阅读说明：本技术 一种尿液中微生物核酸新型保存液 (Novel preservation solution for microbial nucleic acid in urine ) 是由 段志峰 于 2020-08-27 设计创作，主要内容包括：本发明公开了一种尿液中微生物核酸新型保存液,其包括如下组份：核酸酶抑制剂、三羟甲基氨基甲烷-盐酸缓冲液、防腐剂、甲醛猝灭剂、渗透剂、代谢抑制剂、丙三醇,所述尿液中微生物核酸新型保存液的pH为7～8。所述保存液能有效抑制尿液中的白细胞或者是上皮细胞裂解,同时抑制尿液中的游离核酸降解,维持游离核酸的稳定性,从而可以提高尿液检测的准确性,且所述的核酸新型保存液成分相对简单、成本低廉,同时还能够在室温条件下保持游离核酸的稳定性,非常适合在临床广泛使用。(The invention discloses a novel preservation solution for microbial nucleic acid in urine, which comprises the following components: the novel urine preservation solution comprises a nuclease inhibitor, a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, a preservative, a formaldehyde quencher, a penetrating agent, a metabolic inhibitor and glycerol, wherein the pH value of the novel urine preservation solution for microbial nucleic acid is 7-8. The novel preservation solution for the nucleic acid has the advantages that the preservation solution can effectively inhibit the lysis of leucocytes or epithelial cells in urine, simultaneously inhibit the degradation of free nucleic acid in the urine, and maintain the stability of the free nucleic acid, so that the accuracy of urine detection can be improved, the components of the novel preservation solution for the nucleic acid are relatively simple, the cost is low, simultaneously, the stability of the free nucleic acid can be kept at room temperature, and the novel preservation solution is very suitable for being widely used clinically.)

1. A novel preservation solution for microbial nucleic acid in urine is characterized by comprising the following components: the novel urine preservation solution comprises a nuclease inhibitor, a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution, a preservative, a formaldehyde quencher, a penetrating agent, a metabolic inhibitor and glycerol, wherein the pH value of the novel urine preservation solution for microbial nucleic acid is 7-8.

2. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the nuclease inhibitor is ethylenediaminetetraacetic acid or ethylene glycol tetraacetic acid, and the concentration is 5 to 10 mmol/L.

3. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the concentration of the tris-hcl buffer solution is 1 to 3 mmol/L.

4. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the preservative is one or more of dimethylol urea, 2-bromo-2-nitropropane-1, 3-diol, sodium citrate and sodium hydroxymethyl glycinate, and the concentration of the preservative is 2-5 mmol/L.

5. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the formaldehyde quencher is a mixture of an amino acid and an amine-containing compound.

6. The novel preservation solution for microbial nucleic acid in urine according to claim 5, wherein the formaldehyde quencher is a mixture of arginine, lysine, glycine and ethylenediamine, and the mixing concentration is 20 to 30 mmol.

7. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the penetrant is one or more of glucose, sodium chloride, potassium chloride and sodium propionate, and the concentration is 20 to 30 mmol.

8. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the metabolic inhibitor is glyceraldehyde, glyceraldehyde-3-phosphate or 3-phosphate-2-phosphoglyceride, and the concentration is 1 to 2 mmol/L.

9. The novel preservation solution for microbial nucleic acid in urine according to claim 1, wherein the glycerol is contained in an amount of 5 to 10% by mass.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a novel preservation solution for microbial nucleic acid in urine.

Background

Liquid biopsy is a method of obtaining biomarkers directly from body fluids (e.g. blood or urine) rather than solid tissue, and is a simple non-invasive surgical biopsy alternative. Urine test is one of the in vitro samples commonly used in molecular biology tests, and urine is liquid which is generated by kidney and is discharged through ureter and bladder and contains a large amount of metabolic end products. The relationship between various body fluids and organs in the human body is rarely closely related to and easily obtained like urine and kidneys, and can dynamically reflect changes of the kidneys and urinary system and abnormal or disease information of other parts. Nucleic acid extraction is the basis of various molecular biology detection methods, and efficient and complete extraction of DNA is the basis of PCR amplification, library construction, sequencing and other works. Urine contains nucleic acid, and for human body, the test of urine nucleic acid is more non-invasive than that of blood nucleic acid. However, urine contains components such as urea and uric acid which can destroy intracellular organelles and degrade nucleic acid, so that the detection result is directly influenced by the storage time of the urine. In addition, urine has higher nuclease content, more metabolites and larger pH range fluctuation than peripheral blood, so the biological environment of free nucleic acid in urine is more complicated compared with blood. If leucocytes or epithelial cell rupture genome nucleic acid in urine is degraded into the urine, or if nuclease in the urine is not inhibited, the secreted nuclease can cause DNA chain breakage and degradation, initial free DNA can be degraded and cannot reach the detection level, the background noise of detection can be increased, and a false negative result is brought to subsequent detection.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides a novel preservation solution for microbial nucleic acid in urine, which can effectively inhibit the lysis of leucocytes or epithelial cells in urine, inhibit the degradation of free nucleic acid in urine, and maintain the stability of the free nucleic acid, thereby improving the accuracy of urine detection.

