Urine total DNA (Deoxyribose Nucleic Acid) preserving fluid

Abstract

The invention belongs to the technical field of biological products, and particularly relates to a urine total DNA preservation solution. The urine total DNA preserving fluid is prepared from the following main components: a cationic surface active agent, a bacteriostatic agent, a saccharide protective agent and enzyme-free water. The preserving fluid can stably preserve genome DNA (gDNA) and circulating free DNA (cfDNA) in a urine sample for a long time at room temperature, or independently preserve the cfDNA. The preserving fluid is simple in component, simple and convenient to prepare and non-toxic. The DNA of a urine sample added with the preserving fluid is stable after being preserved at room temperature for at least 21 days (experimental verification), and the preservation requirement for a longer time (such as 30 days) can be expected to be met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological products, and particularly relates to a urine total DNA preservation solution. BACKGROUND

[0002] As an important source of non-invasive liquid biopsy, urine contains genomic DNA (gDNA) and cell-free DNA (cfDNA), which has significant clinical value in early tumor screening, genetic disease diagnosis and infectious disease monitoring. However, the urine environment contains highly active nucleases (such as DNase I) and unstable pH, which leads to rapid degradation of DNA. Studies have shown that the degradation rate of cfDNA in untreated urine is more than 50% at room temperature within 2 hours, and gDNA also undergoes severe fragmentation within 24 hours. Current technology mainly relies on cold chain transportation (2-8℃), but more than 30% of the world's regions are limited by the lack of cold chain infrastructure. In addition, repeated freezing and thawing processes can induce mechanical DNA breakage, significantly reducing detection sensitivity. Therefore, developing a technology that can stably preserve gDNA and cfDNA in urine samples at room temperature for a long time is the key to breaking through the bottleneck of clinical application of molecular diagnosis.

[0003] Existing commercial preservation schemes, such as reagents based on chaotropic salts or protease inhibitors, have obvious limitations. First, the single function is prominent: most products are designed for gDNA or cfDNA single target, and cannot be compatible with the simultaneous preservation of both types of DNA; second, the operation is complex and has biological risks: after adding the preservation solution, immediate freezing treatment is required, and toxic components such as sodium azide are included, increasing the risk of operation; third, the processing capacity of large volume samples is insufficient: to improve the detection rate, clinical needs to collect >40mL of urine, but existing preservation agents change the physicochemical properties of urine, resulting in a DNA adsorption loss rate of more than 35% during the centrifugal concentration process. In addition, chemical cross-linking type preservation agents (such as formaldehyde derivatives) can stabilize nucleic acids, but severely interfere with downstream PCR and sequencing results, making it difficult to meet the in vitro diagnostic standards. These defects highlight the urgent need for an innovative solution that can protect gDNA and cfDNA simultaneously, support room temperature stable storage, and be compatible with large sample concentration and purification.

[0004] For example, Chinese patent CN105524916B provides a DNA-like sample preservation diluent and its preparation method. The preservation solution provided by the patent is configured with DNA preservative as the main component. The main component is sodium azide and streptomycin, which are common preservative / bacteriostatic components in DNA preservation solution. Sodium azide is commonly used in blood preservation solution because there are more nutrients and more bacteria in blood than in urine. A stronger preservative is not needed in urine, and both sodium azide and streptomycin are highly toxic, and the preservation solution needs to be sterilized during preparation. For example, Chinese patent application CN107267500A provides a free DNA preservation solution and its preparation method and application. The main component for DNA preservation in this patent is imidazole alkyl urea, which mainly functions to fix cells and prevent the release of gDNA. Therefore, only free DNA can be preserved in this patent.

