Urine free DNA preservative and urine preservation tube

Abstract

The present application relates to a urine free DNA preservative and a urine preservation tube containing the same, the urine free DNA preservative is a Tris-HCl buffer solution containing the following mass concentration of substances: 8% to 11% of an anti-cell coagulation agent, 1.5% to 25% of a cell fixing agent, 7.5% to 11% of a nuclease inhibitor, 0.5% to 1.5% of a cell metabolism inhibitor, 1% to 5% of a free radical scavenger; the cell anticoagulant is dextran, the cell fixing agent is one or more of imidazole alkyl urea and diazimidazole alkyl urea, the nuclease inhibitor is dipotassium ethylenediaminetetraacetate, the cell metabolism inhibitor is sodium fluoride, and the free radical scavenger is glutathione. The urine preservative provided by the present application can preserve the urine sample at room temperature (25 DEG C) for at least 7 days, and the dosage of the preservative is less than that of the urine sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical testing technology, in particular to a urine free DNA preservative and a urine preservation tube. BACKGROUND

[0002] In recent years, domestic and foreign have successively reported the extraction of free DNA from various types of urine samples. Researchers have extracted DNA of Mycobacterium tuberculosis from urine of patients with pulmonary tuberculosis, Y chromosome from urine samples of pregnant women with male fetuses, and relevant mutant genes from urine of a large number of patients with urinary system diseases, so that the DNA in urine as a biomarker to study various diseases caused by gene mutation has become a demand. Urine samples are easy to obtain, convenient to operate, and belong to non-interventional sample collection mode. Urine is a liquid containing a large amount of metabolic end products excreted through the bladder, and the salt concentration, pH value and specific gravity indexes of the human body are quite different due to the differences between individuals, so the environment of DNA is complex and is prone to degradation. At the same time, urine contains a large number of bacteria, which can easily breed after being isolated, so the urine sample for DNA extraction needs to be separated and extracted within 2 hours after collection.

[0003] However, the actual sample size is large or the detection conditions do not allow, and it cannot be guaranteed that all samples can be processed within 2 hours. In order to facilitate the collection, transportation and preservation of urine, and to provide a stable environment for the exfoliated cells and free DNA in urine, a preservative capable of preserving urine at room temperature needs to be developed. SUMMARY

[0004] To solve the above technical problems, the present application provides a urine free DNA preservative, which is a Tris-HCl buffer solution containing the following substances with the following mass concentrations: 8% to 11% of an anti-cell coagulant, 1.5% to 25% of a cell fixative, 7.5% to 11% of a nuclease inhibitor, 0.5% to 1.5% of a cell metabolism inhibitor, and 1% to 5% of a free radical scavenger.

[0005] Among them, the mass concentrations of each substance are: 8% of an anti-cell coagulant, 15% of a cell fixative, 9.25% of a nuclease inhibitor, 1.5% of a cell metabolism inhibitor, and 5% of a free radical scavenger.

[0006] Among them, the cell anticoagulant is dextran.

[0007] Among them, the cell fixative is one or more of imidazole alkyl urea and diazo imidazole alkyl urea.

[0008] Among them, the cell fixative is imidazole alkyl urea and diazo imidazole alkyl urea, and the ratio is 2:1.

[0009] The nuclease inhibitor is dipotassium ethylenediaminetetraacetate.

[0010] The cell metabolism inhibitor is sodium fluoride.

[0011] The free radical scavenger is glutathione.

[0012] A urine preservation tube containing the urine free NDA preservative.

[0013] The volume of the preservative is A, the total volume of the liquid after the urine is added to the preservation tube is B, and (A / B)*100% is greater than or equal to 2%.

[0014] The urine preservative provided by the application can preserve urine samples at room temperature (25 DEG C) for at least 7 days, and the amount of the preservative is less than that of the urine sample. BRIEF DESCRIPTION OF DRAWINGS

[0015] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0016] Figure 1 is a DNA electrophoresis map of a urine sample group in the verification test one of the application.

