Influenza virus extraction diluent and application thereof

Abstract

The application discloses an influenza virus extraction diluent and application thereof, and the diluent comprises Na2HPO4*12H2O, NaH2PO4*2H2O, NaCl, Proclin 300, ethylenediaminetetraacetic acid disodium salt dihydrate, polyoxyethylene-8-octylphenyl ether and alkylphenol polyoxyethylene ether. The polyoxyethylene-8-octylphenyl ether and the alkylphenol polyoxyethylene ether added in the diluent improve the virus extraction capacity, enhance the stability of the product, save the cost and time, provide a reference value for other products, prolong the product service life for customers, reduce the limitation, are more suitable for the temperature, and have greater market value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and particularly relates to an influenza virus extraction diluent and application thereof. Background Art

[0002] Influenza virus is the pathogen of influenza. There are three main detection methods, namely viral nucleic acid detection, virus isolation, and antigen detection.

[0003] 1. Viral nucleic acid testing: Collect throat swabs, nasal swabs, sputum, etc., and use polymerase chain reaction to detect whether there is influenza virus RNA. If the nucleic acid test is positive, influenza can be diagnosed and the type of pathogen can be determined;

[0004] Second, virus isolation. Virus isolation is the gold standard for diagnosing influenza. If influenza virus is isolated from nasopharyngeal secretions, influenza can be diagnosed.

[0005] 3. Antigen test: Antigen test is a commonly used method for rapid influenza virus detection. Its accuracy is lower than nucleic acid test. If the antigen is positive, influenza can be diagnosed.

[0006] Antigen detection often uses colloidal gold-labeled immunochromatography, a new detection technology that has emerged and developed rapidly in recent years. It is quick and simple to operate during sample extraction and testing, takes a short time, and provides relatively accurate results. It is low-cost, pollution-free, and requires no training. It is less affected by human factors and is more suitable for on-site testing than traditional methods, and is suitable for large-scale testing.

[0007] During sample extraction, a sample diluent is often added. Common sample diluent formulas include: PBS (phosphate-buffered saline) is a commonly used sample diluent. It is typically mixed with potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride, and the pH adjusted to 7.2-7.4. BSA (bovine serum albumin) may be added to enhance stability. Tris buffer, whose primary component is tris(hydroxymethyl)aminomethane, is typically reacted with hydrochloric acid to adjust the pH and maintain a stable pH. A physiological saline solution composed of an appropriate concentration of sodium chloride (NaCl) is also used. Other components, such as glucose and urea, are used to improve the recovery rate of high-concentration samples, address the problem of inaccurate quantification of high-concentration samples, and reduce R&D costs. However, conventional diluents are ineffective for extracting viruses from nasopharyngeal swabs. In some testing scenarios, commonly used sample diluents cannot meet testing requirements, resulting in low sample concentrations and weak colloidal gold detection results, affecting test accuracy. Furthermore, most current in vitro diagnostic reagents lack stability and have a short shelf life. Summary of the Invention

[0008] In order to solve one of the above problems, the application provides an influenza virus extraction diluent and application thereof.

[0009] In order to achieve the above-mentioned purposes, the application adopts the following technical means:

[0010] The first aspect of the application provides an influenza virus extraction diluent, which comprises Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, Proclin300, ethylenediaminetetraacetic acid disodium salt dihydrate, polyoxyethylene-8-octylphenyl ether and alkylphenol polyoxyethylene ether.

[0011] In some embodiments of the application, in the diluent, Na2HPO4·12H2O is 2.5-3g / L; NaH2PO4·2H2O is 0.2-0.3g / L; NaCl is 8.5-9.5g / L; Proclin300 is 200μL / L; ethylenediaminetetraacetic acid disodium salt dihydrate is 1-1.2g / L; polyoxyethylene-8-octylphenyl ether is 2.5-3.5%; and alkylphenol polyoxyethylene ether is 0.5-1.5%.

[0012] In some embodiments of the application, preferably, in the diluent, polyoxyethylene-8-octylphenyl ether is 3% and alkylphenol polyoxyethylene ether is 1%.

[0013] Further preferably, in the diluent, the substances and concentrations are as follows: Na2HPO4·12H2O is 2.8g / L; NaH2PO4·2H2O is 0.24g / L; NaCl is 9.1g / L; Proclin300 is 200μL / L; ethylenediaminetetraacetic acid disodium salt dihydrate is 1.114g / L; polyoxyethylene-8-octylphenyl ether is 3%; and alkylphenol polyoxyethylene ether is 1%.

