coating diluent; dilute the mouse anti-human influenza B virus monoclonal antibody 2 to 0.8 mg / mL; and dilute the goat anti-chicken IgY antibody to 1 mg / mL. These three solutions will be used to spray the influenza A detection line, the influenza B detection line, and the quality control line, respectively.
[0045] Seven, spraying of the conjugate pad and nitrocellulose membrane
[0046] Mix the colloidal gold re-suspension of the prepared monoclonal antibody 1 for influenza A and B viruses in the above step. Measure the original OD value of the colloidal gold-labeled re-suspension of chicken IgY antibody prepared in the above step, then add it to the above mixture, and use the gold-labeled re-suspension to accurately dilute the OD value of the chicken IgY gold-labeled mixture to 25. Uniformly spray the final mixed gold-labeled solution on the pretreated and dried conjugate pad at a rate of 4 μL / cm, then bake it in a 37°C oven until dry, and seal it for use in an environment with a humidity of ≤30%.
[0047] At the same time, use the membrane spray and draw instrument to accurately spray the three coating solutions prepared in the above step on the corresponding positions of the nitrocellulose membrane at a rate of 1 μL / cm: influenza B virus detection line (coated with influenza B monoclonal antibody 2), influenza A virus detection line (coated with influenza A monoclonal antibody 2), and quality control line (coated with goat anti-chicken IgY antibody). After spraying, dry the nitrocellulose membrane in a 37°C oven.
[0048] Eight, assembly of test strips
[0049] Under an environment with a humidity of ≤30%, stack and adhere the pretreated sample pad, the conjugate pad sprayed with gold-labeled antibody, the nitrocellulose membrane coated with detection line and quality control line, and the water absorption pad on the base plate in order. The overlapping relationship of each component on the base plate is as follows: the end of the sample pad overlaps the beginning of the conjugate pad by 2 mm, the end of the conjugate pad overlaps the beginning of the nitrocellulose membrane by 2 mm, and the end of the nitrocellulose membrane overlaps the beginning of the water absorption pad by 2 mm. Cut the assembled stack into test strips of a specified width.
[0050] Nine, preparation of sample diluent
[0051] Prepare the sample diluent with the following components: 44 g / L of sodium chloride (NaCl), 6 g / L of Tris, 0.4 g / L of anhydrous disodium hydrogen phosphate, 10 g / L of bovine serum albumin (BSA), 0.4 g / L of anhydrous potassium hydrogen phosphate, 1 mL / L of surfactant S22, and 0.2 mL / L of ProClin300. Adjust the pH value to 7.4 using hydrochloric acid or sodium hydroxide solution.
[0052] Example 2: use of test strips
[0053] The use method of the test strips prepared in Example 1 is as follows:
[0054] (1) Respectively dilute the influenza A virus antigen to the concentration of 23000 ng / mL, 230 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.05 ng / mL with sample diluent; dilute the influenza B virus antigen to the concentration of 6000 ng / mL, 60 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.25 ng / mL;
[0055] (2) Respectively take 80 μL of the influenza virus solution with different concentrations prepared in step (1) and add to the test strip prepared in Example 1, due to capillary action, the sample will move along the test strip to the conjugate pad and nitrocellulose membrane; observe the results after 10 minutes (note: color development is invalid after 15 minutes);
[0056] (3) Result observation: the minimum detection limit of the influenza A virus of the test strip is 0.05 ng / mL, and the minimum detection limit of the influenza B virus is 0.25 ng / mL.
[0057] Comparative Example 1
[0058] For comparison, a commercially available conventional influenza A and B virus antigen detection colloidal gold test strip was selected. The test strip uses a conventional concentration (usually 10-40 OD) of colloidal gold labeled antibody.
[0059] Comparative Example 2
[0060] The preparation process of this comparative example refers to Example 1, but the dilution concentration of mouse anti-human influenza A virus monoclonal antibody 2 is reduced to 0.3 mg / mL; the dilution concentration of mouse anti-human influenza B virus monoclonal antibody 2 is reduced to 0.6 mg / mL, and the remaining steps and reagent formulations are completely the same as Example 1. This comparative example is used to verify the effect of the amount of detection line coated antibody on improving the high sensitivity of the test strip.
[0061] Comparative Example 3
[0062] The preparation process of this comparative example refers to Example 1, but the rate of the final mixed gold label solution sprayed on the conjugate pad is reduced to 3 μL / cm, and the remaining steps and reagent formulations are completely the same as Example 1. This comparative example is used to verify the effect of the gold label content on the conjugate pad on improving the high sensitivity of the test strip.
[0063] The test paper strip prepared in Example 1 was compared with the test paper strips prepared in Comparative Examples 1-3: 0.03 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL of influenza A virus antigen standard solution and 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2 ng / mL, 5 ng / mL of influenza B virus antigen standard solution were detected respectively, and the results are shown in Table 1 and Table 2.
