A urine sample stabilizing protective solution and its application in biomarker detection

Abstract

The present invention relates to a urine sample stabilization protection solution and its application in biomarker detection, belonging to the technical field of application of urine in biomarker detection after storage and transportation at room temperature. The urine sample stabilization preservation solution is composed of physiological saline, preservatives, co-solvent stabilizers, inhibitor compositions and surfactants in a certain proportion; the inhibitor compositions are protease inhibitors, nuclease inhibitors and cell apoptosis inhibitors. The urine sample stabilization preservation solution is used for room temperature storage of urine samples, which can prevent the degradation of proteins, peptides and nucleic acids in urine under room temperature conditions, prevent enzyme inactivation, prevent cell lysis and inhibit the growth of microorganisms, maintain osmotic pressure and pH stability, and maintain the integrity and activity of cells and biomarkers; after adding the preservation solution, the urine sample is stored at room temperature for no less than 30 days to ensure the accuracy and reproducibility of marker detection.

Description

Technical Field

[0001] The invention relates to a urine sample stabilizing protective solution and application thereof in biomarker detection, and in particular to application of urine in biomarker detection after being stored and transported at room temperature. Background Art

[0002] Human urine is a biological fluid with a very complex composition, mainly composed of water, organic and inorganic compounds, bacteria and various types of cells, including red blood cells, white blood cells, prostate cells, kidney cells and urothelial cells. Cellular and non-cellular compounds can be released into urine directly or through glomerular filtration. Inorganic compounds are composed of various inorganic salts, uric acid, urea, glucose, creatinine, etc., and organic matter includes free DNA, RNA fragments, exosomes (EVs), proteins (such as microglobulin, transferrin, hemoglobin, human chorionic gonadotropin HCG, etc.), enzymes (such as glucosidase, urinary amylase, etc.), peptides and other metabolites. These components make urine an attractive multi-omics sample type, which may be used for genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiome analysis, especially the discovery and analysis of biomarkers therein, which can play an important role in the discovery, screening and diagnosis of various abnormalities or diseases.

[0003] Various components in urine have become detection indicators for biomarkers such as cellular components, cfDNA, glucosidase, microglobulin, transferrin, etc. For example, urine DNA can be used to study tumor-specific changes, including methylation, microsatellite composition disorders and point mutations, as well as the presence of viral DNA.

[0004] Liquid biopsy is a revolutionary method that has been gaining momentum in cancer research and care over the past few decades, and it has important advantages over tissue biopsies. Liquid biopsies can capture the heterogeneity within a tumor, while tissue biopsies can only provide a snapshot of the tumor at a specific location and at a specific time. Liquid biopsies can be obtained through minimally invasive (blood, cerebrospinal fluid) or completely non-invasive methods (urine, saliva, sputum), which provides the possibility of more frequent sampling and close monitoring of patients. Therefore, liquid biopsies are more advantageous for cancer screening. First, they may significantly affect the early detection and prognosis of cancer patients. Second, they are easy to monitor treatment response and third, they are easy to detect tumor recurrence. Urine as a liquid biopsy has attracted attention for non-invasive cancer diagnosis of different cancer types, including but not limited to urological cancers, breast cancer, and lung cancer.

[0005] Due to the non-invasive nature of urine collection, it is easy to collect even at home, eliminating the need to collect urine in hospitals and clinics. It can be collected repeatedly and does not require the limitations often associated with other liquid biopsy sample types, allowing for serial sampling and multi-omics analysis, making urine a promising emerging liquid biopsy sample.

[0006] However, the parameters before urine biopsy analysis, including collection, storage, and preservation, have not yet been clearly standardized. The content of biomarker proteins, enzymes, free nucleic acids, etc. in urine is low and easily degraded, which greatly affects the accuracy of the test results. Therefore, it is necessary to develop new urine stabilizing preservation fluids to improve the accuracy of urine biopsy. Summary of the invention

[0007] In order to overcome the defects in the prior art, the present invention provides a urine sample stabilization protection solution and its application in biomarker detection after storage and transportation at room temperature. The urine sample stabilization preservation solution is used for the room temperature storage of urine samples, which can prevent the degradation of proteins, peptides and nucleic acids in urine under room temperature conditions, prevent enzyme inactivation, prevent cell lysis and inhibit the growth of microorganisms, maintain osmotic pressure and pH stability, and maintain the integrity and activity of cells and biomarkers; after adding the preservation solution, the urine sample is stored at room temperature for no less than 30 days to ensure the accuracy and reproducibility of marker detection.

