Sample processing fluid for respiratory virus antigen detection and method for preparing the same

Abstract

The application relates to a sample processing liquid for respiratory virus antigen detection and a preparation method thereof, which comprises a phosphate buffer, 0.05-0.5 g / L sodium caseinate, 1-20 g / L bovine serum albumin, 10-50 mL / L surfactant, 1-10 g / L guanidine hydrochloride, 3.72-37.2 g / L ethylenediaminetetraacetic acid disodium salt dihydrate solution, 1-5 mL / L CL preservative, 1-5 mL / L BND bacteriostatic agent and 900-988 mL / L sterile deionized water; and the application has the advantages of good sample stability.

Description

Technical Field

[0001] This invention belongs to the field of antigen detection technology, specifically relating to a sample processing solution for respiratory virus antigen detection and its preparation method. Background Technology

[0002] Respiratory multiplex viral antigen detection is a key technology for the rapid diagnosis of infections caused by common pathogens such as influenza virus, respiratory syncytial virus (RSV), adenovirus, and SARS-CoV-2. Its core lies in using methods such as immunochromatography or fluorescence immunoassay to simultaneously detect specific antigens of multiple viruses in clinical samples (such as nasopharyngeal swabs and sputum). As the first step in the detection process, the sample processing solution plays a crucial pretreatment role: it needs to efficiently lyse viral particles in the sample, release and protect the native conformation of the target antigens; simultaneously, it inactivates potential biohazards and removes interfering substances such as mucins and cell debris to ensure the sensitivity and specificity of subsequent detection. An ideal universal sample processing solution should be compatible with multiple sample types and different detection platforms, providing a stable and reliable sample matrix for multiplex antigen detection.

[0003] However, the sample processing solutions in the existing technology are inherently unstable, which may lead to unstable test results after the collected samples have been stored for a period of time. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and to provide a sample processing solution for respiratory virus antigen detection with good sample stability and its preparation method.

[0005] The technical solution of the present invention is as follows:

[0006] A sample processing solution for respiratory virus antigen detection contains the following components at the following concentrations:

[0007] 10-100mM phosphate buffer, 0.05-0.5g / L sodium caseinate, 1-20g / L bovine serum albumin, 10-50mL / L surfactant, 1-10g / L guanidine hydrochloride, 3.72-37.2g / L disodium ethylenediaminetetraacetate dihydrate, 1-5mL / L CL preservative, 1-5mL / L BND antibacterial agent, and 900-988mL / L sterile deionized water.

[0008] Furthermore, the surfactants include, but are not limited to, Triton X-100, NP-40, and Tween-20.

[0009] Furthermore, the phosphate buffer solution is prepared from 8-100 g / L NaCl, 200-2000 mg / L KCl, 1.44-15 g / L Na2HPO4, and 240-2500 mg / L KH2PO4.

[0010] Furthermore, the phosphate buffer can be replaced with 20-100mM Tris-HCl buffer or 20-100mM borate-borax buffer.

[0011] Furthermore, guanidine hydrochloride was replaced with urea.

[0012] This invention also proposes a method for preparing a sample processing solution for respiratory virus antigen detection, comprising the following steps:

[0013] S1. Preparation of disodium ethylenediaminetetraacetate dihydrate: Measure 100mL of water and pour it into a beaker. Place the rotor in the beaker and put it on a heating stirrer. After the water is heated to a boil, add 9.306g of disodium ethylenediaminetetraacetate dihydrate. Continue stirring until dissolved. Cool and dilute to 100mL. Store in a reagent bottle for later use.

[0014] S2, Preparation of 20% Triton X-100: Measure 80mL of deionized water and pour it into an Erlenmeyer flask. Measure 20mL of Triton X-100 and add it to the deionized water. Stir well and mix thoroughly. Transfer to a reagent bottle for later use.

[0015] S3, 50% Guanidine Hydrochloride Preparation: Measure 50mL of deionized water and pour it into an Erlenmeyer flask. Weigh 50g of guanidine hydrochloride and add it to the deionized water. Stir thoroughly to dissolve and bring the volume up to 100mL. Pour the solution into a reagent bottle for later use.