[0005] Recent studies have shown that cationic surfactants (such as CTAB) can embed the DNA phosphate backbone through electrostatic interaction to block enzyme cutting, and sugars (such as trehalose) can form a glassy structure to inhibit hydrolysis. However, the effect of single component application is limited: surfactants are easily inactivated in high ionic strength urine, and sugars alone cannot prevent the degradation of large fragments of gDNA. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the present application provides a urine total DNA preservation solution which can not only preserve cfDNA in urine alone, but also preserve total DNA (including gDNA and cfDNA) in urine at the same time.

[0007] The technical solution of the present application to solve the technical problems is as follows:

[0008] In the first aspect of the present application, a urine total DNA preservation solution is provided, comprising the following main components: cationic surfactant, bacteriostatic agent, sugar protective agent and enzyme-free water:

[0009] 1) Cationic surfactant: selected from any one or more of cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB) and ε-polylysine. Its functions include: for destroying cell membrane permeability, releasing intracellular gDNA; at the same time, through electrostatic interaction with negatively charged nucleic acids (gDNA and cfDNA), forming a complex, effectively preventing nuclease degradation, and facilitating subsequent centrifugal concentration. The mass percentage content of cationic surfactant in the preservation solution is 0.5%-5% (w / v);

[0010] 2) Bacteriostatic agent: to inhibit microbial growth in urine, avoid sample contamination and microbial DNA interference. Any one or more of ε-polylysine, isothiazolinones (e.g. ProClin 300), quaternary ammonium salts (e.g. benzalkonium chloride) can be selected. The mass percentage of bacteriostatic agent in the preservative solution is 0.5%-5% (w / v).

[0011] 3) Sugar protective agent: to prevent nucleic acid degradation. By forming a glassy structure to stabilize cell structure, buffer osmotic pressure, and provide mechanical protection for nucleic acid, further enhance the inhibition of hydrolytic degradation. Any one or more of trehalose, glucose, sucrose, etc. can be selected. The mass percentage of sugar protective agent in the preservative solution is 0.5%-5% (w / v), preferably the total mass percentage of sugar protective agent in the preservative solution is 5% (w / v).

[0012] (w / v). More preferably, trehalose and glucose are used, each with a mass percentage of 2.5% (w / v) in the preservative solution.

[0013]

[0014] 4) Enzyme-free water: to dissolve the components and provide a solution environment.

[0015] Preferably, the urine total DNA preservative solution of the present application comprises the following main components: ε-polylysine, trehalose, glucose, enzyme-free water, and the final concentration of ε-polylysine, trehalose and glucose in the preservative solution is 2.5% (w / v) each. Preferably, ε-polylysine with both surface activity and bacteriostatic effect is selected as the cationic surfactant and bacteriostatic agent, which can simplify the formula, reduce the cost and reduce potential toxicity. Using a mixture of trehalose and glucose as a sugar protective agent can provide more comprehensive protection.

[0016] Preferably, a metal chelator (such as EDTA) can be added in an appropriate amount in the urine total DNA preservative solution, with a concentration range of 10 mM-1 M, preferably 500 mM, to inhibit metal ion-dependent nuclease activity, and a small amount of buffer (such as Tris buffer) can also be added, with a concentration range of 10 mM-1 M, preferably 10 mM, to maintain the stability of the solution pH and optimize the preservation environment.

[0017] Preferably, methylene blue dye can be added to the urine total DNA preservative solution, with a mass percentage of 0.1%-1.5% (w / v), preferably 1% (w / v), which has the following effects: (1) as an indicator, it changes color after mixing with urine, making it easy to observe; (2) it has bacteriostatic ability, which cooperates to inhibit bacterial growth.

[0018] ​In a preferred embodiment of the present invention, the urine total DNA preservation solution comprises the following components: ε-polylysine, trehalose, glucose, and enzyme-free water, wherein the final concentrations of ε-polylysine, trehalose, and glucose in the preservation solution are all 2.5% (w / v). The most significant advantage of this preferred embodiment is that it requires only a minimum of three components—ε-polylysine, trehalose, and glucose—to meet the requirements for preserving urine DNA, and it can preserve either cfDNA or urine total DNA (cfDNA + gDNA) individually.

