Preservation reagent for nucleic acid in saliva sample, preparation method and detection kit

Abstract

The invention relates to the technical field of medicine detection preservation reagents, in particular to a preservation reagent for nucleic acid in a saliva sample, a preparation method and a detection kit. The invention relates to a preservation reagent for nucleic acid in a saliva sample, which comprises the following components: 1.0-5.0% (w / v) of PEG (polyethylene glycol) modified branched chain polyethyleneimine, 0.1-1.0 mM of EDTA (ethylene diamine tetraacetic acid), 0.02-0.1% (w / v) of an antibacterial agent, 10-50 mM of a buffer system, 1.0-5.0% (v / v) of an osmotic pressure regulator, 0.1-0.5% (w / v) of an antioxidant and pure water as a solvent, and the pH value of the solution is stabilized at 7.5-8.0. According to the reagent, PEG-modified branched polyethyleneimine is introduced, and EDTA, an antibacterial agent, a buffer system, an osmotic pressure regulator and an antioxidant are combined, so that a multifunctional comprehensive protection system is formed. The stability and the reliability of nucleic acid preservation are improved, the biocompatibility and the environmental friendliness are also considered, and an efficient and innovative solution is provided for the preservation of saliva sample nucleic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical detection preservation reagents, and in particular to a nucleic acid preservation reagent in a saliva sample, a preparation method and a detection kit. Background Art

[0002] With the rapid development of molecular biology and genomics technologies, nucleic acid detection technology has become increasingly important in medical diagnosis, disease monitoring and scientific research. As a non-invasive biological sample, saliva has gradually become an important source for nucleic acid testing due to its convenient collection, non-invasiveness and high patient acceptance. However, the preservation and stability of nucleic acids in saliva samples face many challenges, especially during sample collection, storage and transportation.

[0003] The detection of saliva samples has the following advantages: 1. The collection of saliva samples does not require acupuncture or surgery, which reduces the pain and psychological burden of patients and is particularly suitable for children, the elderly and special populations. 2. Saliva samples can be collected by simple gargling or sucking, without the need for professional medical personnel, which is convenient for large-scale screening and home testing. 3. Saliva contains rich DNA, RNA, proteins and metabolites, which can reflect the health status and disease information of individuals and have extensive research and application value. At the same time, due to the presence of a variety of nucleases in saliva, it is easy to cause the degradation of DNA and RNA, affecting the accuracy of the test results; in addition, saliva samples are susceptible to contamination by oral microorganisms, which not only interfere with nucleic acid detection, but also may cause nucleic acid degradation; finally, the nucleic acid stability of saliva samples requires high storage conditions. Factors such as temperature, pH, and ionic strength will affect the integrity of nucleic acids, increasing the difficulty of sample storage and transportation.

[0004] Therefore, the protection and extraction of nucleic acids in saliva samples face many challenges. Although traditional methods can alleviate these problems to a certain extent, they still have obvious limitations. PEG-modified branched polyethyleneimine (Chinese invention patent applied for by the applicant, application number: 2024108232852) is a new type of nucleic acid protection reagent. Through its unique molecular structure and multifunctional protection mechanism, it provides an innovative solution for the protection and extraction of nucleic acids in saliva samples. Through further research and optimization of this application, PEG-modified branched PEI is expected to play an important role in biomedical research and clinical diagnosis, and promote the development and application of saliva sample analysis technology. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a nucleic acid preservation reagent in saliva samples, which forms a multifunctional and comprehensive protection system by introducing PEG-modified branched polyethyleneimine, combining EDTA, antibacterial agent, buffer system, osmotic pressure regulator and antioxidant. It not only improves the stability and reliability of nucleic acid preservation, but also takes into account biocompatibility and environmental friendliness, providing an efficient and innovative solution for the preservation of nucleic acid in saliva samples.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A nucleic acid preservation reagent in a saliva sample, the preservation reagent comprising the following components: PEG-modified branched polyethyleneimine: 1.0-5.0% (w / v) EDTA: 0.1-1.0 mM Antimicrobial agent: 0.02-0.1% (w / v) Buffer system: 10-50 mM Osmotic pressure regulator: 1.0-5.0% (v / v) Antioxidants: 0.1-0.5% (w / v), As well as pure water as the solvent, the pH of the solution is stable at 7.5-8.0.

[0007] Preferably, the preservation reagent comprises the following components: PEG-modified branched polyethyleneimine: 1.0-3.0% (w / v) EDTA: 0.2-0.5 mM Antimicrobial agent: 0.03-0.08% (w / v) Buffer system: 20-40 mM Osmotic pressure regulator: 2.0-4.0% (v / v) Antioxidant: 0.1-0.5% (w / v).