[0016] S4. Preparation of 20% bovine serum albumin: Measure 60mL of deionized water into an Erlenmeyer flask, weigh 20g of bovine serum albumin and add it to the deionized water, stir thoroughly to dissolve, and make up to 100mL. Then put it into a reagent bottle for later use.

[0017] Preparation of S5, 5% sodium caseinate: Measure 100mL of deionized water into a beaker, put in a rotor, place the beaker on a heating stirrer, weigh it, and slowly stir to add 5g of sodium caseinate. Stir, heat to dissolve, boil, cool and make up to 100mL, and put it into a reagent bottle for later use.

[0018] S6. Take half the total volume of deionized water and pour it into a beaker. Weigh and add the required amounts of NaCl, KCl, Na2HPO4 and KH2PO4. Sequentially measure the various mother liquors, CL preservative and BND antibacterial agent prepared in steps S1-5 above, and stir to mix well.

[0019] Further, after adjusting the pH to 7.2-8.5 with NaOH and HCl solutions, the remaining volume of deionized water is added.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. The phosphate buffer concentration and pH value in the sample processing solution of this invention provide suitable ionic strength and reaction pH for antigen detection, enabling better specific binding of antigen and antibody during the detection process.

[0022] 2. The sodium caseinate and bovine serum albumin in the sample processing solution of this invention can maintain the stability of respiratory pathogen antigens, while reducing the non-specific binding of non-specific proteins to the detection reagents and improving the specificity of the reagents.

[0023] 3. The Triton X-100 or NP-40 and Tween-20 in the sample processing solution of this invention can dissolve lipids on the microbial membrane, increase membrane permeability, and release pathogen antigens into the solution.

[0024] 4. The guanidine hydrochloride or urea in the sample processing solution of this invention can improve the solubility of pathogen antigens in the processing solution. The disodium ethylenediaminetetraacetate dihydrate in the sample processing solution acts as a complexing agent, which can inactivate non-specific enzymes in the sample and improve the specificity of reagent detection. The CL preservative and BND antibacterial agent in the sample processing solution can prevent the sample processing solution and the sample prepared by the sample processing solution from being contaminated by miscellaneous bacteria and improve stability. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] A sample processing solution for respiratory virus antigen detection contains the following components at the following concentrations:

[0028] 10 mM phosphate buffer, 0.05 g / L sodium caseinate, 1 g / L bovine serum albumin, 10 mL / L Triton X-100, 1 g / L guanidine hydrochloride, 3.72 g / L disodium ethylenediaminetetraacetate dihydrate, 1 mL / L CL preservative, 1 mL / L BND antibacterial agent, and 988 mL / L sterile deionized water.

[0029] Phosphate buffer is prepared from 8 g / L NaCl, 200 mg / L KCl, 1.44 g / L Na2HPO4, and 240 mg / L KH2PO4.

[0030] Example 2

[0031] A sample processing solution for respiratory virus antigen detection contains sterile deionized water and components at the following concentrations:

[0032] 100 mM phosphate buffer, 0.5 g / L sodium caseinate, 20 g / L bovine serum albumin, 50 mL / L Triton X-100, 10 g / L guanidine hydrochloride, 37.2 g / L disodium ethylenediaminetetraacetate dihydrate, 5 mL / L CL preservative, 5 mL / L BND antibacterial agent, and 940 mL / L sterile deionized water.

[0033] Phosphate buffer is prepared from 80 g / L NaCl, 2000 mg / L KCl, 14.4 g / L Na2HPO4, and 2400 mg / L KH2PO4.

[0034] Example 3

[0035] A sample processing solution for respiratory virus antigen detection contains sterile deionized water and components at the following concentrations:

[0036] 10 mM phosphate buffer, 0.05 g / L sodium caseinate, 1 g / L bovine serum albumin, 10 mL / L Triton X-100, 5 g / L guanidine hydrochloride, 9.3 g / L disodium EDTA solution, 1 mL / L CL preservative, 1 mL / L BND antibacterial agent, and 988 mL / L sterile deionized water.

[0037] Phosphate buffer is prepared from 8 g / L NaCl, 200 mg / L KCl, 1.44 g / L Na2HPO4, and 240 mg / L KH2PO4.

[0038] Example 4

[0039] A sample processing solution for respiratory virus antigen detection, comprising the proportions and components of Examples 1-3, with Triton X-100 replaced by NP-40 or Tween-20.

