CN114875022B - Urine preservation solution, preservation method and urine preservation tube - Google Patents
Urine preservation solution, preservation method and urine preservation tube Download PDFInfo
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- CN114875022B CN114875022B CN202210607573.5A CN202210607573A CN114875022B CN 114875022 B CN114875022 B CN 114875022B CN 202210607573 A CN202210607573 A CN 202210607573A CN 114875022 B CN114875022 B CN 114875022B
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- 210000002700 urine Anatomy 0.000 title claims abstract description 337
- 239000003761 preservation solution Substances 0.000 title claims abstract description 118
- 238000004321 preservation Methods 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000000243 solution Substances 0.000 claims abstract description 75
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 claims abstract description 38
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000003755 preservative agent Substances 0.000 claims abstract description 27
- 230000002335 preservative effect Effects 0.000 claims abstract description 26
- 239000003161 ribonuclease inhibitor Substances 0.000 claims abstract description 24
- 239000003112 inhibitor Substances 0.000 claims abstract description 21
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 14
- 239000000834 fixative Substances 0.000 claims abstract description 12
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 10
- 102000016911 Deoxyribonucleases Human genes 0.000 claims abstract description 7
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- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims abstract 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 45
- 230000014759 maintenance of location Effects 0.000 claims description 37
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 22
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 20
- 229920002866 paraformaldehyde Polymers 0.000 claims description 20
- SOROIESOUPGGFO-UHFFFAOYSA-N diazolidinylurea Chemical compound OCNC(=O)N(CO)C1N(CO)C(=O)N(CO)C1=O SOROIESOUPGGFO-UHFFFAOYSA-N 0.000 claims description 14
- 229960001083 diazolidinylurea Drugs 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 11
- 239000004471 Glycine Substances 0.000 claims description 11
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 11
- 239000001509 sodium citrate Substances 0.000 claims description 11
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 11
- 229940038773 trisodium citrate Drugs 0.000 claims description 11
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 6
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 4
- ZCTXEAQXZGPWFG-UHFFFAOYSA-N imidurea Chemical compound O=C1NC(=O)N(CO)C1NC(=O)NCNC(=O)NC1C(=O)NC(=O)N1CO ZCTXEAQXZGPWFG-UHFFFAOYSA-N 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 3
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- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 3
- -1 diethyl triamine pentaacetic acid Chemical compound 0.000 claims description 3
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical compound O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 claims description 3
- 229940091173 hydantoin Drugs 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- LZGVDNRJCGPNDS-UHFFFAOYSA-N trinitromethane Chemical compound [O-][N+](=O)C([N+]([O-])=O)[N+]([O-])=O LZGVDNRJCGPNDS-UHFFFAOYSA-N 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 2
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
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- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Mechanical Engineering (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a urine preservation solution which is an aqueous solution containing the following substances in percentage by mass: 0.02% -0.1% of an RNase inhibitor, 2% -10% of a DNase inhibitor, 0.2% -0.8% of a nonionic surfactant, 1% -5% of a cell fixative, 1% -5% of a preservative and 1% -5% of an aldehyde quencher, wherein the RNase inhibitor comprises at least diethyl pyrocarbonate and the DNase inhibitor comprises at least ethylenediamine tetraacetic acid. Also discloses a urine preservation method and a urine preservation tube. After urine is isolated and preserved by adopting the urine preservation solution provided by the invention, more accurate free DNA and RNA information can be provided, the degradation of nucleic acid substances is inhibited, interference signals including but not limited to genome DNA and microorganism interference are reduced, and the whole detection solution result still has higher sensitivity and higher accuracy. Simple operation and long storage period.
Description
Technical Field
The invention relates to the technical field of biological sample preservation, in particular to a urine preservation solution, a preservation method and a urine preservation tube.
Background
Urine is a final metabolite formed by filtering and reabsorption of blood through kidneys, and contains not only excretions in the body such as inorganic salts, uric acid, urea, glucose and the like, but also metabolic substances and genetic variation information of other organs of the body such as free deoxyribonucleic acid (cfDNA), circulating tumor deoxyribonucleic acid (ctDNA) and the like, so that the urine can be used as a biological sample for detecting and diagnosing some urinary system diseases and other system diseases, for example, the cfDNA in the urine can be used for early screening and diagnosis of tumors. Urine collection is completely noninvasive, and can realize home collection, so that the method is a very valuable sample type for early screening and early diagnosis of tumors, such as early screening and early diagnosis of urothelial cancer and other diseases. However, due to the complex urine components, a certain time interval is required from sample collection to sample detection clinically, and cfDNA content in urine is low and easy to degrade, so that the accuracy of the detection result is not ideal, for example, the urine components of patients suffering from haematuria and diabetics are very complex. Therefore, there is a need to develop new urine preservation products to improve the accuracy of urine detection.