[0008] Preferably, the antibacterial agent is sodium azide; the antioxidant is glutathione and ascorbic acid or a mixture of two thereof; and the osmotic regulator is a mixture of one or more of glycerol, mannitol, trehalose, sucrose and glucose.

[0009] Preferably, the antioxidants are 0.2-1.0% glutathione and 0.2-1.0% ascorbic acid.

[0010] Preferably, the osmotic regulators are 0.5-1.0% mannitol and 0.5-1.0% trehalose.

[0011] Preferably, the preservation reagent also includes albumin BSA: 0.05-0.2%, 10-20 μL of protease inhibitor per milliliter of reaction system, and 20-40 units of RNase inhibitor per milliliter of reaction system.

[0012] Preferably, the polyethylene glycol-modified branched polyethyleneimine is prepared by using a branched polyethyleneimine having the following structural formula and a polyvinyl alcohol of 1000-3000 Da; the structural formula of the branched polyethyleneimine is as follows: ; n is 10-100.

[0013] Preferably, the preparation method of polyethylene glycol modified branched polyethyleneimine is as follows: 1) Dissolve 100g branched polyethyleneimine in 10L PBS and stir thoroughly until completely dissolved; dissolve 100g polyvinyl alcohol in 10L DMSO and stir thoroughly until completely dissolved; 2) Add 50 g EDC and 50 g NHS to the branched polyethyleneimine solution and stir for 2 hours to activate the amino groups; 3) Mix the activated branched polyethyleneimine solution with the PVA solution and stir at room temperature for 12-18 hours; 4) Perform dialysis through a dialysis bag to remove unreacted reagents and solvents. The dialysis process is carried out at 4°C. The dialysis solution is PBS. The dialysis lasts for 3 days, and the dialysis solution is replaced every day. 5) After dialysis is completed, the solution is freeze-dried to obtain bPEI-PVA copolymer.

[0014] Furthermore, the present invention also discloses a method for preparing a nucleic acid preservation reagent in a saliva sample, the method comprising the following steps: 1) Weigh an appropriate amount of Tris-HCl and dissolve it in a certain amount of pure water to prepare a solution of the required concentration; use sodium hydroxide or hydrochloric acid to adjust the pH of the solution to 7.5-8.0; 2) Add EDTA, antimicrobial agent, osmotic pressure regulator and antioxidant to the buffer and stir until completely dissolved; 3) Slowly add an appropriate amount of PEG-modified branched polyethyleneimine into the solution and stir evenly; 4) Use pure water to make up the solution to the required volume, ensuring that the concentration of each component meets the formula requirements; filter through a 0.22 μm filter membrane to ensure that the solution is sterile; 5) Dispense the prepared nucleic acid preservation reagent into sterile sample bottles; seal and store at 4°C, away from light and high temperature.

[0015] Furthermore, the present invention also discloses a nucleic acid detection kit in a saliva sample, the kit comprising a preservation reagent tube and a preservation reagent packaged in the preservation reagent tube.

[0016] The present invention forms a multifunctional and comprehensive protection system by introducing PEG-modified branched polyethyleneimine, combined with EDTA, antimicrobial agents, buffer systems, osmotic pressure regulators and antioxidants. It not only improves the stability and reliability of nucleic acid storage, but also takes into account biocompatibility and environmental friendliness, providing an efficient and innovative solution for the storage of nucleic acids in saliva samples.

[0017] 1. Enhance nucleic acid protection ability PEG-bPEI binds to nucleic acids through electrostatic forces to form stable complexes, preventing nucleic acid degradation in saliva samples. The application of PEG-modified branched polyethyleneimine significantly improves the stability of nucleic acids, especially in complex biological samples such as saliva. In addition, PEG modification also improves the water solubility and biocompatibility of polyethyleneimine, reduces toxicity, and ensures the stability of nucleic acids during sample storage.

[0018] 2. Implementation of anti-degradation mechanism EDTA: effectively inhibits the activity of nucleases by chelating metal ions, preventing the degradation of nucleic acids in saliva samples. Antimicrobial agents: such as sodium azide, can inhibit the growth of microorganisms in samples and prevent them from degrading nucleic acids. Antioxidants: such as ascorbic acid, can effectively prevent oxidative stress in samples and protect nucleic acids from oxidative damage.

[0019] 3. Comprehensive protection of buffer system and osmotic pressure regulation Buffer system: Tris-HCl can maintain the pH of the solution at 7.5-8.0, providing an ideal environment to maintain the integrity of nucleic acids. Osmotic pressure regulators: such as mannitol, protect the integrity of nucleic acids during freezing and thawing by regulating the osmotic pressure of the solution.