[0008] In order to solve the technical problem of the present invention, the technical solution proposed is: a stable preservation solution for urine samples, which is composed of physiological saline, preservatives, co-solvent stabilizers, inhibitor compositions and surfactants in a certain proportion; the inhibitor composition is a protease inhibitor, a nuclease inhibitor and a cell apoptosis inhibitor, and the components are composed in a certain proportion. The main components of the preservation solution are: 0.5-5% V / V of preservatives, 10-50% V / V of co-solvent stabilizers, 0.05-10mM of protease inhibitors, 0.005-0.75M of nuclease inhibitors, 0.05-5mM of cell apoptosis inhibitors, and 0.1-5% W / V of surfactants.

[0009] Preferably, the preservation solution is composed of the following:

[0010] Preservatives: Phenoxyethanol,

[0011] Co-solvent stabilizer: polyethylene glycol PEG 6000-8000, glycerol

[0012] Protease inhibitors: Nafamostat mesylate, Upamostat, DTT

[0013] Nuclease inhibitors: ammonium sulfate, K3-EDTA

[0014] Apoptosis inhibitor: ATA

[0015] Surfactant: Poloxamer 188

[0016] The rest was saline.

[0017] Preferably, the preservative is phenoxyethanol.

[0018] Preferably, the co-solvent stabilizer is a combination of polyethylene glycol and glycerol.

[0019] Preferably, the protease inhibitor in the inhibitor composition is one or more of nafamostat mesylate, Upamostat, and dithiothreitol (DTT).

[0020] Preferably, the nuclease inhibitor in the inhibitor composition is one or more of ammonium sulfate and EDTA.

[0021] Preferably, the cell apoptosis inhibitor in the inhibitor composition is one or more of aurintricarboxylic acid (ATA) and enricasin. The surfactant is polyoxypropylene polyoxyethylene copolymer (Poloxamer 188).

[0022] Preferably, the preservation solution is composed of the following:

[0023] Preservative: 0.5-5% V / V phenoxyethanol,

[0024] Co-solvent stabilizer: 0.5% W / V polyethylene glycol PEG 6000, 25% V / V glycerol

[0025] Protease inhibitors: 0.75-2.5 mM Nafamostat Mesylate, 0.05-1 mM Upamostat, 5-25 mM DTT

[0026] Nuclease inhibitors: 0.25-0.75M ammonium sulfate, 5-50mM K3-EDTA

[0027] Apoptosis inhibitor: 0.05-0.5mM ATA

[0028] Surfactant: 0.5% W / V Poloxamer 188

[0029] The rest was saline.

[0030] Preferably, the preservation solution is composed of the following:

[0031] Preservative: 5% V / V Phenoxyethanol,

[0032] Co-solvent stabilizer: 0.5% W / V polyethylene glycol PEG 6000, 25% V / V glycerol

[0033] Protease inhibitors: 0.75 mM Nafamostat Mesylate, 0.5 mM Upamostat, 5 mM DTT

[0034] Nuclease inhibitors: 0.5M ammonium sulfate, 5mM K3-EDTA

[0035] Apoptosis inhibitor: 0.5 mM ATA

[0036] Surfactant: 0.5% W / V Poloxamer 188.

[0037] The rest was saline.

[0038] Preferably, the stabilizing preservation solution for urine samples is used for the preservation of urine samples at room temperature, which can prevent the degradation of proteins, peptides and nucleic acids in urine under room temperature conditions, prevent enzyme inactivation, prevent cell lysis and inhibit the growth of microorganisms, maintain osmotic pressure and pH stability, and maintain the integrity and activity of cells and biomarkers; after adding the preservation solution, the urine sample is stored at room temperature for not less than 30 days to ensure the accuracy and reproducibility of marker detection.