Disclosure of Invention
Based on this, it is necessary to provide a urine preservation solution capable of protecting both DNA and RNA, and further to provide a related preservation method and urine preservation tube. The urine preservation solution can still effectively preserve DNA and RNA information in urine after storage, and achieves higher detection accuracy.
The first object of the invention is to provide a urine preservation solution which is an aqueous solution containing the following substances in percentage by mass: 0.02% -1% of an rnase inhibitor, 2% -10% of a dnase inhibitor, 0.2% -0.8% of a nonionic surfactant, 1% -5% of a cell fixative, 1% -5% of a preservative and 1% -5% of an aldehyde quencher, wherein the rnase inhibitor comprises at least diethyl pyrocarbonate and the dnase inhibitor comprises at least ethylenediamine tetraacetic acid.
In one embodiment, the mass percentage of the diethyl pyrocarbonate in the urine preservation solution is 0.02% -0.1%.
In one embodiment, the mass percentage of the diethyl pyrocarbonate in the urine preservation solution is 0.05% -0.1%.
In one embodiment, the mass percentage of the ethylenediamine tetraacetic acid in the urine preservation solution is 4% -10%.
In one embodiment, the mass percentage of the ethylenediamine tetraacetic acid in the urine preservation solution is 6% -10%.
In one embodiment, the rnase inhibitor further comprises one or both of ammonium sulfate and guanidine isothiocyanate; and/or the number of the groups of groups,
the dnase inhibitor further comprises one or both of diethyl triamine pentaacetic acid and citric acid.
In one embodiment, the nonionic surfactant is selected from one or both of tween 20 and Triton X-100; preferably, the nonionic surfactant comprises at least tween 20.
In one embodiment, the urine retention solution further comprises a pH buffer, the pH of the urine retention solution being selected from the group consisting of 7-8.
In one embodiment, the cell fixative is selected from one or both of paraformaldehyde and imidazolidinyl urea; and/or the number of the groups of groups,
the preservative is selected from one or more of diazolidinyl urea, paraformaldehyde, formalin, hydantoin and trinitromethane; and/or the number of the groups of groups,
the aldehyde quencher is selected from one or more of glycine, arginine and lysine; and/or the number of the groups of groups,
the pH buffer is selected from one or more of Tris-HCl, citric acid, trisodium citrate, phosphate, acetate, borate and carbonate.
In one embodiment, the urine retention solution is an aqueous solution comprising the following mass percent: 0.08-0.1% of diethyl pyrocarbonate, 0.4-0.5% of tween 20, 5-6% of ethylenediamine tetraacetic acid, 4-5% of glycine, 1.3-1.5% of paraformaldehyde and 1.3-1.5% of diazolidinyl urea, wherein the pH value of the urine preservation solution is selected from 7.5-8.
The second object of the present invention is to provide a urine preservation method, comprising the steps of: mixing the urine to be preserved with the urine preservation solution.
A third object of the present invention is to provide a urine retention tube, which is a sample collection tube containing the urine retention solution.
The urine contains a plurality of components, and factors influencing the accuracy of the urine detection result are complex, so that not only the preservation capacity of free cfDNA is related, but also the interference of other factors is required to be eliminated. The large number of microorganisms in urine can be rapidly propagated after the urine is isolated, DNA degradation can be caused by high-content nuclease, a large amount of genome DNA can be released by cell rupture, and the multidimensional factors mutually interfere, so that the difficulty in accurately acquiring free cfDNA information in the urine exists, for example, the preservation of urine components of patients suffering from haematuria and diabetics is very challenging. The inventor also found that unstable urine pH environment is also one of factors affecting the accuracy of urine detection results. In view of this, the detection accuracy can be improved through multiple groups of study, so as to solve the problem that the detection performance of a single dimension cannot meet the performance requirements of most cancers for early screening and early diagnosis, such as early tumor diagnosis. The inventor of the application finds that at present, related researchers mainly focus on the protection of free DNA in urine and less focuses on RNA information, mainly because RNA is unstable and is difficult to store, urine can not be detected in real time in vitro, and screening diagnosis by utilizing the RNA information in urine is not easy to realize.
The urine preservation solution provided by the invention is a reagent for preserving DNA and RNA of urine samples together, and can provide a basis for related researches such as urine multiple-study and the like. According to the urine preservation solution provided by the invention, proper component combinations are screened through a large number of researches, and the cooperative control of various parameters such as the content of each component and the pH value of a system is carried out, so that the components act cooperatively, and the good effect of DNA and RNA co-preservation is realized.