[0020] 4. Long-term storage and transportation The formula of the present invention can significantly prolong the storage time of nucleic acids at both room temperature and low temperature conditions, ensuring the accuracy and reliability of subsequent nucleic acid analysis. During long-term transportation, the reagent can effectively protect nucleic acids and ensure the integrity and quality of the samples.

[0021] In summary, the nucleic acid preservation reagent of the present invention can effectively protect the nucleic acid in the saliva sample to prevent degradation during collection, storage and transportation. The introduction of PEG-bPEI combined with multiple functional components provides a comprehensive protection mechanism and improves the stability and reliability of nucleic acid preservation. The present invention provides an efficient and innovative solution for biomedical research and clinical diagnosis, and promotes the development and application of saliva sample analysis technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the agarose gel electrophoresis analysis diagram of Example 2, Comparative Example 1 and Comparative Example 2 at room temperature. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The preparation method of polyethylene glycol modified branched polyethyleneimine (bPEI-PVA copolymer) is as follows: 1) Dissolve 100g branched polyethyleneimine (molecular weight less than 25000 Da) in 10L PBS and stir thoroughly until completely dissolved; dissolve 100g polyvinyl alcohol (1000-3000Da) in 10L DMSO and stir thoroughly until completely dissolved; 2) Add 50 g EDC and 50 g NHS to the branched polyethyleneimine solution and stir for 2 hours to activate the amino groups; 3) Mix the activated branched polyethyleneimine solution with the PVA solution and stir at room temperature for 12-18 hours; 4) Perform dialysis through a dialysis bag to remove unreacted reagents and solvents. The dialysis process is carried out at 4°C. The dialysis solution is PBS. The dialysis lasts for 3 days, and the dialysis solution is replaced every day. 5) After dialysis is completed, the solution is freeze-dried to obtain bPEI-PVA copolymer.

[0025] Example 1 formula: PEG-modified branched polyethyleneimine (PEG-bPEI): 2.0% (w / v) EDTA: 0.2 mM Antimicrobial agent (sodium azide): 0.05% (w / v) Buffer system (Tris-HCl): 20 mM Osmotic pressure regulator (glycerol): 2.0% (v / v) Antioxidant (ascorbic acid): 0.2% (w / v) Pure water, pH 7.5-8.0; The preparation method of the above-mentioned formula comprises the following steps: 1) Weigh an appropriate amount of Tris-HCl and dissolve it in a certain amount of pure water to prepare a solution of the required concentration; use sodium hydroxide or hydrochloric acid to adjust the pH of the solution to 7.5-8.0; 2) Add EDTA, antimicrobial agent, osmotic pressure regulator and antioxidant to the buffer and stir until completely dissolved; 3) Slowly add an appropriate amount of PEG-modified branched polyethyleneimine into the solution and stir evenly; 4) Use pure water to make up the solution to the required volume, ensuring that the concentration of each component meets the formula requirements; filter through a 0.22 μm filter membrane to ensure that the solution is sterile; 5) Dispense the prepared nucleic acid preservation reagent into sterile sample bottles; seal and store at 4°C, away from light and high temperature.

[0026] Experimental methods 1. Sample collection: Prepare 5 mL of preservation reagent for each formula and mix it with an equal amount of saliva sample.

[0027] 2. Sample storage: Store the mixed samples at room temperature, 4°C and -20°C respectively.

[0028] 3. Nucleic acid extraction: Sample nucleic acid extraction is performed on the 0th, 7th, 14th and 30th days of storage.

[0029] 4. Nucleic acid concentration and purity determination: Use NanoDrop to determine nucleic acid concentration and A260 / A280 ratio.

[0030] 5. Nucleic acid integrity detection: Analyze nucleic acid integrity by agarose gel electrophoresis.

[0031] Experimental results: Storage conditions time Nucleic acid concentration (ng / µL) A260 / A280 Ratio Nucleic acid integrity (%) Room temperature Day 0 50 1.8 100 Day 7 45 1.8 90.8 Day 14 42 1.8 85.7 Day 30 38 1.78 80.4 4°C Day 0 50 1.8 100 Day 7 48 1.8 95.5 Day 14 45 1.79 90.4 Day 30 40 1.78 88.7 -20°C Day 0 50 1.8 100 Day 7 49 1.8 98.6 Day 14 48 1.8 95.7 Day 30 47 1.79 94.2 Example 2 formula: PEG-modified branched polyethyleneimine (PEG-bPEI): 4.0% (w / v) EDTA: 0.1mM Antimicrobial agent (sodium azide): 0.05% (w / v) Buffer system (Tris-HCl): 30 mM Mannitol: 0.50% (w / v) Trehalose: 1.0% (w / v) Antioxidant (ascorbic acid): 0.2% (w / v) Pure water, pH 7.5-8.0; The preparation method and experimental method refer to Example 1.

