CN119086896B - Influenza virus extraction diluent and application thereof - Google Patents
Influenza virus extraction diluent and application thereofInfo
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- CN119086896B CN119086896B CN202411568174.8A CN202411568174A CN119086896B CN 119086896 B CN119086896 B CN 119086896B CN 202411568174 A CN202411568174 A CN 202411568174A CN 119086896 B CN119086896 B CN 119086896B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G01N33/587—Nanoparticles
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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Abstract
本发明公开了一种流感病毒提取稀释液及其应用，稀释液包括Na2HPO4·12H2O，NaH2PO4·2H2O，NaCl，Proclin300，乙二胺四乙酸二钠盐二水合物，聚氧乙烯‑8‑辛基苯基醚，烷基酚聚氧乙烯醚。稀释液中加入物质聚氧乙烯‑8‑辛基苯基醚、烷基酚聚氧乙烯醚，既提高了病毒提取能力又增强了产品的稳定性，节约成本和时间，也给其他产品提供了参考价值，对于客户来说产品使用期限延长，局限性减少，温度适宜性更高，市场价值更大。
The present invention discloses an influenza virus extraction diluent and its application. The diluent includes Na₂HPO₄ · 12H₂O , NaH₂PO₄ · 2H₂O , NaCl, Proclin 300, disodium ethylenediaminetetraacetic acid dihydrate, polyoxyethylene-8-octylphenyl ether, and alkylphenol polyoxyethylene ether. Adding polyoxyethylene-8-octylphenyl ether and alkylphenol polyoxyethylene ether to the diluent improves virus extraction capability and enhances product stability, saving cost and time. The diluent also provides a reference value for other products. For customers, the product has an extended shelf life, reduced limitations, higher temperature adaptability, and greater market value.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to an influenza virus extraction diluent and application thereof.
Background
Influenza viruses are pathogens of influenza, and three detection methods are mainly used, namely virus nucleic acid detection, virus separation and antigen detection.
1. Viral nucleic acid detection, collecting throat swab, nose swab, sputum and the like, and detecting whether RNA of influenza virus exists or not through polymerase chain reaction, wherein if the nucleic acid detection is positive, the influenza can be diagnosed and the type of pathogen can be defined;
2. virus isolation, which is a gold standard for diagnosing influenza, such as isolating influenza virus from nasopharyngeal secretions, i.e. influenza can be diagnosed;
3. antigen detection is a common influenza virus rapid detection method, the accuracy is lower than that of nucleic acid detection, and if the antigen is positive, influenza can be diagnosed.
The antigen detection usually adopts a colloidal gold-labeled immunochromatography, which is a novel detection technology that is rising and rapidly developed in recent years, and has the advantages of rapid and simple operation, short time, accurate result, low cost, no pollution, no need of training, less interference from human factors, suitability for field detection compared with the traditional method, and suitability for large-scale detection.
For sample extraction, a sample extraction diluent is usually added, and a common sample diluent is generally formulated by adding PBS (phosphate buffered saline) , which is a common sample diluent, mixing it with potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride, adjusting the pH to 7.2-7.4, and possibly adding BSA (bovine serum albumin) to increase stability. Tris buffer , the main component of which is Tris, is usually reacted with hydrochloric acid to adjust the pH value for maintaining the pH of the solution stable. Physiological saline composed of sodium chloride (NaCl) with proper concentration. Other components also comprise glucose, urea and the like, are used for improving the recovery rate of the high-concentration sample, solving the problem that the high-concentration sample cannot be accurately fixed, and reducing the research and development cost. However, the conventional diluent has an insignificant effect on the virus extraction capacity of nasopharyngeal swabs, and in certain detection occasions, the conventional sample diluent cannot meet the detection requirements, the concentration of the processed sample is low, the colloidal gold detection result is not strong, the accuracy of the detection result is affected, and most of the existing in-vitro diagnostic reagent products have poor stability and short shelf life.
Disclosure of Invention
In order to solve one of the problems, the invention provides an influenza virus extraction diluent and application thereof.