[0017] Figure 2 is a DNA electrophoresis map of a urine sample group in the verification test two of the application. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the application will be described clearly and completely below in combination with the drawings in the embodiments of the application. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the application, but the application can also be implemented in other ways different from those described herein, and a person skilled in the art can make similar generalizations without departing from the connotation of the application, so the application is not limited by the specific embodiments disclosed below.

[0019] The application provides a urine free DNA preservative and a urine preservation tube containing the preservative.

[0020] The preservative contains the following components with the following mass concentrations:

[0021] 8% to 11% of an anti-cell coagulation agent, 1.5% to 25% of a cell fixing agent, 7.5% to 11% of a nuclease inhibitor, 0.5% to 1.5% of a cell metabolism inhibitor, and 1% to 5% of a free radical scavenger.

[0022] The remaining component is 1.25 mol / L Tris-HCl buffer.

[0023] The pH of the preservative is 7.5, and HCL is used to adjust the pH.

[0024] The preparation method is that Tris is dissolved in purified water, and the pH is adjusted to 7.5 by using HCl to prepare a Tris-HCl solution; each component is added to the buffer solution respectively, and finally the pH is adjusted to 7.5, and then filtration is performed to obtain the buffer solution.

[0025] The use amount of the urine preservation tube in-situ preservative provided by the application follows the following principles:

[0026] The volume of the preservative is A, the total volume of the liquid after the urine is added into the preservation tube is B, and then (A / B)*100%≥2%.

[0027] Example 1

[0028] Preservative components:

[0029] Anti-cell coagulation agent: dextran (mass concentration 8%);

[0030] Cell fixation agent: imidazole alkyl urea (mass concentration 10%), diazo imidazole alkyl urea (mass concentration 5%);

[0031] Cell metabolism inhibitor: sodium fluoride (mass concentration 1.5%);

[0032] Free radical scavenger: glutathione (mass concentration 5%);

[0033] Nuclease inhibitor: ethylenediaminetetraacetic acid dipotassium (mass concentration 9.25%);

[0034] The remaining component is 1.25 mol / L Tris-HCl buffer solution.

[0035] The pH of the protective agent is 7.5.

[0036] Example 2

[0037] Preservative components:

[0038] Anti-cell coagulation agent: dextran (mass concentration 9%);

[0039] Cell fixation agent: imidazole alkyl urea (mass concentration 1.5%);

[0040] Cell metabolism inhibitor: sodium fluoride (mass concentration 0.5%);

[0041] Free radical scavenger: glutathione (mass concentration 1%);

[0042] Nuclease inhibitor: ethylenediaminetetraacetic acid dipotassium (mass concentration 7%);

[0043] The remaining component is 1.25 mol / L Tris-HCl buffer solution.

[0044] Protectant pH 7.5.

[0045] Example Three

[0046] Preservative components:

[0047] Anti-cellular coagulant: Dextran (mass concentration 11%);

[0048] Cell fixation agent: Imidazole alkyl urea (mass concentration 25%);

[0049] Cell metabolism inhibitor: Sodium fluoride (1%);

[0050] Free radical scavenger: Glutathione (mass concentration 2.5%);

[0051] Nuclease inhibitor: Ethylenediaminetetraacetic acid dipotassium (mass concentration 11%);

[0052] The remaining component is: 1.25 mol / L Tris-HCl buffer.

[0053] Protectant pH 7.5.

[0054] Verification Test One

[0055] 1. Cell preservation effect test:

[0056] The protectant (0.2 ml) in the above examples 1 to 3 was added to the collection tube, and vacuumed to a suction volume of 9.8 ml, and the morning urine samples of volunteers A and B were collected, and the samples of each volunteer were respectively loaded into the collection tubes corresponding to examples 1-3, and each example was 6 tubes.

[0057] The above collection tubes containing samples (a total of 36) were stored at room temperature (25°C) for 0 days, 4 days, and 7 days, and the DNA of the samples was extracted and tested at each corresponding time point, and the nucleic acid concentration was tested, and two parallel tests were taken.

