A preservation solution for storing human cellular nucleic acid DNA and RNA in saliva samples at room temperature and at 60°C, and its preparation method.

Abstract

This invention relates to the field of biotechnology, specifically to a preservation solution and its preparation method for preserving human cellular nucleic acid DNA and RNA in saliva samples at both room temperature and 60°C. The preservation solution of this invention, capable of preserving human cellular nucleic acid DNA and RNA in saliva samples at both room temperature and 60°C, comprises the following components: 1,2-cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylpiperazine-2-ethanesulfonic acid, SDS, sodium chloride, ethanol, and Tween-20. This invention offers the following technical advantages: The saliva DNA & RNA preservation solution provided by this invention can stably preserve nucleic acid DNA in saliva samples at 37°C for at least 2 years. The saliva DNA & RNA preservation solution provided by this invention can stably preserve nucleic acid in saliva samples at 37°C for at least 30 days. The saliva DNA & RNA preservation solution provided by this invention can stably preserve nucleic acid in saliva samples at 60°C for at least 7 days, facilitating sample transport to third-party testing institutions for analysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a preservation solution and its preparation method for preserving human cellular nucleic acid DNA and RNA in saliva samples that can be stored at room temperature and at a high temperature of 60°C. Background Technology

[0002] Deoxyribonucleic acid (DNA) is the main chemical component of chromosomes. DNA can form genetic instructions to guide biological development and life functions. Ribonucleic acid (RNA) is the intermediate carrier of genetic information in cellular organisms, participating in protein synthesis and gene expression regulation. RNA is a long-chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds. Unlike DNA, RNA is generally a single-stranded long molecule, making it highly susceptible to degradation during extraction and preservation. Therefore, RNA extraction and preservation are challenging, requiring rigorous and accurate procedures to avoid specific or non-specific degradation. DNA and RNA samples play a crucial role not only in molecular biology research but also in medical identification, detection, and treatment. Therefore, the quality of DNA and RNA significantly affects the accuracy of test results. Currently, blood samples are often used for PCR amplification, identification, and detection of nucleic acids. However, using blood samples has some drawbacks. For example, blood can agglutinate after prolonged storage, affecting extraction efficiency; secondly, blood sampling is an invasive procedure, requiring skilled operators.

[0003] Saliva is a mixed fluid composed of salivary gland secretions, gingival crevicular fluid, and mucosal exudate. It stores a wealth of complex biological information about human oral microbiota, local oral tissues, and the diversity of DNA, RNA, and proteins. Extracted salivary DNA or RNA can be combined with numerous downstream studies, including PCR, Southern blotting, sequencing, and microarray analysis. Compared to blood, saliva is a relatively easy-to-collect and obtainable sample that is safe and non-invasive for the human body, reducing patient anxiety and making it more acceptable. Therefore, it can maximize the sampling scope for genetic research. However, because saliva contains substances such as amylase, mucin, and lysozyme, which accelerate the degradation of DNA or RNA in saliva, developing preservation solutions that prevent the degradation of nucleic acids in saliva is crucial.

[0004] Several existing patents involve the preservation of DNA or RNA in saliva. Patent CN112438253A, for example, uses a preservation solution containing preservatives to inhibit the growth and activity of bacteria, mold, and other microorganisms, extending the preservation time of DNA or RNA without the need for freezing. However, because of the added preservatives, caution is required when using it, as it may be harmful to the human body and should not come into contact with the skin.

[0005] Patent CN111020001A describes a solid preservation solution. In production, it is pre-preserved at the bottom of a saliva collection tube using high-pressure spraying. After sampling, it is rapidly dried under high pressure in a dust-free dryer, transforming the liquid saliva preservation solution into a solid saliva preservative that accumulates at the bottom of the collection tube. Its advantages include better protection against microbial contamination, preservation of DNA fragment integrity, and safety for users, preventing accidental ingestion by children, compared to liquid saliva preservation solutions. However, because it is a solid preservation solution, the sample needs to be high-pressure dried after sampling, requiring specific equipment, significantly increasing costs and complicating the sampling process, causing inconvenience for operators.

[0006] The saliva preservation solution described in patent CN106561631A incorporates DNA stabilizers, disodium EDTA, and tartaric acid; along with microbial inhibitors and penicillin, which both maintain cell morphology, preventing lysis, and inhibit microbial growth. This preservation solution primarily targets the integrity of oral epithelial cells and other human-derived cells in saliva to prevent DNA degradation. It can be stored long-term at room temperature but should not be used for low- or high-temperature preservation. Furthermore, subsequent nucleic acid extraction requires additional cell lysis to obtain the nucleic acids, increasing extraction time and procedures.