In order to achieve the above purpose, the invention adopts the following technical means:
In a first aspect, the invention provides an influenza virus extraction diluent, which comprises Na 2HPO4·12H2O,NaH2PO4·2H2 O, naCl, proclin300, ethylenediamine tetraacetic acid disodium salt dihydrate, polyoxyethylene-8-octylphenyl ether and alkylphenol polyoxyethylene.
In some embodiments of the invention, the diluent comprises ,Na2HPO4·12H2O 2.5-3g/L;NaH2PO4·2H2O 0.2-0.3g/L;NaCl 8.5-9.5g/L;Proclin300 200μL/L; ethylene diamine tetraacetic acid disodium salt dihydrate 1-1.2g/L, polyoxyethylene-8-octyl phenyl ether 2.5-3.5% and alkylphenol polyoxyethylene 0.5-1.5%.
In some embodiments of the present invention, preferably, the diluent comprises 3% polyoxyethylene-8-octylphenyl ether and 1% alkylphenol polyoxyethylene.
Further preferably, the concentration of the substances in the diluent is ：Na2HPO4·12H2O 2.8g/L;NaH2PO4·2H2O 0.24g/L;Nacl 9.1g/L;Proclin300 200μL/L; ethylene diamine tetraacetic acid disodium salt dihydrate 1.114g/L, polyoxyethylene-8-octyl phenyl ether 3% and alkylphenol polyoxyethylene 1%.
In a second aspect, the invention provides the use of an influenza virus extraction diluent according to the first aspect for the preparation of a product for influenza virus extraction and detection.
In some embodiments of the invention, the influenza virus comprises influenza a virus, influenza b virus.
In some embodiments of the invention, related products for influenza virus detection include, but are not limited to, extraction reagents, kits.
In a third aspect the invention provides a kit for influenza virus detection comprising an influenza virus extraction diluent according to the first aspect.
In a fourth aspect, the present invention provides a method for detecting influenza virus using a colloidal gold detection reagent strip, comprising the steps of,
(1) Fully extracting the biological sample by using the influenza virus extraction diluent in the first aspect;
(2) And (3) vertically dripping the extracted liquid in the step (1) at a sample pad of the colloidal gold detection test strip, standing at room temperature for 5-10min, and judging a detection result.
In some embodiments of the invention, the biological sample is a nasopharyngeal secretion sample, an oropharyngeal secretion sample, or other body fluid sample.
The beneficial effects of the invention are that
Compared with the prior art, the invention has the beneficial effects that the invention provides the influenza virus extraction diluent and the application thereof, and the addition of the polyoxyethylene-8-octyl phenyl ether and the alkylphenol ethoxylate into the diluent not only improves the extraction capacity, but also enhances the stability of the product performance, namely, the stability is accelerated for 35 days at the temperature of 45 ℃, the result is good, and the performance is not obviously different from that of 0 day. Saving cost and time, providing reference value for other products, prolonging the service life of the products for customers, reducing limitation, having higher temperature suitability and having higher market value. The operation is simple and convenient, the operation is convenient, is suitable for being used as a conventional detection means.
The diluent is applied to the colloidal gold labeled immunoassay method for detecting influenza virus, has no pollution, does not need training, has short color development time, is convenient and quick, can detect whether the biological sample contains influenza antigen in a short time, does not need expensive instrument and equipment, is suitable for on-site detection, and has wide market prospect and application value.
Drawings
FIG. 1 shows a machine-readable and colorimetric standard chart of the detection result of the invention;
FIG. 2 shows a comparison of T1 values obtained from 0-35 days of testing under 45℃acceleration for the old formulation;
FIG. 3 shows a comparison of T1 values obtained from a 0-35 day test under 45℃acceleration conditions for the new formulation;
FIG. 4 shows a comparison of T2 values from 0-35 days of testing under 45℃acceleration for the old formulation;
figure 5 shows a comparison of the results of the T2 values from the 0-35 day test of the new formulation at 45 ℃ acceleration.
Detailed Description
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
The technical scheme of the patent is further described in detail below with reference to the specific embodiments.
