Whole blood preserving fluid for stably preserving free miRNA as well as preparation method and application of whole blood preserving fluid

Abstract

The invention discloses a whole blood preservation solution for stably preserving free miRNA and a preparation method and application of the whole blood preservation solution. The whole blood preservation solution comprises the following solutes in parts by mass and volume: a cell fixing agent, 0.5%-5% of a cell membrane protective agent, 3.8%-10% of a nuclease inhibitor, 0.1%-3% of a cell metabolism inhibitor, 0.04%-0.5% of a reducing agent and a pH buffering agent, the cell fixing agent is prepared from 0.5%-10% of diazoalkyl urea or 5%-10% of 5, 5-dimethyl hydantoin. The combination of all the components in the whole blood preservation solution can achieve an excellent stable effect, and miRNA detection is not inhibited; erythrocytes in a blood sample are stable, and whole blood is not hemolyzed after being stored at room temperature for 5 days; the free miRNA expression quantity of the plasma is stable, and the whole blood does not change after being stored at room temperature for 5 days.

Description

Technical Field

[0001] The present invention belongs to the technical field of whole blood room temperature storage, and specifically relates to a whole blood storage solution for stably storing free miRNA, and a preparation method and application thereof. Background Art

[0002] Circulating free miRNA is a cell-free extracellular miRNA that exists in human blood. In recent years, the clinical application of circulating free miRNA in peripheral blood has gradually increased: a large number of literatures show that peripheral blood free miRNA can be a marker for the diagnosis of diseases such as tumors and cardiovascular diseases. At the same time, foreign countries have developed a kit based on peripheral blood miRNA detection to assist in the diagnosis of gastric cancer.

[0003] Blood collected from animals or humans is called whole blood. The liquid obtained by separating whole blood without anticoagulation is called serum. The liquid obtained by adding anticoagulants to whole blood and separating it is called plasma. In the actual detection process, the amount of free miRNA in peripheral blood is a key indicator. Whole blood, serum, and plasma all have a certain shelf life and are easily ineffective in liquid form at room temperature. If red blood cells, platelets, or white blood cells rupture in the peripheral blood during storage, the cellular miRNA will enter the plasma and contaminate the free miRNA, thereby seriously affecting the test results. Therefore, it is very necessary to provide a whole blood preservation solution that can stably preserve free miRNA. Summary of the invention

[0004] In order to solve one of the above problems, the present invention provides a whole blood preservation solution for stably preserving free miRNA and its preparation method and application. The whole blood preservation solution solves the problems that free miRNA in peripheral blood is easily contaminated, requires low-temperature transportation, and has high economic costs when using existing whole blood preservation.

[0005] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a whole blood preservation solution for stably preserving free miRNA, comprising the following solutes in mass volume fractions: a cell fixative, 0.5%-5% cell membrane protectant, 3.8%-10% nuclease inhibitor, 0.1%-3% cell metabolism inhibitor, 0.04%-0.5% reducing agent and pH buffer, wherein the cell fixative is 0.5%-10% diazolidinyl urea or 5%-10% 5,5-dimethylhydantoin.

[0006] In some embodiments of the present invention, the cell membrane protective agent is selected from one of trehalose, glucose, sorbitol, rhamnose and sucrose. In some embodiments of the present invention, the cell membrane protective agent is used in an amount of 3%.

[0007] In some embodiments of the present invention, the nuclease inhibitor is selected from one of K3EDTA, sodium citrate, heparin, guanidine salts, SDS, urea, oxovanadyl ribonucleoside complex, and diatomaceous earth. In some embodiments of the present invention, the dosage of the nuclease inhibitor is 5%.

[0008] In some embodiments of the present invention, the cell metabolism inhibitor is selected from one of glyceraldehyde, sodium fluoride, and glyceraldehyde-3-phosphate. In some embodiments of the present invention, the dosage of the cell metabolism inhibitor is 0.2%.

[0009] In some embodiments of the present invention, the reducing agent is selected from one of dithiothreitol, thioglycerol, and tricarboxyethylphosphine. In some embodiments of the present invention, the preferred dosage of the reducing agent is 0.1%.

[0010] In some embodiments of the present invention, the pH buffer is one or more of sodium citrate and citric acid, and the solution is adjusted to a pH range of 5.0 - 6.5.

[0011] In some embodiments of the present invention, it further includes a solvent, and the solvent is nuclease-free water.