[0039] Preferably, the preservation solution and urine are used in a volume ratio of 1 to 4; the protective effect of the preservation solution means that within 30 days at room temperature, the preservation solution can maintain the integrity of proteins, nucleic acids, and cells in urine, maintain the activity of enzymes, and inhibit the growth of bacteria.

[0040] The stable preservation solution of the present invention is mainly composed of a preservative, a co-solvent stabilizer, an inhibitor composition (a protease inhibitor, a nuclease inhibitor and a cell apoptosis inhibitor) and a surfactant in a certain proportion.

[0041] Preservatives are a large class of bactericidal and preservative compounds that can kill or inhibit the growth of microorganisms such as bacteria, molds, yeasts, etc. in urine. There are many types of preservatives, such as antibiotics such as penicillin, streptomycin, gentamicin, etc., and also include compounds such as chlorhexidine, salicylates, parabens, phenoxyethanol, and cationic quaternary ammonium salts. The present invention selects phenoxyethanol, which can inhibit bacteria without destroying cells and protein nucleic acid structures and does not affect enzyme activity, as a component of the preservative in the urine sample preservation solution.

[0042] Co-solvent stabilizers are a large class of substances that reduce the activity of hydrolases based on the principle of size exclusion, avoid protein degradation and enzyme inactivation, and also rely on their solubilizing properties to increase protein solubility and prevent precipitation denaturation and enzyme inactivation caused by protein aggregation. Many compounds have this function, such as dextran, hydroxyethyl starch, water-soluble starch, polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl pyrrolidone, glycerol, sugars, cellulose, etc. The present invention preferably selects a combination of polyethylene glycol and glycerol as a component of the co-solvent stabilizer in the urine sample storage solution.

[0043] Protease inhibitors can inhibit the degradation of proteins in urine and maintain the integrity and correctness of normal peptide chains. Protease inhibitors include PMSF, β-glycerophosphate, leupeptin, APROTININ, pepstantin, AEBSF-HCl, E64, etc. The present invention preferably selects a composition of nafamostat mesylate, Upamostat and dithiothreitol (DTT) as a component of the protease inhibitor in the urine sample preservation solution.

[0044] Nuclease inhibitors can inhibit the degradation of DNA and RNA in samples by nucleases, maintain the integrity of nucleic acid chains and the accuracy of test results. Nuclease inhibitors include a variety of compounds such as guanidine isothiocyanate, ammonium sulfate, RNAsin, EDTA, EGTA and other metal ion chelators. The present invention preferably selects a combination of ammonium sulfate and EDTA as a component of the nuclease inhibitor in the urine sample preservation solution.

[0045] Apoptosis inhibitors protect the integrity and stability of the morphological structure of various cells in urine, prevent apoptosis, and avoid mutation of the markers to be tested, which may affect the accuracy of the test. In the past, cells were mainly preserved with cross-linking fixatives such as paraformaldehyde, but the fixatives can cause irreversible denaturation of proteins and inactivation of enzymes, which are not suitable for the preservation of samples with proteins and enzymes as biomarkers. Therefore, the present invention can preserve cell morphology and protein structure while also preserving enzyme activity at room temperature, and add apoptosis inhibitors. Apoptosis inhibitors include Caspase inhibitors such as Z-VAD-FMK , Q-VD- OP , Ac-DEVD-CMK, IAPs proteins, vincristine and other plant extracts and some anti-tumor drugs, etc., from which the present invention preferably selects aurintricarboxylic acid (ATA) alone or in combination with enricasin as the component of the cell apoptosis inhibitor in the urine sample preservation solution.