[0014] The second aspect of the application provides application of the influenza virus extraction diluent of the first aspect in preparation of products for influenza virus extraction and detection.

[0015] In some embodiments of the application, the influenza virus comprises influenza A virus and influenza B virus.

[0016] In some embodiments of the application, the products for influenza virus detection include but are not limited to extraction reagents and kits.

[0017] The third aspect of the application provides a kit for influenza virus detection, which comprises the influenza virus extraction diluent of the first aspect.

[0018] The fourth aspect of the application provides a method for detecting influenza virus by using a colloidal gold detection reagent strip, which comprises the following steps:

[0019] (1) using the influenza virus extraction diluent of the first aspect to extract the biological sample sufficiently;

[0020] (2) vertically dropping the extracted liquid in step (1) on the sample pad of the colloidal gold detection test strip, and determining the detection result after placing at room temperature for 5-10 min.

[0021] In some embodiments of the present application, the biological sample is a nasopharyngeal secretion sample, an oropharyngeal secretion sample, or other body fluid sample.

[0022] Advantages of the present application

[0023] Compared with the prior art, the present application has the following advantages: the present application provides an influenza virus extraction diluent and its application, and the addition of a substance, polyoxyethylene-8-octylphenyl ether, alkylphenol polyoxyethylene ether, in the diluent improves the extraction capacity and enhances the stability of product performance: the accelerated stability test is done at 45℃ for 35 days, and the results are all good, and the performance has no obvious difference with that at 0 day. The cost and time are saved, and reference value is provided for other products. For customers, the product use period is extended, the limitations are reduced, the temperature suitability is higher, the market value is greater. The operation is simple, and it is suitable as a conventional detection means.

[0024] The diluent is applied in colloidal gold labeled immunoassay for detecting influenza virus, and has no pollution, does not need training, has short color development time, is convenient and fast, can detect whether the biological sample contains influenza antigen in a short time, does not need expensive instrument equipment, is suitable for on-site detection, has broad market prospect and application value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The detection result machine reading and colorimetric standard chart of the present application are shown;

[0026] Figure 2 The T1 value result comparison of the old formula under 45℃ acceleration condition for 0-35 days is shown;

[0027] Figure 3 The T1 value result comparison of the new formula under 45℃ acceleration condition for 0-35 days is shown;

[0028] Figure 4 The T2 value result comparison of the old formula under 45℃ acceleration condition for 0-35 days is shown;

[0029] Figure 5 The T2 value result comparison of the new formula under 45℃ acceleration condition for 0-35 days is shown. DETAILED DESCRIPTION

[0030] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0032] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0033] Preparation:

[0034] 1. Prepare diluent:

[0035] (1) The old formula before improvement: Na2HPO4·12H2O 3 g / L; NaH2PO4·2H2O 0.528 g / L; NaCl 12.8 g / L; Proclin 300 200 μL / L; ethylenediaminetetraacetic acid disodium salt dihydrate 1.234 g / L.

[0036] (2) Improved new formula: Na2HPO4·12H2O 2.8 g / L; NaH2PO4·2H2O 0.24 g / L; NaCl 9.1 g / L; Proclin 300 200 μL / L; ethylenediaminetetraacetic acid disodium salt dihydrate 1.114 g / L; polyoxyethylene-8-octylphenyl ether 3%; alkylphenol polyoxyethylene ether 1%.

[0037] (3) Calculate the dosage of each component based on the old formula before improvement and the new formula after improvement according to the preparation volume of 50 mL of dilution solution, pour the above substances into purified water in turn, stir and dissolve, and then adjust the volume;

[0038] Place the fully dissolved diluted solution into a clean centrifuge tube or container and seal it.

[0039] (4) Dispense the prepared diluent into sampling tubes, 500 μL per tube, and wait for use.

[0040] 2. Preparation of Colloidal Gold Test Strips

[0041] 1. Preparation of gold label pad:

[0042] Place 100 mL of colloidal gold solution in a beaker, stir, and adjust the pH of the gold solution to 9.5 by adding 0.2 M K₂CO₃. After stirring, add 1 mg of influenza A antibody and stir at room temperature for 15 minutes. Add 1 mL of 10% BSA solution to block nonspecific adsorption sites. Stir at room temperature for 15 minutes, then centrifuge at 10,000 rpm for 20 minutes. Discard the supernatant and dilute the precipitate to 1 mL with gold conjugate diluent (Tris 2.5 g / L, casein 2.5 g / L, 0.03% Proclin 300). Label influenza B antibody and mouse IgG for the C-line system in the same manner.