[0012] The second aspect of the present invention provides a method for preparing the whole blood preservative solution described in the first aspect. Add nuclease-free water to a container and vortex, then add a cell fixative, a cell membrane protectant, a nuclease inhibitor, a cell metabolism inhibitor, and a reducing agent, and stir and mix evenly. Adjust the pH value to 5.0 - 6.5 with a pH buffer, and make up the volume with nuclease-free water to obtain the whole blood preservative solution. In some embodiments of the present invention, the cell fixative: 0.5% - 10% diazalkyl urea or 5% - 10% 5,5-dimethylhydantoin; 0.5% - 5% cell membrane protectant, 3.8% - 10% nuclease inhibitor, 0.1% - 3% cell metabolism inhibitor, 0.04% - 0.5% reducing agent.

[0013] In some embodiments of the present invention, the whole blood preservative solution is filtered through a 0.22 μm filter membrane.

[0014] The present invention also provides a method for preserving a blood sample using the whole blood preservative solution described in the first aspect. Inject the whole blood preservative solution described in the first aspect into a blood collection tube without additives, and then evacuate to complete the preparation of a vacuum blood collection tube containing the preservative solution. Use this blood collection tube to collect blood, and immediately invert it 5 - 8 times to mix evenly after blood collection.

[0015] The third aspect of the present invention provides an application of the whole blood preservation solution described in the first aspect in the preparation of a product for stably preserving whole blood. Or provides an application of the whole blood preservation solution described in the first aspect in stably preserving free miRNAs in whole blood. Or provides an application of the whole blood preservation solution described in the first aspect in the preparation of a product for improving the stability of whole blood preservation at room temperature.

[0016] The present invention also provides a whole blood preservation kit, which contains the reagents used for preparing the whole blood preservation solution described in the first aspect.

[0017] The present invention also provides an application of the whole blood preservation kit in stably preserving free miRNAs in whole blood.

[0018] Advantages of the present invention Compared with the prior art, the present invention has the following advantages: The present invention provides a whole blood preservation solution for stably preserving free miRNAs. The combination of the components in the whole blood preservation solution can achieve an excellent stabilizing effect and does not inhibit miRNA detection; the red blood cells in the blood sample are stable, and the whole blood does not hemolyze after being stored at room temperature for 5 days; the expression level of plasma free miRNAs is stable, and there is no change after the whole blood is stored at room temperature for 5 days. Detailed implementation manners

[0019] The following examples are used here to demonstrate the preferred implementation schemes of the present invention. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies discovered by the inventors that can be used to implement the present invention, and thus can be regarded as the preferred solutions for implementing the present invention. However, those skilled in the art should understand according to this specification that many modifications can be made to the specific embodiments disclosed here, and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0020] The numerical ranges in this application are approximate values. Therefore, unless otherwise specified, they may include values outside the ranges. The numerical ranges include all values from the lower limit value to the upper limit value increasing by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if it is stated that a component, physical or other property (such as molecular weight, melt index, etc.) is from 100 to 1000, it means that all individual values such as 100, 101, 102, etc. are clearly listed, as well as all sub-ranges such as 100 to 166, 155 to 170, 198 to 200, etc. For ranges containing values less than 1 or fractions greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For ranges containing single-digit numbers less than 10 (such as 1 to 5), 1 unit is usually regarded as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of the values between the lowest and highest values listed are considered to be clearly recorded in this application. The examples in the embodiments are only for illustrating the technical solutions and do not represent a limitation of the range.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All materials cited herein and the materials they cite will be incorporated by reference. Those skilled in the art will recognize or be able to learn through routine experimentation many equivalent technologies to the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0022] A whole blood preservative solution, comprising a cell fixative; a cell membrane protectant; a nuclease inhibitor, an anticoagulant; a cell metabolism inhibitor; a reducing agent; a pH buffer; and nuclease-free water.

[0023] Among them, the cell fixative is diazolidinyl urea with a mass-volume concentration of 0.5% - 10% and dimethylhydantoin with a mass-volume concentration of 5% - 10%; The cell membrane protectant is selected from one of trehalose, glucose, sorbitol, rhamnose, sucrose, and has a mass-volume concentration of 0.5% - 5%; The nuclease inhibitor is selected from one of K3EDTA, sodium citrate, heparin, guanidine salts, SDS, urea, oxovanadyl ribonucleoside complex, diatomaceous earth, and has a mass-volume concentration of 3.8% - 10%; The cell metabolism inhibitor is selected from one of glyceraldehyde, sodium fluoride, glyceraldehyde-3-phosphate, and has a mass-volume concentration of 0.1% - 3%; The reducing agent is selected from one of dithiothreitol, thioglycerol, tricarboxyethylphosphine, and has a mass-volume concentration of 0.04% - 0.5%; The pH buffer is one or both of sodium citrate and citric acid, and the solution is adjusted to a pH range of 5.0 - 6.5.