[0046] Surfactants can prevent biomolecules from forming aggregates and producing precipitation or adhesion, which affects the sensitivity and accuracy of detection. Surfactants are divided into ionic, nonionic and zwitterionic types. In order to protect the stability of biomarkers, the present invention selects mild nonionic and zwitterionic types, including Tween, Triton X-100, nonylphenol polyoxyethylene ether NONIDET P-40, dodecyl-β-D-maltoside (DDM), etc. The present invention preferably selects mild surfactant polyoxypropylene polyoxyethylene copolymer (poloxamer 188) as the component of surfactant in urine sample preservation solution.

[0047] Beneficial effects:

[0048] The function of the urine sample protection solution prepared by the above-mentioned preferred components is to prevent the degradation of proteins and nucleic acids in urine, prevent enzyme inactivation, prevent cell lysis and prevent the growth of microorganisms, and maintain the integrity and activity of cells and biomarkers.

[0049] After adding the preservation solution, the urine sample is stored at room temperature for at least 30 days to ensure the accuracy and reproducibility of marker detection. During the storage period at room temperature, it can prevent the degradation of protein and nucleic acid in urine, prevent enzyme inactivation, prevent cell apoptosis and inhibit the growth of microorganisms.

[0050] Group A and Group B had basically no effect on NAGase activity, while Group C had a certain inhibitory effect on NAGase activity. Through the screening of urine sample preservation solutions containing three different preservatives, the inhibition rate of solution A on bacteria was 11.1%, the number of red blood cells and white blood cells decreased by 8.3% and 9.5%, respectively, and the NAGase activity decreased by 1.3%; the inhibition rate of solution B on bacteria was 29.3%, the number of red blood cells and white blood cells decreased by 18.2% and 10.5%, respectively, and the NAGase activity decreased by 1.4%; the inhibition rate of solution C on bacteria was 41.2%, the number of red blood cells and white blood cells decreased by 50% and 44.4%, respectively, and the NAGase activity decreased by 23.2%. After comprehensive comparison, solution A, i.e. phenoxyethanol, was selected as the preservative component of the urine sample preservation solution. Although C had the best antibacterial effect, it also significantly affected the number of cells, so it was not selected as the preservative component of the urine sample preservation solution.

[0051] Through Example 1 and Example 2, a combination of phenoxyethanol as a preservative and nafamostat mesylate, Upamostat, DTT, ammonium sulfate, EDTA, and ATA as inhibitors was obtained, and orthogonal tests of different composition concentrations were performed on the above-screened compositions to determine the optimal dosage.

[0052] The results of the detection of bacteria and cell counts showed that the four groups of STVW preservation solutions could inhibit bacterial growth within 30 days, with the T (-56.3%) and W (-52.2%) groups being the best, while the number of cells changed slightly, with the S (-9.95%) and T (-9.97%) groups showing the smallest changes ( Figure 5 ). All four groups of STVW played a role in maintaining the stability of urine samples, among which the preservative was best at a concentration of 5%, and the high concentration group of the inhibitor performed better than the low concentration group. However, considering comprehensive factors such as cost, a moderate concentration can be appropriately used to achieve the best effect, such as group A. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below in conjunction with the accompanying drawings.

[0054] Figure 1 Changes in urine cell and bacterial counts in group ABC ( / μL)

[0055] Figure 2Changes of NAGase activity in ABC group (U / L)

[0056] Figure 3 Changes of NAGase activity in DEF group (U / L)

[0057] Figure 4 PAGE electrophoresis of urine protein in three groups of DEF samples before and after storage

[0058] Figure 5 Changes in the number of bacteria and cells in four groups of STVW samples after 30 days of storage

[0059] Figure 6 Urine protein electrophoresis of four groups of STVW samples stored for 30 days

[0060] Figure 7 Changes of NAG activity in four groups of STVW samples after 30 days of storage

[0061] Figure 8 ctDNA content of four groups of STVW samples stored for 30 days

[0062] Fig. 9 Amplification curves of GAPDH gene in four groups of STVW samples before and after 30 days of storage DETAILED DESCRIPTION

[0063] Example 1 Protective effect of urine sample storage solutions composed of different preservatives on urine samples

[0064] Preparation of urine sample storage solutions composed of three different preservatives