Preparation:
1. preparing a diluent:
(1) The old formulation ：Na2HPO4·12H2O 3g/L;NaH2PO4·2H2O 0.528g/L;Nacl 12.8g/L;Proclin300 200μL/L; ethylenediamine tetraacetic acid disodium salt dihydrate 1.234g/L before modification.
(2) The improved new formulation ：Na2HPO4·12H2O 2.8g/L;NaH2PO4·2H2O 0.24g/L;Nacl 9.1g/L;Proclin300 200μL/L; of ethylenediamine tetraacetic acid disodium salt dihydrate 1.114g/L, polyoxyethylene-8-octylphenyl ether 3% and alkylphenol polyoxyethylene 1%.
(3) Calculating the dosage of each component according to the old formula before improvement and the new formula after improvement and the diluent with the volume of 50mL, and sequentially pouring the materials into purified water for stirring and dissolving and fixing the volume;
and (5) filling the fully dissolved and prepared diluent into a clean centrifuge tube or container, and sealing.
(4) And (5) subpackaging the prepared diluent into sampling tubes, wherein each sampling tube is filled with 500 mu L and waiting for use.
2. Preparation of colloidal gold test strip
1. Preparing a gold mark pad:
100mL of colloidal gold solution is taken and put into a beaker, 0.2M K 2CO3 is added to adjust the pH of gold water to 9.5 by stirring, 1 mg influenza A antibody is added after stirring, 15min is stirred at room temperature, 1mL of 10% BSA solution is added to block non-specific adsorption sites, 10000rpm is used for centrifugation for 20min after 15min stirring at room temperature, the supernatant is discarded, and gold standard diluent (Tris 2.5g/L, casein 2.5g/L and 0.03% Proclin 300) for precipitation is taken to be fixed to 1mL for standby. The same procedure was used to label murine IgG for influenza B antibodies and C-line systems.
And mixing the marked influenza A antibody and the marked influenza B antibody, uniformly spraying the mixture on glass fibers by a metal spraying instrument, and drying overnight to finish the preparation of the gold-labeled pad.
2. Preparation of nitrocellulose membranes
Diluting another influenza A antibody by using a spot membrane diluent （2.9g/L Na2HPO4.12H2O,0.282g/L NaH2PO4.2H2O,8.775g/L Nacl,200μL/L Proclin300）, and spot-plating the other influenza B antibody on a nitrocellulose membrane by using a spot membrane instrument to obtain a T1 line, wherein the spot membrane of the other influenza B antibody is a T2 line, and the spot membrane of the goat anti-mouse IgG is a C line.
3. And assembling the prepared gold-labeled pad, nitrocellulose membrane, water-absorbing filter paper, sample pad, cartridge and the like into the influenza virus detection colloidal gold reagent strip.
3. The colloidal gold detection judgment criteria are as follows:
① The negative is that only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;
② T1 positive, wherein two color reaction lines appear, one in the C area (quality control area) and one in the T1 area;
T2 positive, wherein two color reaction lines appear, one in the C area (quality control area) and one in the T2 area;
T1/T2 positive, three color reaction lines appear, one in the C region (quality control region), one in the T1 region and one in the T2 region.
③ And the test strip is invalid, namely a strip does not appear on the quality control line, so that the test strip is damaged, and a new test strip is replaced for retesting.
The colloidal gold immunoassay analyzer is adopted to judge the numerical value, the color card line intensity displays G1-G10, the relation between the machine reading value range and the color card line intensity is 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 numerical value is, the stronger the color development and the positive value are.
The color chart judgment and the machine-readable judgment methods are shown in the following table 1.
Table 1 color card judging and machine-readable judging method
EXAMPLE 1 accelerated experiments at 45℃with diluent
1. Colloidal gold method for detecting diluent for 0 day
1. And (3) preparing a sample detection liquid, namely respectively placing the swabs with different virus concentrations into sampling tubes with just prepared and packaged diluents, fully extracting, uniformly stirring, and taking out the swabs, wherein the liquid part in the sampling tubes is the sample detection liquid.
2. The effect of the colloidal gold method for detecting the diluent is that 3-5 drops of sample detection liquid (each concentration is repeatedly detected for 3 times) are respectively and vertically dripped to the sample pad of the prepared colloidal gold detection reagent strip, and the detection result is judged as shown in the table 2 after the sample detection reagent strip is placed at room temperature for 10 min.