[0024] The technical solutions of this patent will be further described in detail below in conjunction with specific implementation examples, and the examples shall not be construed as limiting the scope of protection.

[0025] Example 1 Taking the preparation of 1 L of whole blood preservative solution as an example, prepare raw materials according to the following mass - volume fractions: 15% diazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0026] The preparation process is as follows: First, add 500 mL of nuclease - free water to a beaker, vortex with a magnetic stirrer, and sequentially add diazolidinyl urea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease - free water.

[0027] The prepared whole blood preservative solution is filtered through a 0.22 - um filter membrane and reserved for use.

[0028] Example 2 Taking the preparation of 1 L of whole blood preservative solution as an example, prepare raw materials according to the following mass - volume fractions: 10% diazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0029] The preparation process is as follows: First, add 500 mL of nuclease - free water to a beaker, vortex with a magnetic stirrer, and sequentially add diazolidinyl urea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease - free water.

[0030] The prepared whole blood preservative solution is filtered through a 0.22 - um filter membrane and reserved for use.

[0031] Example 3 Taking the preparation of 1 L of whole blood preservative solution as an example, prepare raw materials according to the following mass - volume fractions: 5% diazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0032] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex it with a magnetic stirrer, and sequentially add diazalkylurea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0033] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0034] Example 4 Taking the preparation of 1 L of whole blood preservative solution as an example, prepare the raw materials according to the following mass-volume fractions: 2.5% diazalkylurea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0035] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex it with a magnetic stirrer, and sequentially add diazalkylurea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0036] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0037] Example 5 Taking the preparation of 1 L of whole blood preservative solution as an example, prepare the raw materials according to the following mass-volume fractions: 1% diazalkylurea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0038] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex it with a magnetic stirrer, and sequentially add diazalkylurea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0039] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0040] Example 6 Taking the preparation of 1 L of whole blood preservative solution as an example, prepare the raw materials according to the following mass-volume fractions: 0.5% diazalkylurea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0041] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add diazalkylurea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0042] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0043] Example 7 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 0.1% diazalkylurea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0044] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add diazalkylurea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0045] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0046] Example 8 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 15% 5,5-dimethylhydantoin, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0047] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add 5,5-dimethylhydantoin, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0048] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0049] Example 9 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 10% 5,5-dimethylhydantoin, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0050] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add 5,5-dimethylhydantoin, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0051] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and set aside.

[0052] Example 10 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 5% 5,5-dimethylhydantoin, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0053] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add 5,5-dimethylhydantoin, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0054] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and set aside.

[0055] Example 11 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 2.5% 5,5-dimethylhydantoin, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0056] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add 5,5-dimethylhydantoin, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0057] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and set aside.

[0058] Example 12 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 1% 5,5-dimethylhydantoin, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0059] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add 5,5-dimethylhydantoin, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0060] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0061] Example 13 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 10% imidazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0062] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add imidazolidinyl urea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0063] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0064] Example 14 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 5% imidazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0065] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add imidazolidinyl urea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0066] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0067] Example 15 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 2.5% imidazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0068] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add imidazolidinyl urea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0069] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0070] Example 16 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 1% imidazolidinyl urea, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0071] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add imidazolidinyl urea, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0072] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0073] Example 17 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 10% paraformaldehyde, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0074] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add paraformaldehyde, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0075] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0076] Example 18 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 1% paraformaldehyde, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0077] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex it with a magnetic stirrer, and sequentially add paraformaldehyde, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0078] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0079] Example 19 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 10% formaldehyde, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0080] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex it with a magnetic stirrer, and sequentially add formaldehyde, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0081] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0082] Example 20 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 1% formaldehyde, 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0083] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex it with a magnetic stirrer, and sequentially add formaldehyde, glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0084] The prepared whole blood preservative solution is filtered through a 0.22 μm filter membrane and reserved for use.

[0085] Comparative Example 1 Use a conventional K3EDTA tube (BD, 367863).

[0086] Comparative Example 2 Taking the preparation of 1 L of whole blood preservative solution as an example, weigh the raw materials according to the following mass-volume fractions: 3% glucose, 5% sodium citrate, 0.2% sodium fluoride, 0.1% dithiothreitol, 0.1 mol / L citric acid solution.