[0032] Experimental results: Storage conditions time Nucleic acid concentration (ng / µL) A260 / A280 Ratio Nucleic acid integrity (%) Room temperature Day 0 52 1.8 100 Day 7 48 1.8 92.5 Day 14 45 1.8 89.7 Day 30 42 1.78 85.6 4°C Day 0 52 1.8 100 Day 7 50 1.8 96.4 Day 14 48 1.8 92.6 Day 30 45 1.79 90.8 -20°C Day 0 52 1.8 100 Day 7 51 1.8 98.5 Day 14 50 1.8 95.4 Day 30 49 1.79 94.6 Example 3 formula: PEG-modified branched polyethyleneimine (PEG-bPEI): 2.0% (w / v) EDTA: 0.2 mM Antimicrobial agent (sodium azide): 0.05% (w / v) Buffer system (Tris-HCl): 20 mM Osmotic pressure regulator (glycerol): 2.0% (v / v) Antioxidant (ascorbic acid): 0.2% (w / v) Albumin BSA: 0.1% (w / v) Pure water, pH 7.5-8.0; The preparation method and experimental method refer to Example 1.

[0033] Experimental results: Storage conditions time Nucleic acid concentration (ng / µL) A260 / A280 Ratio Nucleic acid integrity (%) Room temperature Day 0 51 1.8 100 Day 7 48 1.8 94.8 Day 14 46 1.8 90.4 Day 30 44 1.79 86.7 4°C Day 0 51 1.8 100 Day 7 50 1.8 96.7 Day 14 48 1.8 92.4 Day 30 46 1.79 90.6 -20°C Day 0 51 1.8 100 Day 7 50 1.8 98.7 Day 14 50 1.8 96.4 Day 30 49 1.79 94.2 Comparative Example 1 Branched polyethyleneimine (bPEI): 2.0% (w / v) EDTA: 0.2 mM Antimicrobial agent (sodium azide): 0.05% (w / v) Buffer system (Tris-HCl): 20 mM Osmotic pressure regulator (glycerol): 2.0% (v / v) Antioxidant (ascorbic acid): 0.2% (w / v) Pure water, pH 7.5-8.0; The preparation method and experimental method refer to Example 1.

[0034] Experimental results: Storage conditions time Nucleic acid concentration (ng / µL) A260 / A280 Ratio Nucleic acid integrity (%) Room temperature Day 0 50 1.8 100 Day 7 45 1.78 82.5 Day 14 38 1.74 76.6 Day 30 32 1.70 68.2 4°C Day 0 50 1.8 100 Day 7 46 1.78 85.3 Day 14 42 1.77 80.4 Day 30 38 1.75 75.7 -20°C Day 0 50 1.8 100 Day 7 48 1.8 98.2 Day 14 46 1.8 94.4 Day 30 44 1.78 88.7 Comparative Example 2 EDTA: 0.2 mM Antimicrobial agent (sodium azide): 0.05% (w / v) Buffer system (Tris-HCl): 20 mM Osmotic pressure regulator (glycerol): 2.0% (v / v) Antioxidant (ascorbic acid): 0.2% (w / v) Pure water, pH 7.5-8.0; The preparation method and experimental method refer to Example 1.

[0035] Experimental results: Storage conditions time Nucleic acid concentration (ng / µL) A260 / A280 Ratio Nucleic acid integrity (%) Room temperature Day 0 49 1.8 100 Day 7 32 1.65 60.4 Day 14 23 1.51 43.4 Day 30 11 1.46 21.7 4°C Day 0 49 1.8 100 Day 7 35 1.72 75.8 Day 14 25 1.63 56.4 Day 30 15 1.51 35.9 -20°C Day 0 49 1.8 100 Day 7 48 1.8 98.1 Day 14 45 1.8 95.4 Day 30 42 1.78 85.3 Each embodiment of the present invention shows the effect of preserving nucleic acid under different temperature conditions. Figure 1 It is an agarose gel electrophoresis analysis diagram of Example 2, Comparative Example 1 and Comparative Example 2 at room temperature; the main band is clear and does not show degradation when stored at room temperature for 30 days using the preservation solution of Example 2, the concentration of Comparative Example 1 decreases significantly, and the main band of Comparative Example 2 degrades.