[0065] Liquid A consists of: Preservative: 5% (V / V) phenoxyethanol,

[0066] Co-solvent stabilizer: 0.5% (W / V) PEG 6000, 25% (V / V) glycerol

[0067] Protease inhibitors: 0.75 mM Nafamostat Mesylate, 0.5 mM Upamostat, 5 mM DTT

[0068] Nuclease inhibitors: 0.5M ammonium sulfate, 5mM K3-EDTA

[0069] Apoptosis inhibitor: 0.5 mM ATA

[0070] Surfactant: 0.5% (W / V) Poloxamer 188

[0071] The composition of liquid B is as follows: 0.25% (V / V) methylisothiazolinone is used to replace 5% phenoxyethanol, and the rest is the same as liquid A.

[0072] The composition of liquid C is as follows: 5% phenoxyethanol is replaced by 1.75% (W / V) imidazolidinyl urea, and the rest is the same as liquid A.

[0073] Taking the preparation of 1000 mL of solution A as an example, the preparation method is briefly described as follows: 500 mL of normal saline is mixed with 250 mL of glycerol, 5 g of PEG 6000, 5 g of poloxamer 188 and 66 g of ammonium sulfate are weighed, added to the above mixture, and then 10 mL of 0.5 M K3-EDTA is added respectively, and then the volume is made up to 940 mL with normal saline, sterilized at high temperature and high pressure, and after being allowed to stand at room temperature, 5 mL of inhibitor mixture (DMSO solution of 0.15 M nafamostat mesylate, 0.1 M Upamostat and 0.1 MATA), 5 mL of 1 M DTT and 50 mL of phenoxyethanol are added to obtain solution A.

[0074] Fresh human urine samples were added with liquid A, liquid B, and liquid C in a volume ratio of 4:1, that is, 4 volumes of urine samples were added with 1 volume of preservation solution, and three groups of urine samples, group A, group B, and group C, were obtained. After being stored at 37 degrees on the same day and for 7 days, the following three indicators were tested twice: total bacterial count, cell count, and N-acetyl-β-D-glucosaminidase NAG activity. The detection methods and test results are briefly described as follows:

[0075] In this embodiment, a urine analyzer is used to detect the total bacterial count and cell count of fresh or stored urine to examine and compare the inhibitory effects of three different preservative components on microorganisms in urine samples and the stabilizing effects on cell integrity, so as to select the preservative component with the best effect. Figure 1 Note: AB C represent urine samples preserved with different preservatives, 0 represents fresh urine samples tested on the same day, 7 represents urine samples stored at 37 degrees for 7 days, among the three groups of urine samples, group C has better antibacterial effect than group B and group A, and group A has better cell protection effect than group B and group C. Figure 1 As shown, no bacterial growth was observed in the urine samples of Group A, Group B, and Group C after being stored at 37 degrees on the same day and for 7 days. The antibacterial effect of Group C was better than that of Group B and better than that of Group A. However, from the change in cell number, Group A was better than Group B and better than Group C. Comprehensively comparing, A is the preferred preservative component of the preservation solution. Although C has the best antibacterial effect, it also significantly affects the cell number. Therefore, it was not selected as the preservative component of the urine sample preservation solution.

[0076] This example uses a biochemical colorimetric method to detect the activity of N-acetyl-β-D-glucosaminidase NAG, in order to select the type of preservative that has a mild effect on NAG enzyme and has the least effect on its activity. N-acetyl-β-D-glucosaminidase is a high-molecular glycoprotein widely present in the human body, and is also an important lysosomal hydrolase. It is not filtered by the glomerulus in the urine circulation. When the renal parenchyma is damaged, the urine NAG increases. It is currently considered to be a more sensitive indicator of early kidney damage. N-acetyl-β-D-glucosaminidase can be used for auxiliary diagnosis of renal tumors. It is reported that the urine NAG of renal malignant tumors is significantly increased, and its level is related to the size of the tumor and the degree of renal parenchymal damage. For renal transplant patients, urine NAG determination can detect rejection reactions early. Generally, N-acetyl-β-D-glucosaminidase increases 1 to 3 days before the onset of various clinical indications, and NAG of diabetic nephropathy is significantly increased.