[0040] Example 5

[0041] A sample processing solution for respiratory virus antigen detection, comprising the proportions and components of Examples 1-3, wherein the phosphate buffer prepared from 8-100 g / L NaCl, 200-2000 mg / L KCl, 1.44-15 g / L Na2HPO4, and 240-2500 mg / L KH2PO4 is replaced with 20-100 mM Tris-HCl buffer or 20-100 mM borate-borax buffer.

[0042] Example 6

[0043] A sample processing solution for respiratory virus antigen detection, comprising the proportions and components of Examples 1-3, with guanidine hydrochloride replaced by urea.

[0044] Example 7

[0045] A method for preparing a sample processing solution for respiratory virus antigen detection includes the following steps:

[0046] S1. Preparation of disodium ethylenediaminetetraacetate dihydrate: Measure 100mL of water and pour it into a beaker. Place the rotor in the beaker and put it on a heating stirrer. After the water is heated to a boil, add 9.306g of disodium ethylenediaminetetraacetate dihydrate. Continue stirring until dissolved. Cool and dilute to 100mL. Store in a reagent bottle for later use.

[0047] S2, Preparation of 20% Triton X-100: Measure 80mL of deionized water and pour it into an Erlenmeyer flask. Measure 20mL of Triton X-100 and add it to the deionized water. Stir well and mix thoroughly. Transfer to a reagent bottle for later use.

[0048] S3, 50% Guanidine Hydrochloride Preparation: Measure 50mL of deionized water and pour it into an Erlenmeyer flask. Weigh 50g of guanidine hydrochloride and add it to the deionized water. Stir thoroughly to dissolve and bring the volume up to 100mL. Pour the solution into a reagent bottle for later use.

[0049] S4. Preparation of 20% bovine serum albumin: Measure 60mL of deionized water into an Erlenmeyer flask, weigh 20g of bovine serum albumin and add it to the deionized water, stir thoroughly to dissolve, and make up to 100mL. Then put it into a reagent bottle for later use.

[0050] Preparation of S5, 5% sodium caseinate: Measure 100mL of deionized water into a beaker, put in a rotor, place the beaker on a heating stirrer, weigh it, and slowly stir to add 5g of sodium caseinate. Stir, heat to dissolve, boil, cool and make up to 100mL, and put it into a reagent bottle for later use.

[0051] S6. Take half of the total prepared volume of deionized water and pour it into a beaker. Weigh and add the required amounts of NaCl, KCl, Na2HPO4, and KH2PO4. Sequentially measure the various mother liquors, CL preservative, and BND antibacterial agent prepared in steps S1-5 above, stir and mix well. Adjust the pH to 7.2-8.5 with NaOH solution and HCl solution, and then add the remaining volume of deionized water.

[0052] Study on the stability of samples prepared based on the three sample processing solutions in Examples 1-3:

[0053] Healthy volunteers who were not infected with the respiratory viruses to be tested were recruited. Three pharyngeal swab samples were collected from each volunteer. The samples were treated with different sample processing solutions, and then the samples prepared with the same sample processing solution were mixed. The pharyngeal swab mixture treated with sample processing solution one was called negative matrix solution one, the pharyngeal swab mixture treated with sample processing solution two was called negative matrix solution two, and the pharyngeal swab mixture treated with sample processing solution three was called negative matrix solution three. A series of concentrations of samples were prepared by diluting inactivated cultures of influenza A virus (FluA, source: ATCCVR-1894), influenza B virus (FluB, source: ATCCVR-1804PQ), respiratory syncytial virus (RSV, source: ATCCVR-1540), adenovirus (ADV, source: ATCCVR-7), and Mycoplasma pneumoniae (MP, source: ATCC-15531) with the three negative matrix solutions. The prepared series of concentrations of samples were tested using a respiratory pathogen antigen multiplex detection reagent. The detection limit concentrations of different cultures diluted with different negative matrix solutions were observed and recorded. Samples were prepared by diluting various viral cultures at twice the detection limit concentration using different negative matrix solutions. The prepared samples were aliquoted and stored at 25℃, 2-8℃, and -20℃ respectively, and tested at different times. The test results are recorded below:

[0054] Results of Sample Stability Study on Sample Processing Solution Preparation

[0055]

[0056] Results of the stability study of sample preparation solution II

[0057]

[0058] Results of the stability study of sample preparation solution III

[0059]

[0060] The above results indicate that sample treatment solution three has the best stability. The reason for this may be that sample treatment solution one contains less protein protectant, while sample treatment solution two contains too much of various substances.