[0087] The preparation process is as follows: First, add 500 mL of nuclease-free water to a beaker, vortex with a magnetic stirrer, and sequentially add glucose, sodium citrate, dithiothreitol, and sodium fluoride. After stirring and mixing evenly, adjust the pH value to 5.5 with 0.1 mol / L citric acid, and then make up the volume to 1 L with nuclease-free water.

[0088] The prepared whole blood preservative solution is filtered through a 0.22-μm filter membrane and reserved for use.

[0089] Perform blood preservation experiments and performance tests on the whole blood preservative solutions prepared in Examples 1 - 20 and Comparative Example 2, and the conventional K3EDTA tube in Comparative Example 1 respectively for comparison.

[0090] The test method is as follows: (1)Judgment of the inhibition degree of preservative solution components Collect the blood of volunteers with a conventional K3EDTA tube. Immediately invert it 5 - 8 times after blood collection to mix evenly, and prepare platelet-poor plasma by secondary centrifugation. Take the platelet-poor plasma prepared with the K3EDTA tube, and only add the preservative solutions obtained in Examples 1 - 20 and Comparative Example 1 during extraction. Mix them according to the ratio of platelet-poor plasma:preservative solution = 7:1 as samples to judge whether the components in the preservative solution will inhibit miRNA detection.

[0091] In the inhibition degree test, we tested 9 targets including miR-let-7a-5p, miR-16-5p, miR-20a-5p, miR21-5p, miR-23a-3p, miR-103a-3p, miR-191-5p, miR-146a-5p, and miR-451a.

[0092] The test results of the inhibition degree of different preservative solutions on different targets in miRNA detection are shown in Table 1.

[0093] Table 1 Test results of the inhibition degree of different preservative solutions on different targets in miRNA detection

[0094] (2)Preservation performance test Collect the blood of volunteers with blood collection tubes added with the preservative solutions obtained in Examples 1 - 20 and Comparative Example 2 respectively. Immediately invert it 5 - 8 times after blood collection to mix evenly, take out 1 mL of whole blood and prepare platelet-poor plasma by secondary centrifugation as the sample on Day 0; place the remaining whole blood samples on a shaker at room temperature of 22°C at 100 rpm / min for transportation simulation test, and take samples at 3 days and 5 days respectively to prepare platelet-poor plasma on Day 3 and Day 5.

[0095] The plasma sample was serially diluted 5000-fold to be used as a sample for detecting plasma hemoglobin content. The commercial kit Human Hemoglobin ELISA Kit (abcam, ab157707) was used to detect the plasma hemoglobin content.

[0096] The commercial kit Maxwell® RSC miRNA from Tissue or Plasma and Serum Kits (Promega, AS1680) was used for plasma miRNA extraction.

[0097] The MiRXES system was used to detect three targets, miR-16-5P, miR-20a-5p, and miR-23a-3p, in the extracted nucleic acid.

[0098] The specific operation procedure is as follows: The components in the RT kit were pre-dissolved on ice in advance.

[0099] A reaction system was configured using a 15-μL RT system as shown in Table 2.

[0100] Table 2 RT reaction system

[0101] All experiments were carried out on ice. First, all components in the RT reaction system except template RNA were mixed to form RT Mix. 5 μL of each sample was added to an 8-well strip, and 10 μL of the prepared RT Mix was added to each reaction well. The 8-well strip was centrifuged briefly, shaken thoroughly to mix, centrifuged briefly again, and placed in a PCR instrument, and the reaction was carried out according to the program in Table 3 below: Table 3 RT reaction program

[0102] The components in the qPCR kit were pre-dissolved on ice in advance.

[0103] A reaction system was configured using a 15-μL qPCR system as shown in Table 4.

[0104] Table 4 qPCR reaction system

[0105] All experiments were carried out on ice. The RT product was diluted 10-fold to be used as the template for qPCR. 5 μL of each sample was added to a 384-well plate, and 10 μL of the prepared qPCR Mix (a mixture of other reactants except template cDNA) was added to each reaction well. The sealed 384-well plate was centrifuged and placed in a qPCR instrument, and the reaction was carried out according to the program in Table 5 below: Table 5 qPCR reaction procedure

[0106] The results of miRNA stability tests for samples with different preservation solutions and different preservation durations at room temperature are shown in Table 6.