[0077] The detection principle of acetyl-β-D-glucosaminidase (NAG) activity is that N-acetyl-β-D-glucosaminidase catalyzes the reaction of substrate MPT-NAG to generate N-acetyl-BD-glucosaminidase and MPT. The absorbance of MPT at 340nm increases, and the rate of increase is determined by the concentration of N-acetyl-β-D-glucosaminidase. The content of N-acetyl-β-D-glucosaminidase (NAG) in the sample can be measured according to the standard curve. Using the N-acetyl-β-D-glucosaminidase (NAG) assay kit (MPT-NAG method) (Engene Biotech), the OD value is detected at 340nm by a microplate reader according to the method in the kit manual. By comparing the OD values ​​measured in fresh and stored urine samples, the effects of three different preservative components on enzyme activity can be determined, and the mildest preservative that has no effect on enzyme activity can be selected. Figure 2 Note: AB C represent urine samples stored with different preservatives, 0 represents fresh urine samples tested on the same day, 7 represents urine samples stored at 37 degrees for 7 days and tested. Among the urine samples of groups A and B, group A and B had no effect on NAGase activity, while group C had an inhibitory effect. Figure 2 As shown in the figure, the urine samples of groups A, B, and C were stored at 37 degrees on the same day and for 7 days. The results of NAGase activity test showed that groups A and B had basically no effect on NAGase activity, while group C had a certain inhibitory effect on NAGase activity.

[0078] Summary: Through the screening of urine sample preservation solutions containing three different preservatives, groups A and B had basically no effect on NAGase activity, while group C had a certain inhibitory effect on NAGase activity. The inhibition rate of solution A on bacteria was 11.1%, the number of red blood cells and white blood cells decreased by 8.3% and 9.5%, respectively, and the NAGase activity decreased by 1.3%; the inhibition rate of solution B on bacteria was 29.3%, the number of red blood cells and white blood cells decreased by 18.2% and 10.5%, respectively, and the NAGase activity decreased by 1.4%; the inhibition rate of solution C on bacteria was 41.2%, the number of red blood cells and white blood cells decreased by 50% and 44.4%, respectively, and the NAGase activity decreased by 23.2%. After comprehensive comparison, A, i.e., phenoxyethanol, was preferred as the preservative component of the urine sample preservation solution. Although C had the best antibacterial effect, it also significantly affected the number of cells, so it was not selected as the preservative component of the urine sample preservation solution.

[0079] Example 2 Protective effect of urine sample storage solutions composed of different inhibitors on urine samples

[0080] Preparation of urine sample storage solution for groups D, E, and F:

[0081] Solution D consists of 0.75 mM nafamostat mesylate, 0.5 mM ATA, 5% phenoxyethanol, 0.5% PEG 6000, 25% glycerol, 0.5% poloxamer 188, 0.5 M ammonium sulfate, 5 mM DTT, and 5 mM K3-EDTA in physiological saline.

[0082] Solution E is composed of Solution D plus 0.5 mM Upamostat and 2.5 mM Enlicasin

[0083] Solution F is composed of Solution D plus 0.5mM Upamostat (equivalent to Solution A)

[0084] Fresh human urine samples were taken and added with liquid D, liquid E and liquid F in a volume ratio of 4:1, that is, 4 volumes of urine samples were added with 1 volume of preservation solution to obtain three groups of urine samples, group D, group E and group F. After being stored at 37 degrees for 7 days on the same day, the following two indicators were tested twice: glucosidase NAG activity and urine protein.

[0085] Figure 3 Note: DEF represents urine samples stored with different inhibitors, 0 represents fresh urine samples tested on the same day, 7 represents urine samples stored at 37 degrees for 7 days, and the three groups of urine samples with DEF had no significant effect on NAG enzyme activity.

[0086] The method for detecting the activity of glucosidase NAG is the same as in Example 1. The detection results of groups D, E, and F are shown in Figure 3 After being stored at 37 degrees for 7 days, the three groups had little effect on the activity of NAGase. Figure 3 Changes in NAGase activity of urine samples from the DEF group after fresh storage and storage at 37°C for 7 days).