The technical scheme adopted by the invention is as follows:

a novel preservation solution for microbial nucleic acid in urine comprises the following components: the novel urine preservation solution comprises a nuclease inhibitor, a tris-hydroxy methyl aminomethane-hydrochloric acid buffer solution, a preservative, a formaldehyde quencher, a penetrant and a metabolism inhibitor, wherein the pH value of the novel urine preservation solution for microbial nucleic acid is 7-8.

The novel preservation solution for microbial nucleic acid in urine can inhibit the activity of nuclease in a system by using the nuclease inhibitor, prevent the nucleic acid in a urine sample from being degraded, and simultaneously can maintain the stability of leucocytes or epithelial cells and avoid cell lysis; the tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution can keep the urine sample neutral or weakly alkaline and provide a relatively stable pH value environment for nucleic acid in the urine sample; the preservative has the function of anticoagulation, ensures that blood cells are not broken, and maintains the stability of the blood cells; the formaldehyde quencher mainly reacts with free aldehyde to quench free formaldehyde and other free aldehydes, so that damage to free nucleic acid is avoided, the nucleic acid in urine can maintain the stability of the structure of the nucleic acid, and the inhibition of free aldehyde on polymerase chain reaction and enzyme reaction in a sequencing process is avoided; the osmotic agent can better maintain the osmotic pressure of the cells, and the cell lysis is further avoided by the lysis; the metabolism inhibitor can further inhibit the degradation of free nucleic acid and stabilize the free nucleic acid in urine; the glycerol can make the preservation solution more stable. According to the invention, through reasonable use of the components, the prepared novel preservation solution for the microbial nucleic acid in the urine can quickly stabilize the urine sample, effectively inhibit the activity of nuclease, prevent cell lysis, well protect free nucleic acid in the urine and prolong the preservation time.

Preferably, the nuclease inhibitor is ethylene diamine tetraacetic acid or ethylene glycol tetraacetic acid, and the concentration is 5-10 mmol/L.

Preferably, the concentration of the tris-hydrochloride buffer is 1-3 mmol/L.

Preferably, the preservative is one or more of dimethylol urea, 2-bromo-2-nitropropane-1, 3-diol, sodium citrate and sodium hydroxymethyl glycinate, and the concentration is 2-5 mmol/L.

Preferably, the formaldehyde quencher is a mixture of an amino acid and an amine-containing compound. Further preferably, the formaldehyde quencher is a mixture of arginine, lysine, glycine and ethylenediamine, and the mixing concentration is 20-30 mmol.

Preferably, the penetrating agent is one or more of glucose, sodium chloride, potassium chloride and sodium propionate, and the concentration is 20-30 mmol.

Preferably, the metabolic inhibitor is glyceraldehyde, glyceraldehyde-3-phosphate or 3-phosphate-2-phosphoglyceride, and the concentration is 1-2 mmol/L.

Preferably, the glycerol accounts for 5-10% by mass.

Compared with the prior art, the invention has the beneficial effects that: the novel preservation solution for the microbial nucleic acid in the urine can quickly and effectively inhibit the activity of nuclease, prevent the nucleic acid in a urine sample from being degraded, ensure the content of free nucleic acid, avoid that the detection level cannot be reached due to the degradation of the free nucleic acid, simultaneously maintain the stability of leucocytes or epithelial cells, avoid cell cracking, reduce the detection background noise and improve the accuracy of urine detection; the novel nucleic acid preservation solution disclosed by the invention is relatively simple in components and low in cost, can keep the stability of free nucleic acid at room temperature, and is very suitable for being widely used clinically; the novel nucleic acid storage solution of the present invention can be stored for a long time and can provide sufficient time for examination and receiving.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail with reference to specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

The experimental raw materials, reagent materials and the like used in the following examples are all common commercial products unless otherwise specified.