Sample treating fluid for respiratory virus antigen detection and preparation method thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of antigen detection, and in particular relates to a sample processing liquid for respiratory virus antigen detection and a preparation method thereof. Background Art

[0002] Respiratory multiple virus 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 novel coronavirus (SARS-CoV-2). Its core lies in the simultaneous detection of specific antigens of multiple viruses in clinical samples (such as nasopharyngeal swabs and sputum) using methods such as immunochromatography or fluorescent immunoassay. As the first step in the detection process, the sample processing fluid undertakes a vital pretreatment function: it needs to efficiently lyse the viral particles in the sample, release and protect the natural conformation of the target antigen; at the same time, it inactivates potential biological hazards, removes interfering substances such as mucin and cell debris, and ensures the sensitivity and specificity of subsequent detection. The ideal universal sample processing fluid should be compatible with a variety of sample types and different detection platforms, providing a stable and reliable sample matrix for multiple antigen detection.

[0003] However, due to the insufficient stability of the sample processing solution in the prior art, the test results of the collected samples may become unstable after being stored for a period of time. Summary of the Invention

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

[0005] The technical solutions of the present invention are as follows:

[0006] A sample processing solution for respiratory virus antigen detection, containing the following components in the following concentrations:

[0007] 10-100 mM 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, 1-5 mL / L CL preservative, 1-5 mL / L BND antibacterial agent, and 900-988 mL / L sterile deionized water.

[0008] Furthermore, the surfactant includes but is not limited to Triton X-100, NP-40 and Tween-20.

[0009] Furthermore, the phosphate buffer 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 was replaced with 20-100 mM Tris-HCl buffer or 20-100 mM boric acid-borax buffer.

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

[0012] The present invention also provides a method for preparing a sample processing liquid for respiratory virus antigen detection, comprising the following steps:

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

[0014] S2. Preparation of 20% Triton X-100: Pour 80 mL of deionized water into a conical flask, add 20 mL of Triton X-100 into the deionized water, stir thoroughly, and transfer to a reagent bottle for later use;

[0015] S3. Preparation of 50% guanidine hydrochloride: Pour 50 mL of deionized water into a conical flask, weigh 50 g of guanidine hydrochloride and add it to the deionized water. Stir thoroughly to dissolve, dilute to 100 mL, and transfer to a reagent bottle for later use.

[0016] S4. Preparation of 20% bovine serum albumin: Measure 60 mL of deionized water and pour it into a conical flask. Weigh 20 g of bovine serum albumin and add it to the deionized water. Stir thoroughly to dissolve. Make up to 100 mL and transfer to a reagent bottle for later use.

[0017] S5. Preparation of 5% sodium caseinate: Measure 100 mL of deionized water and pour it into a beaker. Place a rotor in the beaker and place it on a heated stirrer. After weighing, slowly stir and add 5 g of sodium caseinate. Stir and heat to dissolve and boil. Cool and dilute to 100 mL. Pour into a reagent bottle and set aside.

[0018] S6. Take half of the total preparation volume of deionized water and pour it into a beaker. Weigh and add the required amount of NaCl, KCl, Na2HPO4 and KH2PO4. Then weigh the various mother liquors, CL preservatives and BND antibacterial agents prepared in steps S1-5 above and stir to mix.

[0019] Furthermore, the pH was adjusted to 7.2-8.5 with NaOH solution and HCl solution, and then the remaining volume of deionized water was added.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The concentration and pH value of the phosphate buffer in the sample treatment solution of the present invention provide suitable ionic strength and reaction pH for antigen detection, so that the antigen and antibody in the detection process can be better specifically bound.

[0022] 2. Sodium caseinate and bovine serum albumin in the sample treatment solution of the present invention can maintain the stability of respiratory pathogen antigens, while reducing the nonspecific binding of nonspecific proteins to the detection reagent and improving the specificity of the reagent.