TABLE 20 day colloidal gold assay of dilutions
2. Colloidal gold method for detecting diluent for 7 days
1. And (3) preparing a sample detection liquid, namely respectively placing the swabs with different virus concentrations into a sampling tube which is prepared and packaged, and storing the swab in a 45 ℃ environment for 7 days, fully extracting, uniformly stirring, and taking out the swab, wherein the liquid part in the sampling tube is the sample detection liquid.
2. The effect of the colloidal gold method for detecting the diluent is that 3-5 drops of sample detection liquid are respectively dripped to sample pads of the prepared colloidal gold detection reagent strips vertically, and the detection results are shown in the table 3 after the sample detection liquid is placed for 10min at room temperature.
TABLE 3 colloidal gold assay results for 7 days on diluent
3. Colloidal gold method for detecting diluted solution for 14 days
1. And (3) preparing a sample detection liquid, namely respectively placing the swabs with different virus concentrations into a sampling tube which is prepared and packaged, and storing the swab in a 45 ℃ environment for 14 days, fully extracting, uniformly stirring, and taking out the swab, wherein the liquid part in the sampling tube is the sample detection liquid.
2. The effect of the colloidal gold method for detecting the diluent is that 3-5 drops of sample detection liquid are respectively dripped to sample pads of the prepared colloidal gold detection reagent strips vertically, and the detection results are shown in the table 4 after the sample pads are placed for 10min at room temperature.
TABLE 4 14 day colloidal gold assay results for dilutions
4. Colloidal gold method for detecting diluent for 21 days
1. And (3) preparing a sample detection liquid, namely respectively placing the swabs with different virus concentrations into a sampling tube which is prepared and packaged, and storing the swab in a 45 ℃ environment for 21 days, fully extracting, uniformly stirring, and taking out the swab, wherein the liquid part in the sampling tube is the sample detection liquid.
2. The effect of the colloidal gold method for detecting the diluent is that 3-5 drops of sample detection liquid are respectively dripped to sample pads of the prepared colloidal gold detection reagent strips vertically, and the detection results are shown in table 5 after the sample detection liquid is placed for 10min at room temperature.
TABLE 5 colloidal gold assay results for 21 days on diluent
5. Colloidal gold method for detecting diluent for 28 days
1. And (3) preparing a sample detection liquid, namely respectively placing the swabs with different virus concentrations into a sampling tube which is prepared and packaged, and storing the swab in a 45 ℃ environment for 28 days, fully extracting, uniformly stirring, and taking out the swab, wherein the liquid part in the sampling tube is the sample detection liquid.
2. The effect of the colloidal gold method for detecting the diluent is that 3-5 drops of sample detection liquid are respectively dripped to sample pads of the prepared colloidal gold detection reagent strips vertically, and the detection results are shown in table 6 after the sample detection liquid is placed for 10min at room temperature.
TABLE 6 28 day colloidal gold assay results for dilutions
6. 35-Day colloidal gold method for detecting diluent
1. And (3) preparing a sample detection liquid, namely respectively placing the swabs with different virus concentrations into a sampling tube which is prepared and packaged, and storing the swab in a 45 ℃ environment for 35 days, fully extracting, uniformly stirring, and taking out the swab, wherein the liquid part in the sampling tube is the sample detection liquid.
2. The effect of the colloidal gold method for detecting the diluent is that 3-5 drops of sample detection liquid are respectively dripped to sample pads of the prepared colloidal gold detection reagent strips vertically, and the detection results are shown in the table 7 after the sample detection liquid is placed for 10min at room temperature.
TABLE 7 35 day colloidal gold assay of the dilutions
The average numbers of the data of examples 1 to 6 are compared with each other as shown in tables 8, 9 and 2 and 3, and with the average numbers of the old and new formulations as shown in tables 10, 11 and 4 and 5.