[0019] The urine total DNA preservation solution of the present invention is added to the preservation sample in the following ratio: urine:preservation solution = 1 mL: 100 μL.

[0020] In a second aspect of the present invention, a method for preparing a urine total DNA preservation solution as described in the first aspect is provided, the specific operation of which is as follows: dissolve the weighed components in enzyme-free water and mix them evenly.

[0021] In a third aspect of the invention, the use of the urine total DNA preservation solution as described in the first aspect in the preservation of urine total DNA, urine cfDNA or urine gDNA is provided.

[0022] The urine total DNA preservation solution of the present invention can be directly added to the original urine to preserve total DNA (gDNA+cfDNA).

[0023] The urine total DNA preservation solution of the present invention can also be used to preserve cfDNA alone: ​​after centrifuging the original urine (e.g., 3000×g, 15min) to remove the cell precipitate, take the supernatant and add it to the preservation solution.

[0024] In a fourth aspect of the invention, a urine preservation tube is provided, the tube containing a total urine DNA preservation solution as described in the first aspect.

[0025] In a fifth aspect of the invention, a kit for preserving urine at room temperature is provided, the kit comprising a preservation tube as described in the fourth aspect or a total urine DNA preservation solution as described in the first aspect.

[0026] The present invention has the following technical effects:

[0027] Addressing the technical bottlenecks of existing technologies, the inventors innovatively proposed a cationic surfactant-carbohydrate synergistic protection system. This system constructs dynamic protective micelles through a hydrogen bond network between the two: the surfactant provides primary electrostatic shielding, while the carbohydrate enhances three-dimensional spatial stability. This enables simultaneous and efficient preservation of urinary gDNA and cfDNA at room temperature for up to 14 days, while ensuring that the preserved samples can be directly centrifuged and concentrated. Furthermore, the components are simplified, non-toxic, and low-cost. This technology fills the gap in the field of urinary DNA preservation by providing a comprehensive solution encompassing "multi-target, cold chain-free, and concentration capabilities," offering crucial technical support for screening in remote areas and large-scale populations. The most significant feature of this invention is that it requires only a minimum of three components—ε-polylysine, trehalose, and glucose—to meet the requirements for preserving urinary DNA, and can preserve cfDNA or total urinary DNA (cfDNA + gDNA) separately. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0029] ε-polylysine, trehalose, and glucose were all purchased from Aladdin.

[0030] Example 1: Composition and Preparation of Preservative Solution

[0031] The preservation solution of this invention has a simplified formulation as follows: the components are ε-polylysine, trehalose, glucose, and enzyme-free water. The final concentration of all solute components (ε-polylysine, trehalose, and glucose) in the solution is 2.5% (w / v).

[0032] Preparation method: Dissolve the weighed components in enzyme-free water and mix well. Take 2.5g of ε-polylysine, 2.5g of trehalose, and 2.5g of glucose, dissolve them in 92.5mL of enzyme-free water, and mix well.

[0033] Example 2: Validation of the preservation solution's effectiveness (total DNA preservation)

[0034] Prepare the preservation solution according to the scheme in Example 1: Take 2.5g of ε-polylysine, 2.5g of trehalose and 2.5g of glucose, dissolve them in 92.5mL of enzyme-free water and mix well.

[0035] Verification method:

[0036] 1. Randomly collect 100mL of morning urine or second morning urine samples from 7 volunteers and divide them equally.

[0037] 2. Immediately add the prepared preservation solution (ratio: 1 mL urine : 100 μL preservation solution) to one sample and mix well. The other sample is left untreated (original urine control).

[0038] 3. Both samples were stored at room temperature.

[0039] 4. On day 0 and day 7 of storage, take 5 mL of equal quantum sample from each sample and extract total DNA using the Zymo Quick-DNAUrine Kit (D3061).