[0087] Urine protein detection: The polyacrylamide PAGE electrophoresis method is used to detect the urine protein content in the urine sample. The urine protein content of normal people is about 100 mg / L, mainly composed of albumin, a small amount of microglobulin, transferrin, etc., but the urine protein of kidney disease or some tumor patients increases to 150 mg / L, and the composition is diverse, such as immunoglobulin K chain (Bence Jones protein M), etc. When detecting biomarkers, it is very necessary to maintain the integrity of the protein in the urine sample. This embodiment uses electrophoresis to detect whether the fresh and stored urine protein (with albumin as the target protein for detection) is degraded or the content is reduced, so as to select inhibitors of anti-protein hydrolysis as components of the urine sample protection solution.

[0088] Figure 4 Note: DEF represents urine samples stored with different inhibitors, 0 represents fresh samples tested on the same day, 7 represents samples stored at 37 degrees for 7 days and tested. There is no significant difference in the PAGE electrophoresis of urine protein before and after storage in the three groups of DEF, indicating that all three groups have protective effects. The test results of groups D, E, and F are shown in Figure 4 After being stored at 37 degrees for 7 days, the three groups of DEF samples were concentrated and then subjected to PAGE electrophoresis. The results showed that all three groups of preservation solutions had a protective effect on urine protein, namely, anti-hydrolysis effect, and the protective effects of the three groups were similar without significant differences.

[0089] Summary: After screening three different inhibitor combinations for DEF, the three groups of inhibitors had no obvious effects on NAGase activity and urine protein content. For example, NAGase activity decreased by 0.69%, 1.99% and 0.52% respectively. Therefore, all of them can be used as inhibitor components. After comprehensive consideration, the best one is F, i.e. A liquid.

[0090] Example 3 Screening of preservative and inhibitor concentrations in urine sample storage solution

[0091] Prepare four groups of urine sample storage solutions: S, T, V, and W, with the following compositions (Table 1):

[0092] Table 1

[0093]

[0094] Note: Liquid A is for reference.

[0095] Prerequisite description: Through Example 1 and Example 2, phenoxyethanol is used as a preservative, and nafamostat mesylate, Upamostat, DTT, ammonium sulfate, EDTA, and ATA are used as inhibitor combinations. Orthogonal tests of different composition concentrations are performed on the above-screened compositions to determine the optimal dosage.

[0096] Fresh human urine samples were taken in a volume ratio of 4:1 and added with S solution, T solution, V solution and W solution, respectively, that is, 4 volumes of urine samples were added with 1 volume of preservation solution to obtain four groups of urine samples, namely, group S, group T, group V and group W. The samples were stored at 25 degrees for 30 days. During the storage period, samples were taken twice (day 0 and day 30) to detect the following five indicators: bacterial and cell counts, NAGase activity, urine protein, urine cfDNA content and total RNA content. The detection methods of the first three items were the same as those in Examples 1 and 2.

[0097] Figure 5 Note: STVW represents stored urine samples with different proportions of components, 0 represents fresh testing on the same day, 30 represents testing at 25 degrees for 30 days. The four groups of stored samples can inhibit bacterial growth within 30 days, and the T and W groups have the best inhibition, while the cell number changes slightly, with S and T groups being the best.

[0098] The results of the detection of bacteria and cell counts showed that the four groups of STVW preservation solutions could inhibit bacterial growth within 30 days, with the T (-56.3%) and W (-52.2%) groups being the best, while the number of cells changed slightly, with the S (-9.95%) and T (-9.97%) groups showing the smallest changes ( Figure 5 ).

[0099] Figure 6 Note: STVW represents stored urine samples with different proportions of components. 0 represents fresh testing on the same day, and 30 represents testing at 25 degrees for 30 days. The urine protein in the four groups of stored samples did not degrade within 30 days.