TABLE 8T 1 mean data for old formulations
TABLE 9 mean data for novel formulation T1
TABLE 10T 2 mean data for old formulations
TABLE 11T 2 mean data for novel formulations
Meanwhile, the detection rate of the enterprise quality control product is also tested, and the comparison result of the performance of the old formula and the new formula is shown in the following table 12, wherein the detection rate is obtained by testing 100 parts of the enterprise quality control product.
Table 12 comparison of the Performance of the old formulation with the New formulation
The overall results show that the old formulation showed a rapid decrease in both T1 and T2 mean values measured at 7 to 14 days, indicating instability, by accelerated experiments at 45 ℃. The new formula accelerates the experiment at 45 ℃, the T1 average value and the T2 average value of 0-35 days have no obvious difference from the result of 0 day, and the stability of the product performance is changed, and the stability brings about the extension of the service life.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the above teachings of the application, and such equivalents are intended to fall within the scope of the application as defined by the claims.
Claims (6)
1. An influenza virus extraction diluent is characterized in that the diluent comprises Na 2HPO4·12H2O,NaH2PO4·2H2 O, naCl, proclin300, ethylenediamine tetraacetic acid disodium salt dihydrate, polyoxyethylene-8-octylphenyl ether and alkylphenol polyoxyethylene;
the dilution liquid comprises 1-1.2g/L of ,Na2HPO4·12H2O 2.5-3g/L;NaH2PO4·2H2O 0.2-0.3g/L;NaCl 8.5-9.5g/L;Proclin300 200μL/L; ethylene diamine tetraacetic acid disodium salt dihydrate, 2.5-3.5% of polyoxyethylene-8-octyl phenyl ether and 0.5-1.5% of alkylphenol polyoxyethylene.
2. The influenza virus extraction diluent according to claim 1, wherein said diluent comprises 3% of polyoxyethylene-8-octylphenyl ether and 1% of alkylphenol ethoxylates.
3. Use of an influenza virus extraction dilution according to any one of claims 1-2 in the preparation of a product for influenza virus extraction and detection.
4. The method of claim 3, wherein the influenza virus comprises influenza A virus or influenza B virus.
5. The method according to claim 3, wherein the product related to influenza virus detection comprises an extraction reagent, a kit and a colloidal gold detection reagent strip.
6. A kit for influenza virus extraction and detection, characterized by comprising the influenza virus extraction diluent according to any one of claims 1-2.
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| CN104251908A (en) * | 2013-06-28 | 2014-12-31 | 广州万孚生物技术股份有限公司 | Sample pad processing liquid, H7 subtype avian influenza virus colloidal gold test strip and preparation method thereof |
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| US20040265800A1 (en) * | 2003-06-30 | 2004-12-30 | Sysmex Corporation | Sample pretreatment solution for immunological test and method for using the same |
| CN107561297B (en) * | 2017-08-25 | 2019-12-10 | 宁波美康盛德医学检验所有限公司 | Reagent for blood fat typing detection |
| CN109142725A (en) * | 2018-08-29 | 2019-01-04 | 陕西省人民医院 | A kind of A type is general and the preparation method of the colloidal gold combined Test paper of H7N9 subtype influenza virus |
| CN111579792B (en) * | 2020-04-30 | 2020-11-27 | 杨小军 | Influenza virus and 2019 novel coronavirus antibody combined detection device and preparation method thereof |
| JP7714330B2 (en) * | 2020-09-08 | 2025-07-29 | デンカ株式会社 | Test kit with improved specificity by suppressing false positives |
| CN117233381A (en) * | 2023-08-25 | 2023-12-15 | 可孚医疗科技股份有限公司 | Sample pad treatment fluid for detecting respiratory viruses and application thereof |
| CN118501433A (en) * | 2024-06-03 | 2024-08-16 | 广州华澳生物科技有限公司 | A color latex chromatography detection method and detection kit for respiratory syncytial virus and adenovirus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2005024323A (en) * | 2003-06-30 | 2005-01-27 | Sysmex Corp | Specimen pretreatment liquid for inspecting immunity, and treatment method |
| CN104251908A (en) * | 2013-06-28 | 2014-12-31 | 广州万孚生物技术股份有限公司 | Sample pad processing liquid, H7 subtype avian influenza virus colloidal gold test strip and preparation method thereof |
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