[0040] 5. Quantitative real-time PCR was used to detect the content of the human internal reference gene Line 1 in extracted DNA. The primer sequences and amplification conditions were referenced from patent CN113373031B. The amplification cycle threshold (Ct value) reflects the expression level of Line 1 in the sample, indirectly reflecting the relative DNA content; a lower Ct value indicates a higher initial DNA content. The differences in Ct values ​​between samples with preservation solution added at the same time point and primary urine were compared, as well as the changes in Ct values ​​of the same sample at different time points.

[0041] Experimental results:

[0042] The amplification Ct values ​​when the samples were stored for 0 days are shown in Table 1:

[0043] Table 1

[0044]

[0045]

[0046] The amplification Ct values ​​after 7 days of sample storage are shown in Table 2:

[0047] Table 2

[0048]

[0049] Note: Urine samples 4 and 5 were clearly contaminated with bacteria on day 7, and the extracted DNA could not be amplified (Ct value recorded as N / A). However, the corresponding samples with the preservation solution of this invention could still be successfully amplified.

[0050] Experimental conclusion:

[0051] The preservation solution of this invention can effectively protect total DNA in urine samples. After 7 days of storage at room temperature, the Ct values ​​of samples with the preservation solution were significantly lower than (samples 1, 2, 3, 6, 7) or close to (samples 4, 5) their day 0 levels, and far lower than those of untreated primary urine samples during the same period (Ct values ​​increased significantly). In contrast, untreated primary urine samples showed severe degradation (significantly increased Ct values) or bacterial inactivation within 7 days.

[0052] Example 3: Comparison of Long-Term Preservation Effects

[0053] To evaluate the long-term preservation effect of the preservation solution of the present invention (simplified formulation of Example 1), it was compared with commercially available urine preservation tubes (Jiangsu Kangjian Medical Supplies Co., Ltd., product number: KJ759-1, hereinafter referred to as "K preservation solution"). Urine samples from the seven volunteers in Example 2 (A: primary urine, B: primary urine + K preservation solution, C: primary urine + preservation solution of the present invention) were used and stored at room temperature. Samples were taken on days 0, 7, 14, and 21. Total DNA was extracted according to the method described in Example 2 (same batch of reagents and primers), and the Ct value of Line 1 was detected.

[0054] The experimental results are shown in Table 3-6:

[0055] Table 3

[0056]

[0057] Table 4

[0058]

[0059]

[0060] Table 5

[0061]

[0062] Table 6

[0063]

[0064]

[0065] Experimental conclusion:

[0066] 1. The preservation solution of this invention has excellent performance: During the 21-day preservation period, the Ct values ​​of samples treated with the preservation solution of this invention (Group C) remained highly stable. The average Ct value on day 21 (13.38) differed from the average Ct value on day 0 (12.86) by only 0.52, indicating that the total DNA preservation effect was excellent.

[0067] 2. Significantly superior to commercially available products: On day 7 of storage, the average Ct value of the preservation solution group of this invention (13.16) was significantly lower than that of the K preservation solution group (14.31). By day 14 and day 21, the Ct values ​​of almost all samples (group C) were lower than those of the corresponding K preservation solution samples (group B), and the advantage in average Ct value continued to widen (day 14: 13.00 vs 14.17; day 21: 13.38 vs 14.70).

[0068] 3. The original urine control group failed: The untreated original urine (Group A) was severely degraded, and the Ct value increased sharply over time. Some samples (4,5) failed to be detected early due to bacterial contamination (N / A).

[0069] The above examples verify the excellent preservation effect of the preservation solution of the present invention at room temperature for at least 21 days. Based on the stability of its protective mechanism (cationic surfactant-carbohydrate synergistic micelles) and the trends of existing experimental data, it is expected that the preservation solution of the present invention can stabilize urine DNA for 30 days or longer under reasonable storage conditions (such as avoiding extreme temperatures, light, and contamination).