[0007] Patent CN114467919A discloses a preservation solution for nucleic acids in saliva that can be stored at both room temperature and low temperature, and remains liquid even at low temperatures, preventing degradation of the nucleic acids due to freeze-thaw cycles. However, this invention only states that its preservation solution is for room temperature and low temperature storage. In some special circumstances, such as during high-temperature transportation in summer when there are no vehicles available for refrigerated transport, the high temperature environment may cause degradation of the nucleic acids in saliva, and refrigerated transport increases costs. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a preservation solution and its preparation method for human cellular nucleic acid DNA and RNA in saliva samples that can be preserved at room temperature and at a high temperature of 60°C. When mixed with saliva samples, this preservation solution can rapidly lyse human cells in the saliva and inhibit bacterial and other microbial contamination. It can preserve human cellular DNA or RNA in saliva samples for extended periods at room temperature, maintaining a stable state and avoiding the various nucleic acid degradation risks that may occur during sample transport and testing when samples cannot be preserved at low temperatures.

[0009] In order to achieve the above objectives,

[0010] In a first aspect of the present invention, a preservation solution is provided that can preserve human cellular nucleic acid DNA and RNA in saliva samples at room temperature and at a high temperature of 60°C.

[0011] The preservation solution comprises the following components: 1,2-cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylpiperazine-2-ethanesulfonic acid, SDS, sodium chloride, ethanol, and Tween-20.

[0012] In the preservation solution,

[0013] The molar concentration of the 1,2-cyclohexanediaminetetraacetic acid is 0.1-10 M;

[0014] The molar concentration of the hydroxyethylpiperazine-2-ethanesulfonic acid is 0.01-1M;

[0015] The SDS content is 0.1%-20% v / v;

[0016] The sodium chloride content is 0.1%–1% w / v;

[0017] The percentage content of the ethanol is 5%-80% v / v;

[0018] The molar concentration of tween-20 is 0.5–1 M.

[0019] Furthermore, the pH of the preservation solution is 6.0-10.0.

[0020] Furthermore, the molar concentration of the 1,2-cyclohexanediaminetetraacetic acid (CDTA) is 1-6 M.

[0021] Furthermore, the molar concentration of the hydroxyethylpiperazine-2-ethanesulfonic acid is 0.01-0.5M.

[0022] Furthermore, the percentage content of the SDS is 0.1%-10% v / v.

[0023] Furthermore, the sodium chloride content is 0.2%–0.8% w / v.

[0024] Furthermore, the percentage content of the ethanol is 10%–70% v / v.

[0025] Furthermore, the molar concentration of the tween-20 is 0.5-0.8 M.

[0026] Furthermore, the saliva originates from the oral cavity of a mammal.

[0027] Furthermore, the RNA is viral RNA, oral microbial RNA, or human cell RNA.

[0028] Furthermore, the DNA is viral DNA, oral microbial DNA, or human cell DNA.

[0029] In a preferred embodiment of the present invention, the preservation solution contains the following components: 1,2-cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylpiperazine-2-ethanesulfonic acid, SDS, sodium chloride, ethanol, and Tween-20.

[0030] In the preservation solution,

[0031] The molar concentration of the 1,2-cyclohexanediaminetetraacetic acid is 2M;

[0032] The molar concentration of the hydroxyethylpiperazine-2-ethanesulfonic acid is 0.05 M;

[0033] The SDS content is 0.5% v / v;

[0034] The sodium chloride content is 0.5% w / v;

[0035] The percentage content of the ethanol is 20% v / v;

[0036] The molar concentration of the tween-20 is 0.5 M;

[0037] The pH of the preservation solution is 7.0.

[0038] In another preferred embodiment of the present invention, the preservation solution contains the following components: 1,2-cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylpiperazine-2-ethanesulfonic acid, SDS, sodium chloride, ethanol, and Tween-20.

[0039] In the preservation solution,

[0040] The molar concentration of the 1,2-cyclohexanediaminetetraacetic acid is 3M;

[0041] The molar concentration of the hydroxyethylpiperazine-2-ethanesulfonic acid is 0.1 M;

[0042] The SDS content is 1% v / v;

[0043] The sodium chloride content is 0.8% w / v;

[0044] The percentage content of the ethanol is 40% v / v;

[0045] The molar concentration of the tween-20 is 0.7 M;

[0046] The pH of the preservation solution is 7.5.