[0100] Figure 7 Note: STVW represents stored urine samples with different proportions of components, 0 represents fresh testing on the same day, 30 represents testing at 25 degrees for 30 days. The NAG activity of the four groups of stored samples decreased after 30 days, but the decrease between the groups was not significant.

[0101] The results of urine protein and NAG enzyme activity test showed that the urine protein of the four groups of STVW storage solutions did not degrade significantly after 30 days. Figure 6 ), the NAG enzyme activity changes were between 4.89-7.38U / L, and the absolute values ​​were within the standard range of 0.3-11.5U / L, with small differences among the groups ( Figure 7 ).

[0102] Figure 8 Note: STVW represents stored urine samples with different proportions of components, 0 represents fresh testing on the same day, 30 represents testing at 25 degrees for 30 days, and there is no significant decrease in ctDNA in the four groups of samples stored for 30 days.

[0103] DNA content, i.e., urine circulating free DNA (cfDNA), was detected by ultraviolet method. Fresh samples and samples stored at 25°C for 30 days were extracted using circulating free DNA (cfDNA) extraction kit (Qiagen) according to the instructions. cfDNA was then extracted by OD 260 The results of ultraviolet detection of nucleic acid content showed that the cfDNA of the four groups of STVW storage solution did not decrease significantly after 30 days, with the largest decrease of 3.31% in the W group. The S group only decreased by 0.69%, indicating that the four groups of STVW had a protective effect, namely, anti-DNA enzyme degradation ( Figure 8 ).

[0104] Fig. 9 Note: STVW represents stored urine samples with different proportions of components, 0 represents fresh testing on the same day, 30 represents testing at 25 degrees for 30 days, and there is no obvious change in the GAPDH gene amplification curves of the four groups of samples stored for 30 days, indicating that the preservation effect of the STVW group is equivalent and the RNA is stable and not degraded.

[0105] Total RNA content was determined by RT-PCR. Fresh samples and samples stored at 25°C for 30 days were used to extract total RNA using Trizol extraction reagent. The housekeeping gene GAPDH was amplified using an RT-PCR kit (Qiagen). The results of the ct values ​​of each group are shown in Table 2 and Fig. 9 ,The results showed that the preservation effects of the four STVW groups were equivalent, and the RNA was stable and not degraded.

[0106] Summary: All four groups of STVW played a role in maintaining the stability of urine samples. Among them, the preservative concentration of 5% was the best, and the high concentration group of the inhibitor performed better than the low concentration group. However, considering comprehensive factors such as cost, a moderate concentration can be appropriately used to achieve the best effect, such as group A.

[0107] Table 2 ct values ​​of GAPDH gene amplification in four groups of STVW samples before and after 30 days of storage

[0108]

[0109] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A stable storage solution for urine samples, characterized in that: The preservation solution is composed of: Preservative: 0.5-5% V / V phenoxyethanol, Co-solvent stabilizer: 0.5% W / V polyethylene glycol PEG 6000 and 25% V / V glycerol, Protease inhibitors: 0.75-2.5 mM Nafamostat Mesylate, 0.05-1 mM Upamostat and 5-25 mM DTT, Nuclease inhibitors: 0.25-0.75M ammonium sulfate and 5-50mM K3-EDTA, Apoptosis inhibitor: 0.05-0.5mM ATA, Surfactant: 0.5% W / V Poloxamer 188, The rest was saline.

2. The stable storage solution for urine samples according to claim 1, characterized in that: The preservation solution is composed of: The preservation solution is composed of: Preservative: 5% V / V Phenoxyethanol, Co-solvent stabilizer: 0.5% W / V polyethylene glycol PEG 6000 and 25% V / V glycerol, Protease inhibitors: 0.75 mM Nafamostat Mesylate, 0.5 mM Upamostat and 5 mM DTT, Nuclease inhibitors: 0.5 M ammonium sulfate and 5 mM K3-EDTA, Apoptosis inhibitor: 0.5 mM ATA, Surfactant: 0.5% W / V Poloxamer 188, The rest was saline.

3. The use of the stable preservation solution for urine samples according to claim 1, characterized in that: The volume ratio of preservation solution to urine was 1:4.

Images