[0036] The addition of PEG-modified branched polyethyleneimine to the formulation significantly improved the stability and integrity of nucleic acids at room temperature, 4°C, and -20°C.

[0037] The results of Comparative Examples 1 and 2 show that the effect of using only the basic formula is far inferior to the formula with PEG-modified branched polyethyleneimine added.

[0038] Example 2 (PEG-modified branched polyethyleneimine 4.0%) exhibited the best nucleic acid stability, especially under low-temperature storage conditions.

[0039] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A reagent for preserving nucleic acid in saliva samples, characterized in that: The preservation reagent includes the following components: PEG-modified branched polyethyleneimine: 1.0-5.0% (w / v) EDTA: 0.1-1.0 mM Antimicrobial agent: 0.02-0.1% (w / v) Buffer system: 10-50 mM Osmotic pressure regulator: 1.0-5.0% (v / v) Antioxidants: 0.1-0.5% (w / v), As well as pure water as the solvent, the pH of the solution is stable at 7.5-8.

0.

2. The nucleic acid preservation reagent in saliva sample according to claim 1, characterized in that: The preservation reagent includes the following components: PEG-modified branched polyethyleneimine: 1.0-3.0% (w / v) EDTA: 0.2-0.5 mM Antimicrobial agent: 0.03-0.08% (w / v) Buffer system: 20-40 mM Osmotic pressure regulator: 2.0-4.0% (v / v) Antioxidant: 0.1-0.5% (w / v).

3. A nucleic acid preservation reagent in a saliva sample according to claim 1 or 2, characterized in that: The antibacterial agent is sodium azide; the antioxidant is glutathione and ascorbic acid or a mixture of two thereof; the osmotic regulator is mannitol, trehalose, sucrose and glucose or a mixture thereof.

4. The nucleic acid preservation reagent in saliva sample according to claim 3, characterized in that: The antioxidants selected are 0.2-1.0% glutathione and 0.2-1.0% ascorbic acid.

5. The nucleic acid preservation reagent in saliva sample according to claim 3, characterized in that: The osmotic regulators used are mannitol 0.5-1.0% and trehalose 0.5-1.0%.

6. A reagent for preserving nucleic acid in saliva samples according to claim 1 or 2, characterized in that: The storage reagent also includes albumin BSA: 0.05-0.2%, protease inhibitors added 10-20 µL per ml reaction system, and RNase inhibitors added 20-40 units per ml reaction system.

7. A reagent for preserving nucleic acid in saliva samples according to claim 1 or 2, characterized in that: It is prepared by using branched polyethyleneimine with the following structural formula and polyvinyl alcohol of 1000-3000Da; The structural formula of the branched polyethyleneimine is as follows: ; n is 10-100.

8. The nucleic acid preservation reagent in saliva sample according to claim 6, characterized in that: The preparation method of polyethylene glycol modified branched polyethyleneimine is as follows: 1) Dissolve 100g branched polyethyleneimine in 10L PBS and stir thoroughly until completely dissolved; dissolve 100g polyvinyl alcohol in 10L DMSO and stir thoroughly until completely dissolved; 2) Add 50 g EDC and 50 g NHS to the branched polyethyleneimine solution and stir for 2 hours to activate the amino groups; 3) Mix the activated branched polyethyleneimine solution with the PVA solution and stir at room temperature for 12-18 hours; 4) Perform dialysis through a dialysis bag to remove unreacted reagents and solvents. The dialysis process is carried out at 4°C. The dialysis solution is PBS. The dialysis lasts for 3 days, and the dialysis solution is replaced every day. 5) After dialysis is completed, the solution is freeze-dried to obtain bPEI-PVA copolymer.

9. The method for preparing a nucleic acid preservation reagent in a saliva sample according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: 1) Weigh an appropriate amount of Tris-HCl and dissolve it in a certain amount of pure water to prepare a solution of the required concentration; use sodium hydroxide or hydrochloric acid to adjust the pH of the solution to 7.5-8.0; 2) Add EDTA, antimicrobial agent, osmotic pressure regulator and antioxidant to the buffer and stir until completely dissolved; 3) Slowly add an appropriate amount of PEG-modified branched polyethyleneimine into the solution and stir evenly; 4) Use pure water to make up the solution to the required volume, ensuring that the concentration of each component meets the formula requirements; filter through a 0.22 μm filter membrane to ensure that the solution is sterile; 5) Dispense the prepared nucleic acid preservation reagent into sterile sample bottles; seal and store at 4°C, away from light and high temperature.

10. A kit for detecting nucleic acid in a saliva sample, comprising a preservation reagent tube and a preservation reagent as claimed in any one of claims 1 to 8 packaged in the preservation reagent tube.

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