[0047] The preservation solution of the present invention has the following functions:

[0048] (a) To maintain the stability of viruses, oral microorganisms or human cellular nucleic acid DNA & RNA in saliva samples;

[0049] (b) Release DNA or RNA nucleic acids from saliva samples.

[0050] When using the preservation solution of the present invention, the volume ratio of the sample to the preservation reagent is 1:1.

[0051] In a second aspect of the invention, a method for preparing the preservation solution as described in the first aspect of the invention is provided.

[0052] The preparation method includes the following steps:

[0053] 1) The components of the first aspect of the present invention are mixed in the following order: sodium chloride, ethanol, 1,2-cyclohexanediaminetetraacetic acid (CDTA), SDS, and Tween-20. The pH is adjusted with the components in the hydroxyethylpiperazine-2-ethanesulfonic acid buffer solution, and the volume is adjusted with deionized water.

[0054] 2) Filtration yields the preservation solution.

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0056] The present invention has the following technical effects:

[0057] 1) This invention's preservation solution does not use toxic or hazardous preservatives such as guanidine salts or sodium azide. It utilizes a buffer system to maintain a stable pH, thereby extending the preservation time. Specific concentrations of solvents such as CDTA, SDS, and Tween-20 are used to lyse and bind various protein impurities, including lysins, in the sample, thus promoting nucleic acid stability. Existing technologies cannot simultaneously preserve DNA and RNA in saliva for extended periods. However, this invention, through optimization of the preservation solution formula, can preserve DNA in saliva for a longer time, as well as easily degradable RNA. This invention solves the problem of nucleic acid degradation caused by the inability to achieve low-temperature preservation due to transportation or other reasons. The preservation solution of this invention can stably preserve nucleic acids in saliva at 60°C for at least 7 days, facilitating sample transfer to third-party testing institutions for analysis.

[0058] 2) This invention offers a wide preservation temperature range, long preservation time, and high nucleic acid integrity. This is primarily reflected in the stable preservation of nucleic acids in saliva samples for at least 7 days at 60℃, facilitating sample transfer to third-party testing institutions. Furthermore, the nucleic acid preservation solution provided by this invention can also stably preserve DNA in saliva samples for at least 2 years at 37℃ and RNA in saliva samples for at least 30 days at 37℃, representing higher temperatures and longer preservation times than existing technologies.

[0059] 3) The main technical contribution of this invention lies in the optimization of the formula, specifically the selection of the types and concentrations of substances within it. The selected chemical substances must be within a certain concentration range to achieve long-term preservation of DNA and RNA in saliva samples; otherwise, the preservation solution cannot preserve DNA in saliva for 2 years or RNA for 1 month. Furthermore, substances such as CDTA, SDS, and Tween 20 bind to or cleave DNases, RNases, and other proteases in saliva samples, thereby preventing enzyme degradation of nucleic acids (DNA and RNA) and inactivating microorganisms in the saliva sample. Hydroxyethylpiperazine-2-ethanesulfonic acid and NaCl buffer the pH and ionic strength of the preservation solution, maintaining a relatively stable pH and ionic strength concentration within a certain range. A certain proportion of ethanol provides some protection for DNA and RNA. Attached Figure Description

[0060] Figure 1 This invention demonstrates the preservation effect of the saliva DNA & RNA preservation solution on nucleic acid DNA in saliva samples.

[0061] Figure 2 This invention demonstrates the short-term preservation effect of saliva DNA & RNA preservation solution and RNase-free water on nucleic acid and RNA in saliva samples.

[0062] Figure 3 This invention demonstrates the long-term preservation effect of the saliva DNA & RNA preservation solution on nucleic acid RNA in saliva samples. Detailed Implementation

[0063] The implementation schemes of the present invention will now be described in detail with reference to specific embodiments.

[0064] Example 1: Saliva Preservation Solution Formulation

[0065] In Example 1, two saliva sample preservation solutions, Preservation Solution No. 1 and Preservation Solution No. 2, were provided, with the following formulations:

[0066] Table 1. Saliva Preservation Solution Formulation

[0067]

[0068] The two preservation solutions are prepared using the same method, including the following steps:

[0069] 1) Mix the components in the following order: sodium chloride, ethanol, 1,2-cyclohexanediaminetetraacetic acid (CDTA), SDS, and Tween-20. Adjust the pH with the components in the hydroxyethylpiperazine-2-ethanesulfonic acid buffer solution and bring the volume to a final volume with deionized water.