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

[0024] 4. The guanidine hydrochloride or urea in the sample treatment solution of the present invention can improve the solubility of pathogen antigens in the treatment solution. The disodium ethylenediaminetetraacetic acid dihydrate in the sample treatment solution acts as a chelating agent, which can inactivate nonspecific enzymes in the sample and improve the detection specificity of the reagent. The CL preservative and BND antibacterial agent in the sample treatment solution can prevent the sample treatment solution and the sample prepared with the sample treatment solution from being contaminated by miscellaneous bacteria, thereby improving stability. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] A sample processing solution for respiratory virus antigen detection, containing the following components in 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 ethylenediaminetetraacetic acid disodium salt dihydrate, 1 mL / L CL preservative, 1 mL / L BND antibacterial agent and 988 mL / L sterile deionized water.

[0029] Phosphate buffer was 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, comprising sterile deionized water and the following components in 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 ethylenediaminetetraacetic acid disodium salt dihydrate, 5 mL / L CL preservative, 5 mL / L BND antibacterial agent and 940 mL / L sterile deionized water.

[0033] Phosphate buffer was 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, comprising sterile deionized water and the following components in 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 ethylenediaminetetraacetic acid disodium salt dihydrate, 1 mL / L CL preservative, 1 mL / L BND antibacterial agent and 988 mL / L sterile deionized water.

[0037] Phosphate buffer was 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 comprises 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 comprises the proportions and components of Examples 1-3, and replaces a 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 with a 20-100 mM Tris-HCl buffer or a 20-100 mM boric acid-borax buffer.

[0042] Example 5

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

[0044] Example 6

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

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

[0047] S2. Preparation of 20% Triton X-100: Pour 80 mL of deionized water into a conical flask, add 20 mL of Triton X-100 into the deionized water, stir thoroughly, and transfer to a reagent bottle for later use;

[0048] S3. Preparation of 50% guanidine hydrochloride: Pour 50 mL of deionized water into a conical flask, weigh 50 g of guanidine hydrochloride and add it to the deionized water. Stir thoroughly to dissolve, dilute to 100 mL, and transfer to a reagent bottle for later use.

[0049] S4. Preparation of 20% bovine serum albumin: Measure 60 mL of deionized water and pour it into a conical flask. Weigh 20 g of bovine serum albumin and add it to the deionized water. Stir thoroughly to dissolve. Make up to 100 mL and transfer to a reagent bottle for later use.

[0050] S5. Preparation of 5% sodium caseinate: Measure 100 mL of deionized water and pour it into a beaker. Place a rotor in the beaker and place it on a heated stirrer. After weighing, slowly stir and add 5 g of sodium caseinate. Stir and heat to dissolve and boil. Cool and dilute to 100 mL. Pour into a reagent bottle and set aside.

[0051] S6. Take half of the total preparation volume of deionized water and pour it into a beaker. Weigh and add the required amounts of NaCl, KCl, Na2HPO4 and KH2PO4. Then weigh the various mother liquors, CL preservatives, and BND antibacterial agents prepared in steps S1-5 above, stir and mix, adjust the pH to 7.2-8.5 with Na0H solution and HCl solution, and then add the remaining volume of deionized water.

[0052] Study on the stability of samples prepared using the three sample treatment solutions of Examples 1-3:

[0053] Normal volunteers who were not infected with the respiratory viruses to be tested were recruited. Three throat swab samples were collected from each person. After being treated with different sample processing solutions, the samples prepared with the same sample processing solution were mixed. The mixed throat swab samples treated with sample processing solution 1 were designated as negative matrix solution 1, the mixed throat swab samples treated with sample processing solution 2 were designated as negative matrix solution 2, and the mixed throat swab samples treated with sample processing solution 3 were designated as negative matrix solution 3. Inactivated cultures of influenza A virus (FluA source: ATCC VR-1894), influenza B virus (FluB source: ATCC VR-1804PQ), respiratory syncytial virus (RSV source: ATCC VR-1540), adenovirus (ADV source: ATCC VR-7), and Mycoplasma pneumoniae (MP source: ATCC-15531) were diluted with the three negative matrix solutions to prepare a series of sample concentrations. The prepared samples were tested using a multiplex detection reagent for respiratory pathogen antigens, and the detection limits of the different cultures diluted with different negative matrix solutions were observed and recorded. Prepare the test samples by diluting different virus cultures to 2 times the detection limit concentration using different negative matrix solutions. After packaging, store the prepared samples at 25°C, 2-8°C, and -20°C. Test them at different times and record the test results as follows:

[0054] Sample treatment solution - Preparation sample stability study results

[0055]

[0056]

[0057] Results of stability study on samples prepared with sample treatment solution 2

[0058]

[0059]

[0060] Results of stability study on samples prepared with sample treatment solution 3

[0061]

[0062]

[0063] The above results show that sample treatment solution three has the best stability. The possible reason is that the content of protein protective agent in sample treatment solution one is relatively low, while the content of various substances in sample treatment solution two is too high.

[0064] It should be noted that when using the sample processing liquid provided in Examples 1-3, the nasopharyngeal swab, oropharyngeal swab, and nasal swab for collecting respiratory samples are placed in the processing liquid, the sampling end of the swab is rotated in the sample tube for 30 seconds, and when squeezing the test tube storing the sample processing liquid, the swab is rotated 5 times, the tube is squeezed to twist out the swab, and the swab is discarded to prepare the test sample required for the respiratory multiple virus antigen detection reagent.

[0065] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sample processing solution for respiratory virus antigen detection, characterized in that: Contains the following ingredients: 10-100 mM 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, 1-5 mL / L CL preservative, 1-5 mL / L BND antibacterial agent, and 900-988 mL / L sterile deionized water.

2. The sample processing liquid for respiratory virus antigen detection according to claim 1, characterized in that: The surfactant includes but is not limited to Triton X-100, NP-40 and Tween-20.

3. The sample processing liquid for respiratory virus antigen detection according to claim 1, characterized in that: The phosphate buffer 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 liquid for respiratory virus antigen detection according to claim 1, characterized in that: The phosphate buffer was replaced with 20-100 mM Tris-HCl buffer or 20-100 mM boric acid-borax buffer.

5. The sample processing liquid 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 claims 1-5, characterized in that: S1. Preparation of ethylenediaminetetraacetic acid disodium salt dihydrate: Measure 100 mL of water and pour it into a beaker. Place a rotor in the beaker and place it on a heated stirrer. After stirring and heating the water to a boil, add 9.306 g of ethylenediaminetetraacetic acid disodium salt dihydrate and continue stirring until dissolved. Cool and dilute to 100 mL and place in a reagent bottle for later use. S2. Preparation of 20% Triton X-100: Pour 80 mL of deionized water into a conical flask, add 20 mL of Triton X-100 into the deionized water, stir thoroughly, and transfer to a reagent bottle for later use; S3. Preparation of 50% guanidine hydrochloride: Pour 50 mL of deionized water into a conical flask, weigh 50 g of guanidine hydrochloride and add it to the deionized water. Stir thoroughly to dissolve, dilute to 100 mL, and transfer to a reagent bottle for later use. S4. Preparation of 20% bovine serum albumin: Measure 60 mL of deionized water and pour it into a conical flask. Weigh 20 g of bovine serum albumin and add it to the deionized water. Stir thoroughly to dissolve. Make up to 100 mL and transfer to a reagent bottle for later use. S5. Preparation of 5% sodium caseinate: Measure 100 mL of deionized water and pour it into a beaker. Place a rotor in the beaker and place it on a heated stirrer. After weighing, slowly stir and add 5 g of sodium caseinate. Stir and heat to dissolve and boil. Cool and dilute to 100 mL. Pour into a reagent bottle and set aside. S6. Take half of the total preparation volume of deionized water and pour it into a beaker. Weigh and add the required amount of NaCl, KCl, Na2HPO4 and KH2PO4. Then weigh the various mother liquors, CL preservatives and BND antibacterial agents prepared in steps S1-S5 above in turn and stir to mix.

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

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