[0070] Example 4: Comparison of the effects of different surfactants

[0071] Based on the simplified formulation in Example 1 (carbohydrates: trehalose 2.5% + glucose 2.5%), with the carbohydrate components fixed, preservation solutions were prepared using different cationic surfactants (all with a final concentration of 2.5%), namely the ε-polylysine group, the tetradecyltrimethylammonium bromide (TTAB) group, the dodecyltrimethylammonium bromide (DTAB) group, and the hexadecyltrimethylammonium bromide (CTAB) group.

[0072] Test method: Take 50 mL of fresh urine from two different volunteers (sample A and sample B), add an equal volume (5 mL) of the four preservation solutions described above, and mix well. Store the treated samples at room temperature for 7 days. After 7 days, extract total DNA according to the method described in Example 2 and detect the Ct value of Line 1.

[0073] The experimental results are shown in Table 7:

[0074] Table 7

[0075]

[0076]

[0077] Experimental conclusions: Under the same carbohydrate protection system and concentration, the preservation effects of different cationic surfactants were as follows: ε-polylysine (average Ct 15.42) > CTAB (average Ct 17.69) > DTAB (average Ct 19.86) > TTAB (average Ct 19.98). Among them, ε-polylysine showed significantly better performance than the other three, verifying its effectiveness as a preferred surfactant.

[0078] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A urine total DNA preservation solution, characterized in that, It contains the following main components: cationic surfactant, antibacterial agent, sugar preservative, and enzyme-free water; wherein: 1) Cationic surfactant: selected from any one or more of hexadecyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB) and ε-polylysine, and the mass percentage of cationic surfactant in the preservation solution is 0.5%-5% (w / v); 2) Antibacterial agent: selected from any one or more of ε-polylysine, isothiazolinones, and quaternary ammonium salts, with the antibacterial agent having a mass percentage of 0.5%-5% (w / v) in the preservation solution; 3) Sugar preservative: selected from any one or more of trehalose, glucose, sucrose, etc.; the mass percentage of sugar preservative in the preservation solution is 0.5%-5% (w / v).

2. The urine total DNA preservation solution according to claim 1, characterized in that, The urine total DNA preservation solution contains the following main components: ε-polylysine, trehalose, glucose, and enzyme-free water, wherein the final concentrations of ε-polylysine, trehalose, and glucose in the preservation solution are all 2.5% (w / v).

3. The urine total DNA preservation solution according to claim 1, characterized in that, The urine total DNA preservation solution contains a metal chelating agent at a concentration of 10 mM to 1 M; it also contains a buffer solution at a concentration of 10 mM to 1 M.

4. The urine total DNA preservation solution according to claim 1, characterized in that, The urine total DNA preservation solution contains methylene blue dye at a mass percentage of 0.1%-1.5% (w / v).

5. The urine total DNA preservation solution according to claim 1, characterized in that, The urine total DNA preservation solution is composed of the following components: ε-polylysine, trehalose, glucose and enzyme-free water, wherein the final concentrations of ε-polylysine, trehalose and glucose in the preservation solution are all 2.5% (w / v).

6. The urine total DNA preservation solution according to claim 1, characterized in that, The ratio of urine total DNA preservation solution to the preserved sample is: urine:preservation solution = 1 mL: 100 μL.

7. The method for preparing the urine total DNA preservation solution according to any one of claims 1-5, characterized in that, The specific steps are as follows: Dissolve the weighed components in enzyme-free water and mix thoroughly.

8. The use of the urine total DNA preservation solution as described in any one of claims 1-5 in the preservation of urine total DNA, urine cfDNA or urine gDNA.

9. A urine storage tube, characterized in that, The preservation tube contains the total urine DNA preservation solution as described in any one of claims 1-5.

10. A reagent kit for preserving urine at room temperature, characterized in that, The kit contains the preservation tube as described in claim 9 or the total urine DNA preservation solution as described in any one of claims 1-5.

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