[0070] 2) Filtration yields the preservation solution.

[0071] Example 2. Study on the stability of human DNA in saliva samples preserved with salivary DNA & RNA preservation solution

[0072] Experimental materials: saliva samples

[0073] Preservation solution: Saliva DNA & RNA preservation solutions No. 1 and No. 2 from Example 1

[0074] Experimental methods:

[0075] Saliva samples were provided by eight different volunteers, numbered 1-8. 2 mL of each saliva sample was placed in 2 mL of either the No. 1 or No. 2 saliva DNA & RNA preservation solution prepared in Example 1, mixed by inversion, and stored at 37°C. Saliva samples numbered 1-4 were preserved in preservation solution No. 1, and saliva samples numbered 5-8 were preserved in preservation solution No. 2.

[0076] Before extraction, shake well to mix. Use the magnetic bead saliva DNA extraction kit (Kangwei Century, catalog number CW2506) to extract DNA and RNA. Use a pipette to draw 350 μL of saliva DNA and RNA preservation solution into column 1 of the deep well plate. For specific operating steps and extraction procedures, please refer to the extraction kit instructions. Store the extracted nucleic acid samples at -20°C.

[0077] The CW2683 Human Genomic DNA Quantification Kit was used to perform qPCR quantitative analysis on nucleic acid samples extracted on days 1-14-30-6 months-12 months-24 months. The basic reaction system of 20 μL is as follows:

[0078] reagents 20μL reaction system 2*GoldStar TaqMan Mixture 10μL Primer Mix 3μL Template 4μL <![CDATA[ddH2O]]> 3μL

[0079] The qPCR reaction temperature conditions are as follows:

[0080]

[0081] Experimental results:

[0082] 1) qPCR reaction results from 8 different volunteers, such as Figure 1 As shown, the results indicate that the Ct values ​​for human genome quantification remained stable and showed no significant changes (p>0.05).

[0083] 2) Volunteer samples numbered 1 and 2 in preservation solution 1 and volunteer samples numbered 5 and 6 in preservation solution 2 were selected to conduct experiments on the preservation performance of the two saliva DNA & RNA preservation solutions on nucleic acids in saliva at different time periods. The results are shown in Table 2. The nucleic acid concentration of saliva samples stored in the two preservation solutions did not change significantly on day 0, month 1, month 6, month 12, month 18 and month 26.

[0084] The above results indicate that both preservation solutions 1 and 2 of Example 1 can preserve human nucleic acid DNA in saliva samples for at least 2 years.

[0085] Table 2. Preservation performance of two salivary DNA & RNA preservation solutions on nucleic acids in saliva at different time periods.

[0086]

[0087]

[0088] Example 3. Study on the stability of human RNA in saliva samples preserved with salivary DNA & RNA preservation solution

[0089] Experimental materials: saliva samples

[0090] Preservation solutions: Saliva DNA & RNA preservation solutions No. 1 and No. 2 from Example 1, and RNase-free water. Experimental method:

[0091] Experimental Group 1: Approximately 2 mL of fresh saliva from 4 volunteers was placed in 2 mL of the No. 1 saliva DNA & RNA preservation solution prepared in Example 1, vortexed to mix, and stored at 37°C. The saliva samples from the 4 volunteers in Experimental Group 1 correspond to... Figure 2 Numbers 1-4 in the list.

[0092] Control group: Approximately 2 mL of fresh saliva from one volunteer was placed in 2 mL of RNase-free water prepared in Example 1, vortexed to mix, and stored at 37°C. The saliva samples from volunteers in the control group corresponded to... Figure 2 Number 5 in the list.

[0093] Experimental Group 2: Approximately 2 mL of fresh saliva from 8 volunteers was placed in 2 mL of saliva DNA & RNA preservation solutions (No. 1 and No. 2) prepared in Example 1, vortexed to mix, and stored at 37°C. The saliva samples from the 8 volunteers in Experimental Group 2 correspond to... Figure 3 The saliva samples numbered 1-8 are as follows: saliva samples numbered 1-4 are preserved in preservation solution No. 1 of Example 1, and saliva samples numbered 5-8 are preserved in preservation solution No. 2 of Example 1.

[0094] Before extraction, shake well to mix. Use the magnetic bead saliva DNA extraction kit (Kangwei Century, catalog number CW2506) to extract DNA and RNA. Use a pipette to draw 350 μL of saliva DNA and RNA preservation solution into column 1 of the deep well plate. For specific operating steps and extraction procedures, please refer to the extraction kit instructions. Store the extracted nucleic acid samples at -20°C.