[0043] After mixing the labeled influenza A antibodies and influenza B antibodies, they were evenly sprayed on the glass fiber using a gold sprayer. After drying overnight, the gold label pad was prepared.

[0044] 2. Preparation of nitrocellulose membrane

[0045] Dilute another influenza A antibody with spotting diluent (2.9 g / L Na2HPO4.12H2O, 0.282 g / L NaH2PO4.2H2O, 8.775 g / L Nacl, 200 μL / L Proclin300) and spot it on the nitrocellulose membrane using a spotter to form the T1 line. Use the same method to spot another influenza B antibody as the T2 line and goat anti-mouse IgG as the C line.

[0046] 3. Assemble the prepared gold label pad, nitrocellulose membrane, absorbent filter paper, sample pad, card shell, etc. into a colloidal gold reagent strip for influenza virus detection.

[0047] 3. The criteria for colloidal gold detection are as follows:

[0048] ① Negative: Only one band appears on the quality control line, and no band appears in the test area, which is negative;

[0049] ②T1 positive: two colored reaction lines appear, one in the C area (quality control area) and the other in the T1 area;

[0050] T2 positive: two colored reaction lines appear, one in the C area (quality control area) and the other in the T2 area;

[0051] T1 / T2 positive: Three colored reaction lines appear, one in area C (quality control area), one in area T1, and one in area T2.

[0052] ③ Invalid: No strip appears on the quality control line, indicating that the test strip is damaged and a new test strip should be replaced and retested.

[0053] The values are judged by colloidal gold immunoassay instrument, the color card line intensity shows G1-G10, and the machine reading value range is related to the color card line intensity as shown in Figure 1 T1 represents the intensity of the influenza A detection result, T2 represents the intensity of the influenza B detection result, and the larger the value represents the stronger the color development and positive value.

[0054] The judgment method of color card reading and machine reading is shown in Table 1.

[0055] Table 1 Judgment method of color card reading and machine reading

[0056]

[0057] Example 1 Accelerated experiment of diluent at 45°C

[0058] I. Colloidal gold method detection of diluent 0 days

[0059] 1. Sample detection liquid preparation: Put swabs with different virus concentration strengths into sampling tubes containing just prepared and packaged diluents, extract and stir thoroughly, then take out the swabs, and the liquid part in the sampling tube is the sample detection liquid.

[0060] 2. Colloidal gold method detection of diluent effect: Add 3-5 drops of sample detection liquid to the sample pad of the prepared colloidal gold detection reagent strip vertically, and judge the detection result after 10 min of room temperature standing as shown in Table 2.

[0061] Table 2 Colloidal gold method detection result of diluent 0 days

[0062]

[0063] II. Colloidal gold method detection of diluent 7 days

[0064] 1. Sample detection liquid preparation: Put swabs with different virus concentration strengths into sampling tubes containing prepared and packaged diluents, and store them in a 45°C environment for 7 days, extract and stir thoroughly, then take out the swabs, and the liquid part in the sampling tube is the sample detection liquid.

[0065] 2. Colloidal gold method detection of diluent effect: Add 3-5 drops of sample detection liquid to the sample pad of the prepared colloidal gold detection reagent strip vertically, and judge the detection result after 10 min of room temperature standing as shown in Table 3.

[0066] Table 3 Colloidal gold method detection result of diluent 7 days

[0067]

[0068] III. Colloidal gold method detection of diluent 14 days

[0069] 1. Sample detection liquid preparation: Put swabs with different virus concentration strengths into the sampling tubes containing diluted liquid prepared and packaged, and placed in a 45℃ environment for 14 days. After fully extracting and stirring, take out the swabs, and the liquid part in the sampling tube is the sample detection liquid.

[0070] 2. Colloidal gold method for detecting diluted liquid effect: Add 3-5 drops of sample detection liquid to the sample pad of the prepared colloidal gold detection reagent strip vertically, and determine the detection results after placing at room temperature for 10 minutes as shown in Table 4.

[0071] Table 4 Diluted liquid 14-day colloidal gold method detection results

[0072]

[0073] Four, 21-day colloidal gold method detection of diluted liquid

[0074] 1. Sample detection liquid preparation: Put swabs with different virus concentration strengths into the sampling tubes containing diluted liquid prepared and packaged, and placed in a 45℃ environment for 21 days. After fully extracting and stirring, take out the swabs, and the liquid part in the sampling tube is the sample detection liquid.