[0064] Table 1 Comparison of detection of influenza A virus antigen standard solution
[0065] Influenza A virus antigen concentration 0.03 ng / mL 0.05 ng / mL 0.1 ng / mL 0.5 ng / mL 1 ng / mL Example 1 Negative Positive Positive Positive Positive Comparative Example 1 Negative Negative Negative Positive Positive Comparative Example 2 Negative Negative Negative Positive Positive Comparative Example 3 Negative Negative Positive Positive Positive
[0066] Table 2 Comparison of detection of influenza B virus antigen standard solution
[0067] Influenza B virus antigen concentration 0.1 ng / mL 0.25 ng / mL 0.5 ng / mL 2 ng / mL 5 ng / mL Example 1 Negative Positive Positive Positive Positive Comparative Example 1 Negative Negative Negative Positive Positive Comparative Example 2 Negative Negative Positive Positive Positive Comparative Example 3 Negative Negative Negative Positive Positive
[0068] Obviously, the above examples are merely examples for the sake of clarity, and are not limiting of the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.
Claims
1. The application of high-concentration colloidal gold in the preparation of influenza virus test strips, characterized by: The test strip uses a high-concentration colloidal gold-labeled monoclonal antibody against influenza virus with an OD value of 100.
2. A test strip for detecting influenza virus, characterized in that, The influenza virus includes influenza A virus and influenza B virus, and the method for preparing the test strip includes the following steps: (1) Prepare colloidal gold-labeled reconstitution solution; (2) Influenza A virus monoclonal antibody 1 was labeled with high-concentration colloidal gold and then reconstituted to obtain a colloidal gold-labeled reconstituted solution of influenza A virus monoclonal antibody 1, wherein the OD value of the high-concentration colloidal gold was 100. (3) Influenza B virus monoclonal antibody 1 was labeled with high-concentration colloidal gold and then reconstituted to obtain a colloidal gold-labeled reconstituted solution of influenza B virus monoclonal antibody 1, wherein the OD value of the high-concentration colloidal gold was 100. (4) Chicken IgY antibody was labeled with high concentration colloidal gold and then reconstituted to obtain chicken IgY antibody colloidal gold labeled reconstituted solution; (5) Prepare coating solutions for influenza A virus monoclonal antibody 2, influenza B virus monoclonal antibody 2 and goat anti-chicken IgY antibody respectively; (6) The colloidal gold-labeled reconstitution solution of influenza A virus monoclonal antibody 1, the colloidal gold-labeled reconstitution solution of influenza B virus monoclonal antibody 1 and the colloidal gold-labeled reconstitution solution of chicken IgY antibody are mixed and sprayed onto the conjugate pad. The coating solution of influenza A virus monoclonal antibody 2, the coating solution of influenza B virus monoclonal antibody 2 and the coating solution of goat anti-chicken IgY antibody are respectively sprayed onto the nitrocellulose membrane to form the influenza A virus detection line, the influenza B virus detection line and the quality control line. (7) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are stacked and adhered to the base plate in sequence, and then cut to obtain the test strip.
3. The test strip according to claim 2, characterized in that: It also includes a step of pretreating the sample pad and the conjugate pad.
4. The test strip according to claim 3, characterized in that, The pretreatment involves immersing the sample pad and conjunctival pad in sample pad pretreatment solution and conjunctival pad pretreatment solution, respectively, wherein: The sample pad pretreatment solution contains 45-60 mM Tris-HCl, 0.4-0.6 wt% casein, 1-2 wt% polyvinylpyrrolidone K30, 1-2 wt% surfactant S9, 0.05-0.08 wt% Tween-20, 0.02-0.1 wt% preservative, and 0.002-0.01 wt% inhibitor, with a pH of 8.0-9.
0. The conjugation pad pretreatment solution contains 40-55 mM Tris-HCl, 0.4-0.6 wt% BSA or casein, 0.8-1.2 wt% polyvinylpyrrolidone K30, 0.04-0.08 wt% Tween-20, and 0.02-0.1 wt% preservative, with a pH of 8.0-9.
0.
5. The test strip according to claim 2, characterized in that: In step (1), the colloidal gold-labeled reconstitution solution contains 50 mM Tris-HCl, 0.5 wt% casein, 20 wt% sucrose, 5 wt% trehalose, and 0.02-0.1 wt% preservative, with a pH of 7.5-8.
5.
6. The test strip according to claim 2, characterized in that: In step (5), the concentration of the monoclonal antibody 2 coating solution for influenza A virus was diluted to 0.5 mg / mL, the concentration of the monoclonal antibody 2 coating solution for influenza B virus was diluted to 0.8 mg / mL, and the concentration of the goat anti-chicken IgY antibody coating solution was diluted to 1 mg / mL.
7. The test strip according to claim 2, characterized in that: In step (6), the OD value of the mixed chicken IgY antibody colloidal gold label is 25.
8. The test strip according to claim 2, characterized in that: The test strip also includes a sample diluent.
9. The test strip according to claim 8, characterized in that: The sample diluent comprises 40-45 g / L NaCl, 6-6.3 g / L Tris, 0.4-0.6 g / L anhydrous disodium hydrogen phosphate, 10 g / L BSA, 0.4-0.6 g / L anhydrous dipotassium hydrogen phosphate, 0.8-1.2 mL / L S22, 0.2 mL / L ProClin 300, and has a pH of 7-8.
10. The application of the test strip according to any one of claims 2-9 in influenza virus detection.
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