[0061] It should be noted that, when using the sample processing solution provided in Examples 1-3, the nasopharyngeal swab, oropharyngeal swab, and nasal swab collected from the respiratory tract are placed into the processing solution, the swab sampling end is rotated in the sample tube for 30 seconds, the swab is rotated 5 times when the test tube containing the sample processing solution is squeezed, the swab is squeezed out and discarded, and the test sample required for the respiratory tract multiple viral antigen detection reagent can be prepared.

[0062] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sample processing solution for detecting respiratory virus antigens, characterized in that: It contains the following components: 10-100mM phosphate buffer, 0.05-0.5g / L sodium caseinate, 1-20g / L bovine serum albumin, 10-50mL / L surfactant, 1-10g / L guanidine hydrochloride, 3.72-37.2g / L disodium ethylenediaminetetraacetate dihydrate, 1-5mL / L CL preservative, 1-5mL / L BND antibacterial agent, and 900-988mL / L sterile deionized water.

2. The sample processing solution for respiratory virus antigen detection according to claim 1, characterized in that: The surfactants include, but are not limited to, Triton X-100, NP-40, and Tween-20.

3. The sample processing solution for respiratory virus antigen detection according to claim 1, characterized in that: The phosphate buffer solution is prepared from 8-100 g / L NaCl, 200-2000 mg / L KCl, 1.44-15 g / L Na2HPO4, and 240-2500 mg / L KH2PO4.

4. The sample processing solution for respiratory virus antigen detection according to claim 1, characterized in that: Replace the phosphate buffer with 20-100mM Tris-HCl buffer or 20-100mM borate-borax buffer.

5. The sample processing solution for respiratory virus antigen detection according to claim 1, characterized in that: Guanidine hydrochloride was replaced with urea.

6. A method for preparing a sample processing solution for respiratory virus antigen detection according to any one of claims 1-5, characterized in that: S1. Preparation of disodium ethylenediaminetetraacetate dihydrate: Measure 100mL of water and pour it into a beaker. Place the rotor in the beaker and put it on a heating stirrer. After the water is heated to a boil, add 9.306g of disodium ethylenediaminetetraacetate dihydrate. Continue stirring until dissolved. Cool and dilute to 100mL. Store in a reagent bottle for later use. S2, Preparation of 20% Triton X-100: Measure 80mL of deionized water and pour it into an Erlenmeyer flask. Measure 20mL of Triton X-100 and add it to the deionized water. Stir well and mix thoroughly. Transfer to a reagent bottle for later use. S3, 50% Guanidine Hydrochloride Preparation: Measure 50mL of deionized water and pour it into an Erlenmeyer flask. Weigh 50g of guanidine hydrochloride and add it to the deionized water. Stir thoroughly to dissolve and bring the volume up to 100mL. Transfer the solution to a reagent bottle for later use. S4. Preparation of 20% bovine serum albumin: Measure 60mL of deionized water into an Erlenmeyer flask, weigh 20g of bovine serum albumin and add it to the deionized water, stir thoroughly to dissolve, and make up to 100mL. Then put it into a reagent bottle for later use. Preparation of S5, 5% sodium caseinate: Measure 100mL of deionized water into a beaker, put in a rotor, place the beaker on a heating stirrer, weigh it, and slowly stir to add 5g of sodium caseinate. Stir, heat to dissolve, boil, cool and make up to 100mL, and put it into a reagent bottle for later use. S6. Take half the total volume of deionized water and pour it into a beaker. Weigh and add the required amounts of NaCl, KCl, Na2HPO4 and KH2PO4. Measure the various mother liquors, CL preservative and BND antibacterial agent prepared in steps S1-S5 in sequence and stir to mix well.

7. The method for preparing a sample processing solution for respiratory virus antigen detection according to claim 6, characterized in that: After adjusting the pH to 7.2-8.5 with NaOH and HCl solutions, add the remaining volume of deionized water.

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