[0075] 2. Colloidal gold method for detecting diluted liquid effect: Add 3-5 drops of sample detection liquid to the sample pad of the prepared colloidal gold detection reagent strip vertically, and determine the detection results after placing at room temperature for 10 minutes as shown in Table 5.

[0076] Table 5 Diluted liquid 21-day colloidal gold method detection results

[0077]

[0078] Five, 28-day colloidal gold method detection of diluted liquid

[0079] 1. Sample detection liquid preparation: Put swabs with different virus concentration strengths into the sampling tubes containing diluted liquid prepared and packaged, and placed in a 45℃ environment for 28 days. After fully extracting and stirring, take out the swabs, and the liquid part in the sampling tube is the sample detection liquid.

[0080] 2. Colloidal gold method for detecting diluted liquid effect: Add 3-5 drops of sample detection liquid to the sample pad of the prepared colloidal gold detection reagent strip vertically, and determine the detection results after placing at room temperature for 10 minutes as shown in Table 6.

[0081] Table 6 Diluted liquid 28-day colloidal gold method detection results

[0082]

[0083] Six, 35-day colloidal gold method detection of diluted liquid

[0084] 1. Sample detection liquid preparation: Put swabs with different virus concentration strengths into dilution liquid sampling tubes that have been configured and subpackaged and stored in a 45°C environment for 35 days, extract and mix thoroughly, then take out the swabs, and the liquid part in the sampling tube is the sample detection liquid.

[0085] 2. Colloidal gold method detection of dilution liquid effect: Add 3-5 drops of sample detection liquid to the sample pad of the prepared colloidal gold detection reagent strip vertically, and determine the detection result after 10 minutes of room temperature placement as shown in Table 7.

[0086] Table 7 Dilution liquid 35-day colloidal gold method detection results

[0087]

[0088] The average number comparison of the data of the above-mentioned Examples 1 to 6 is as follows: the T1 average value data of the old and new formulations are shown in Tables 8, 9 and Figure 2 and Figure 3 , and the T2 average value data of the old and new formulations are shown in Tables 10, 11 and Figure 4 and Figure 5 .

[0089] Table 8 T1 average value data of the old formulation

[0090]

[0091] Table 9 T1 average value data of the new formulation

[0092]

[0093] Table 10 T2 average value data of the old formulation

[0094]

[0095] Table 11 T2 average value data of the new formulation

[0096]

[0097] At the same time, we also tested the detection rate using enterprise quality control products, and the performance comparison results of the old and new formulations are shown in Table 12, and the detection rate is the result obtained by testing 100 enterprise quality control products.

[0098] Table 12 Performance comparison of the old and new formulations

[0099]

[0100] The total results show that the old formula has a rapid decline in the average of T1 and T2 in the accelerated experiment at 45 DEG C for 7 to 14 days, showing instability. The new formula has no significant difference between the average of T1 and T2 in the accelerated experiment at 45 DEG C for 0 to 35 days and the result of 0 day, showing stability of product performance, which prolongs the service life.

[0101] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present application upon reading and understanding the above lecture of the present application, and it is intended that all such alterations and modifications fall within the scope of the present application.

Claims

1. An influenza virus extraction diluent, characterized in that: The diluents include Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, Proclin300, ethylenediaminetetraacetic acid disodium salt dihydrate, polyoxyethylene-8-octylphenyl ether, and alkylphenol polyoxyethylene ether; The diluent comprises 2.5-3 g / L of Na2HPO4·12H2O, 0.2-0.3 g / L of NaH2PO4·2H2O, 8.5-9.5 g / L of NaCl, 200 μL / L of Proclin300, 1-1.2 g / L of disodium ethylenediaminetetraacetic acid dihydrate, 2.5-3.5% of polyoxyethylene-8-octylphenyl ether, and 0.5-1.5% of alkylphenol polyoxyethylene ether.

2. An influenza virus extraction diluent according to claim 1, characterized in that: In the diluent, polyoxyethylene-8-octylphenyl ether is 3%; and alkylphenol polyoxyethylene ether is 1%.

3. Use of the influenza virus extraction diluent according to any one of claims 1-2 in the preparation of products for influenza virus extraction and detection.

4. The use according to claim 3, characterized in that: The influenza virus includes influenza A virus and influenza B virus.

5. The use according to claim 3, characterized in that: Products related to influenza virus detection include extraction reagents, test kits, and colloidal gold detection reagent strips.

6. A kit for extracting and detecting influenza viruses, characterized in that: The invention comprises the influenza virus extract diluent according to any one of claims 1 to 2.

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