[0107] Table 6 Results of miRNA stability tests for samples with different preservation solutions and different preservation durations at room temperature

[0108] The average Ct values of multi-target miRNA inhibition degree, hemoglobin content detection, and miRNA Ct values of preservation performance for the preservation solutions of Examples 1 - 20 and Comparative Examples 1 - 2 are shown in Table 7.

[0109] Table 7 Test results of the performance of the preservation solutions of Examples 1 - 20 and Comparative Examples 1 - 2

[0110] From the above results, it can be seen that when the cell fixatives are diazolidinyl urea (0.5% - 10%) and 5,5 - dimethylhydantoin (5% - 10%), better effects are achieved, which can simultaneously meet the following three effects: (1) The average miRNA Ct value of the inhibition degree is less than or equal to the Ct value of Comparative Example 1 (control group without preservation solution) + 1, indicating no inhibition of miRNA detection; (2) The hemoglobin concentration is less than or equal to 600 μg / mL, indicating stable red blood cells and no hemolysis after 5 - day storage at room temperature; (3) The free miRNA is stably preserved, and the average miRNA Ct value of the preservation stability is greater than or equal to the Ct value of Comparative Example 1 Day0 - 1, indicating stable free miRNA expression and no change after 5 - day storage at room temperature.

[0111] When diazolidinyl urea is used as the cell fixative and 15% is added, hemolysis occurs immediately after addition; when 0.1% is added, hemolysis occurs during storage at room temperature.

[0112] When 5,5 - dimethylhydantoin is used as the cell fixative and 15% is added, hemolysis occurs immediately after addition; when the addition is less than 5%, hemolysis occurs during storage at room temperature.

[0113] When imidazolidinyl urea is used as the cell fixative and 2.5% - 10% is added, it shows inhibition of miRNA detection; when 1% is added, hemolysis occurs during storage at room temperature; When formaldehyde and paraformaldehyde are used as the cell fixatives and 10% is added, the miRNA Ct value is not detected; when 1% is added, it shows inhibition of miRNA detection.

[0114] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A whole blood preservation solution for stably preserving free miRNA, characterized in that, It includes solutes in the following mass-volume fractions: a cell fixative, 0.5% - 5% of a cell membrane protectant, 3.8% - 10% of a nuclease inhibitor, 0.1% - 3% of a cell metabolism inhibitor, 0.04% - 0.5% of a reducing agent, and a pH buffer. The cell fixative is 0.5% - 10% of diazalkyl urea or 5% - 10% of 5,5-dimethylhydantoin.

2. The whole blood preservation solution for stably preserving free miRNA according to claim 1, characterized in that, The cell membrane protectant is selected from one of trehalose, glucose, sorbitol, rhamnose, and sucrose.

3. The whole blood preservative for stably preserving free miRNA according to claim 1, characterized in that, The nuclease inhibitor is selected from one of K3EDTA, sodium citrate, heparin, guanidine salts, SDS, urea, oxovanadyl ribonucleoside complex, and diatomaceous earth.

4. A whole blood preservative solution for stably preserving free miRNA according to claim 1, characterized in that, The cell metabolism inhibitor is selected from one of glyceraldehyde, sodium fluoride, and glyceraldehyde-3-phosphate.

5. A whole blood preservative solution for stably preserving free miRNA according to claim 1, characterized in that, The reducing agent is selected from one of dithiothreitol, thioglycerol, and tricarboxyethylphosphine.

6. The whole blood preservative solution for stably preserving free miRNA according to claim 1, wherein The pH buffer is one or both of sodium citrate and citric acid, adjusting the solution to a pH range of 5.0 - 6.

5.

7. A whole blood preservative solution for stably preserving free miRNA according to any one of claims 1-6, characterized in that It also includes a solvent, and the solvent is nuclease-free water.

8. A method for preparing the whole blood preservative solution according to claim 7, characterized in that, Add nuclease-free water to a container and vortex, then add a cell fixative: 0.5% - 10% of diazalkyl urea or 5% - 10% of 5,5-dimethylhydantoin; 0.5% - 5% of a cell membrane protectant, 3.8% - 10% of a nuclease inhibitor, 0.1% - 3% of a cell metabolism inhibitor, 0.04% - 0.5% of a reducing agent, stir and mix evenly, adjust the pH value to 5.0 - 6.5 with a pH buffer, and make up the volume with nuclease-free water to obtain a whole blood preservative solution.

9. The preparation method according to claim 8, wherein, The whole blood preservative solution is filtered through a 0.22 μm filter membrane.

10. Use of the whole blood preservative solution according to claim 7 in the preparation of a product for stably preserving whole blood and stably preserving free miRNA in whole blood.

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