[0058] Sample processing: 1800g, 4°C centrifugation for 10min, separation of urine cells, DNA extraction was performed according to the requirements of the nucleic acid extraction and purification kit, Quawell Q5000 ultramicro ultraviolet spectrophotometer was used to determine the nucleic acid concentration, and gel electrophoresis analysis was performed.

[0059] The results are shown in Figure 1 and Table 1;

[0060] Figure 1 a and Figure 1 b are the electrophoresis results of volunteer A, wherein lanes 1-3 are the test results of the preservation tubes corresponding to examples 1-3 at 0 days, lanes 4-6 are the test results of the preservation tubes corresponding to examples 1-3 at 4 days, and lanes 7-9 are the test results of the preservation tubes corresponding to examples 1-3 at 7 days.

[0061] Figure 1 c and Figure 1 d is the electrophoresis result of volunteer B, wherein lanes 1-3 are the test results of the preservation tubes corresponding to examples 1-3 at 0 days, lanes 4-6 are the test results of the preservation tubes corresponding to examples 1-3 at 4 days, and lanes 7-9 are the test results of the preservation tubes corresponding to examples 1-3 at 7 days.

[0062] Figure 1 a- Figure 1 d The leftmost lane is a marker.

[0063] Table 1: Nucleic acid concentration test results of verification test one

[0064]

[0065] Table 1 combines Figure 1 a- Figure 1 The results of d show that the extracted nucleic acid concentration did not change significantly, indicating that no cell lysis and release of genomic DNA occurred after normal temperature preservation. The 0-day data can be understood as a control.

[0066] 2. Free DNA preservation effect determination:

[0067] The protective agent (0.2 ml) in the above examples 1 to 3 was added to the collection tube, and vacuumed to a suction volume of 9.8 ml. Morning urine samples of volunteers A and B were collected, and each sample of each volunteer was placed in the collection tube corresponding to examples 1-3, and each example had 6 tubes, and two parallel samples were taken.

[0068] Each collection tube was added with 1000 ng / 10 ml urine of genomic reference gene GAPDH fragment (Shanghai Sunway Biotech), and placed at room temperature. The samples were stored at room temperature (25°C) for 0 days, 4 days and 7 days, respectively. At each time point, the corresponding samples were taken for DNA extraction, and the free DNA (GAPDH) fragment was subjected to fluorescence quantitative analysis.

[0069] Sample pretreatment conditions: 1800g, 4°C centrifugation for 10 min, separation of supernatant into a new centrifuge tube, and further 10000g, 4°C centrifugation of the supernatant for 5 min to obtain the pretreated urine. The cfDNA in the urine was extracted by a free DNA extraction kit (CWBIO CWY007S), and the fluorescence quantitative analysis was performed by a PCR instrument.

[0070] Fluorescence quantitative analysis conditions: The extracted free DNA was used as a template, and the added DNA fragment GAPDH was selected for fluorescence quantitative PCR to determine the remaining amount of added GAPDH in the urine sample, and the results are shown in Table 2.

[0071] Primer:

[0072] GAPDH-F: GGACTGAGGCTCCCAC;

[0073] GAPDH-R: GCATGGACTGTGGTCTGCAA.

[0074] PCR conditions:

[0075]

[0076]

[0077] Table 2 Verification Experiment 1 Free DNA Assay Results

[0078]

[0079] Results: The smaller the Cp value, the more cfDNA, and vice versa.

[0080] Verification Test 2

[0081] During the inventor's experiments, it was found that the preservation solution provided by the application can well preserve urine samples at 45°C for 36 hours within a certain period of time. This function can be applied to remote county or township medical institutions that need to be sent for inspection, and can to some extent reduce the problem of sample temperature rise and cfDNA degradation or cell lysis releasing genomic DNA affecting the test results due to delayed express delivery.

[0082] The protective agent (0.2 ml) in the above Examples 1 to 3 was added to the collection tube, and vacuumed to a suction volume of 9.8 ml. The morning urine sample of volunteer C was collected, two tubes were collected for each example, and two parallels were taken. 1000 ng / 10 ml urine of genomic reference gene GAPDH fragment (Shanghai Biotechnology) was added to each collection tube. After three hours of warming to 45°C, gel electrophoresis, nucleic acid concentration and Cp value determination were performed at 24h, 36h and 48h after warming.