[0095] The CW3149 1.26*HyperProbe OneStep RT-qPCR Kit (UNG) was used to perform qPCR quantification analysis on RNA samples extracted on days 1-7-14. For details on the basic reaction system and reaction temperature conditions, please refer to the kit's instruction manual.

[0096] Experimental results:

[0097] The results are as follows Figure 2 As shown, in the control group, under the saliva preservation background without RNase, human cell nucleic acid RNA was extracted after 7 days. The qPCR results showed a significant increase in Ct value, indicating significant degradation of nucleic acid RNA. Similarly, human cell nucleic acid RNA was extracted from the salivary DNA & RNA preservation solution No. 1 in Example 1 of Experimental Group 1, and the results showed no significant change in Ct value (p > 0.05).

[0098] The results are as follows Figure 3 For samples in Experimental Group 2 preserved using saliva DNA & RNA preservation solutions No. 1 and No. 2 of Example 1, after 30 days of preservation at 37°C, the Ct value on day 30 was less than 0.5 compared to that on day 0 and day 15, indicating no significant difference (p > 0.05). The above results demonstrate that both saliva DNA & RNA preservation solutions can significantly protect nucleic acid RNA stability for at least 30 days at 37°C.

[0099] Example 4. Study on the stability of nucleic acids in saliva preserved with salivary DNA & RNA preservation solution at 60℃

[0100] Experimental materials: saliva samples

[0101] The experimental group used the No. 1 saliva preservation solution from Example 1 above.

[0102] Experimental methods:

[0103] Approximately 2 mL of fresh saliva from 8 volunteers was placed in 2 mL of the No. 1 saliva DNA & RNA preservation solution prepared in Example 1, vortexed to mix, and stored at 60°C. Nucleic acid extraction was performed on the saliva preservation solution stored at 60°C on day 0 and day 7, respectively.

[0104] Before extraction, shake well to mix. Use the magnetic bead saliva DNA extraction kit (Kangwei Century, catalog number CW2506) to extract DNA and RNA. Use a pipette to draw 350 μL of saliva DNA and RNA preservation solution into column 1 of the deep well plate. For specific operating steps and extraction procedures, please refer to the extraction kit instructions. Store the extracted nucleic acid samples at -20°C.

[0105] Experimental results:

[0106] In this embodiment, nucleic acid was extracted from the saliva background of the No. 1 saliva DNA & RNA preservation solution. Compared with the nucleic acid extracted on day 0, the nucleic acid concentration extracted on day 7 showed no significant change. These results indicate that the No. 1 saliva DNA & RNA preservation solution significantly protects the stability of nucleic acid and RNA for at least 7 days at 60°C.

[0107] Table 3. Preservation performance of saliva DNA & RNA preservation solution No. 1 on nucleic acids in saliva at 60℃

[0108]

[0109] 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 preservation solution for storing human cellular nucleic acid DNA and RNA in saliva samples at room temperature and at a high temperature of 60°C, characterized in that: The preservation solution is composed of the following components: 1,2-cyclohexanediaminetetraacetic acid (CDTA), hydroxyethylpiperazine-2-ethanesulfonic acid, SDS, sodium chloride, ethanol, and Tween-20. In the preservation solution, The molar concentration of the 1,2-cyclohexanediaminetetraacetic acid is 2-3 M. The molar concentration of the hydroxyethylpiperazine-2-ethanesulfonic acid is 0.05-0.1 M. The SDS content is 0.5%-1% v / v. The sodium chloride content is 0.5%–0.8% w / v. The percentage content of the ethanol is 20%-40% v / v. The molar concentration of the Tween-20 is 0.5–0.7 M. The pH of the preservation solution is 7-7.

5.

2. The preservation solution according to claim 1, characterized in that, The saliva comes from the oral cavity of mammals.

3. The preservation solution according to claim 1, characterized in that, The RNA is oral microbial RNA or human cell RNA, and the DNA is oral microbial DNA or human cell DNA.

4. The method for preparing the preservation solution according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Mix the components in the following order: sodium chloride, ethanol, 1,2-cyclohexanediaminetetraacetic acid (CDTA), SDS, and Tween-20. Adjust the pH with the components in the hydroxyethylpiperazine-2-ethanesulfonic acid buffer solution and bring the volume to a final volume with deionized water. 2) Filtration yields the preservation solution.

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