[0083] The determination method is the same as Verification Test 1.

[0084] The test results are shown in Figure 2 a, Figure 2 b, Figure 2 c and Tables 3 and 4.

[0085] Figure 2 In lane 1-3 of a, the three tubes of Examples 1-3 correspond to the samples at 24h (the first parallel group), lanes 4-6 are the 24h corresponding samples of the three tubes of Examples 1-3 (the second parallel group), and the rightmost lane is the marker.Figure 2 b is the result of 36h, Figure 2 c is the result of 48h, the lane sample corresponds to Figure 2 a.

[0086] The first group of parallel samples were detected at room temperature for 4 days before heating as a control:

[0087] The nucleic acid concentrations of Examples 1-3 were 143.42 ng / ul, 151.03 ng / ul, and 147.76 ng / ul, respectively.

[0088] The Cp values of Examples 1-3 were 14.24, 15.74, and 14.59, respectively.

[0089] Table 3 Verification Test Two Nucleic Acid Concentration Test Results

[0090]

[0091] Table 4 Verification Test Two Free DNA Determination Results

[0092]

[0093] As shown in Tables 3 and 4, the preservatives of the three examples can maintain intact nucleated cells and prevent degradation of free DNA at 45°C for 36h. However, at 48h, the nucleated cells of Example 3 sample were broken and released DNA, resulting in a decrease in the Cp value of free DNA. The amount of DNA in Example 2 sample changed little, but the Cp value of free DNA increased, and the degradation of free nucleic acid in the sample was obvious.

[0094] As shown in Figure 2, Figure 2 c, it can be seen that the bands of lanes 3 and 6 are dim at 48h, which proves that the amount of DNA in the cells decreases and that the nucleated cells are broken.

[0095] For the verification at 45°C, the remaining four volunteers were also selected for testing, and each volunteer collected three samples corresponding to the three time points for the above tests. Due to space limitations, only the corresponding data are listed, and the details are shown in Tables 5 and 6.

[0096] Table 5 Verification Test Two Nucleic Acid Concentration Test Results of Volunteers D-G

[0097]

[0098] Table 6 Verification Test Two Free DNA Determination Results of Volunteers D-G

[0099]

[0100] In combination with the data of the verification test, the preservative and the preservation tube using the preservative can prolong the preservation period by 36 hours after a temperature rise after being stored at room temperature for a period of time.

[0101] Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

Claims

1. A urine cell-free DNA preservative, characterized by, Tris-HCl buffer solution containing one of the following (1)-(3) in a mass concentration of 8% of an anti-cell coagulant, 15% of a cell fixative, 9.25% of a nuclease inhibitor, 1.5% of a cell metabolism inhibitor, 5% of a free radical scavenger, the cell fixative being imidazole alkyl urea and diazimidazole alkyl urea; (2) 9% of an anti-cell coagulant, 1.5% of a cell fixative, 7% of a nuclease inhibitor, 0.5% of a cell metabolism inhibitor, 1% of a free radical scavenger, the cell fixative being imidazole alkyl urea; (3) 11% of an anti-cell coagulant, 25% of a cell fixative, 11% of a nuclease inhibitor, 1% of a cell metabolism inhibitor, 2.5% of a free radical scavenger, the cell fixative being imidazole alkyl urea; Wherein: the cell anticoagulant is dextran; the free radical scavenger is glutathione; the urine free DNA preservative is stored in a urine storage tube; the volume of the preservative is A, the total volume of the liquid after adding urine to the storage tube is B, then (A / B)*100% = 2%. the nuclease inhibitor is ethylenediaminetetraacetic acid disodium salt.

2. The urine cell-free DNA preservative of claim 1, wherein, the cell metabolism inhibitor is sodium fluoride. 3.The urine cell-free DNA preservative of claim 1 or 2, characterized in that, ​

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