The urine preservation solution provided by the invention comprises an RNase inhibitor, a DNase inhibitor, a nonionic surfactant, a cell fixing agent, a preservative and an aldehyde quencher, and controls the pH value of a proper system (such as pH 7-8), so that degradation of free DNA and RNA in urine can be effectively avoided, interference of microorganisms in urine is avoided, further, interference of genomic DNA of cells can be effectively avoided, effective co-preservation of free DNA and RNA in a complex urine environment can be realized, support of multi-group study information preservation of urine samples can be provided, and foundation is provided for multi-group study early diagnosis and early screening study of urine.
The urine preservation method provided by the invention is simple to operate, and has long preservation period which can be up to 7 days or more. After urine is isolated and preserved by adopting the urine preservation solution provided by the invention, more accurate free DNA and RNA information can be provided, the degradation of nucleic acid substances is inhibited, interference signals including but not limited to genome DNA and microorganism interference are reduced, and the whole detection solution result still has higher sensitivity and higher accuracy.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort to a person skilled in the art.
FIG. 1 is a graph showing comparison of the protection ability of a urine preservation solution to cfDNA of a urine supernatant in the presence or absence of the urine preservation solution according to example 1 of the present invention, wherein the concentration unit on the ordinate is ng/. Mu.L;
FIG. 2 is a graph showing the comparative protection ability of urine sediment genomic DNA by urine preservation solution in example 1 of the present invention, wherein the concentration unit on the ordinate is ng/. Mu.L;
FIG. 3 is a graph showing the comparison of the protection ability of different urine preservation solutions to whole urine RNA in example 2 of the present invention; the abscissa indicates the number of cycles (cycle number), and the ordinate delta Rn (delta Rn) indicates the fluorescence intensity of the PCR amplification product at the nth cycle;
FIG. 4 is a graph showing comparison of the protective capacity of different urine preservation solutions to the cfDNA of the urine supernatant according to example 3 of the present invention, and the concentration unit on the ordinate is ng/. Mu.L.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the invention and are not intended to limit the scope of the invention in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the invention, and equivalents thereof are also intended to fall within the scope of the invention. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention, it being understood that the invention may be practiced without one or more of these details.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection.
The term "plural", and the like in the present invention refers to, unless otherwise specified, a number of 2 or more. For example, "one or more" means one kind or two or more kinds.
As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.
In the present invention, "suitable" in "suitable combination manner", "suitable manner", "any suitable manner", etc., are used to implement the technical scheme of the present invention, solve the technical problem of the present invention, and achieve the technical effects expected by the present invention.
In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. If there are multiple "preferences" in a solution, if there is no particular description and there is no conflict or constraint, then each "preference" is independent of the others.
In the present invention, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the invention.
In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
In the present invention, "first aspect", "second aspect", "third aspect", "first object", "second object", "third object", etc. are for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of the indicated technical features. Moreover, "first," "second," "third," etc. are for non-exhaustive list description purposes only, and it should be understood that no closed limitation on the number is made.
The terms "comprising," "including," and "comprising," as used herein, are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, a numerical range (i.e., a numerical range) is referred to, and, unless otherwise indicated, a distribution of optional values within the numerical range is considered to be continuous and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints. When a numerical range merely points to integers within the numerical range, unless expressly stated otherwise, both endpoints of the numerical range are inclusive of the integer between the two endpoints, and each integer between the two endpoints is equivalent to the integer directly recited. When multiple numerical ranges are provided to describe a feature or characteristic, the numerical ranges may be combined. In other words, unless otherwise indicated, the numerical ranges disclosed herein are to be understood as including any and all subranges subsumed therein. The "numerical value" in the numerical interval may be any quantitative value, such as a number, a percentage, a proportion, or the like. "numerical interval" allows to broadly include quantitative intervals such as percentage intervals, proportion intervals, ratio intervals, etc.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.
In the present invention, the term "normal temperature" generally means 4℃to 35℃and preferably 20.+ -. 5 ℃. In some embodiments of the invention, ambient temperature refers to 20 ℃ to 30 ℃.
In a first aspect of the invention, a urine preservation solution is provided, which can be used as a reagent for co-preserving DNA and RNA of a urine sample, and can provide a basis for related researches such as urine multiple-study.
In some embodiments of the invention, the RNA to be preserved is mRNA. Further, the urine preservation solution can preserve DNA and mRNA of a urine sample at the same time.
In some embodiments of the invention, a urine preservation solution is provided, which comprises an RNase inhibitor, a DNase inhibitor, a nonionic surfactant, a cell fixing agent, a preservative and an aldehyde group quencher, and controls a proper system pH value (such as pH 7-8), so that degradation of free DNA and RNA in urine can be effectively avoided, interference of microorganisms in urine is avoided, further, interference of genomic DNA of cells can be effectively avoided, effective co-preservation of free DNA and RNA in a complex urine environment can be realized, support of multi-group study of urine samples for multi-group study information preservation can be provided, and a foundation is provided for multi-group study early diagnosis and early screening study of urine.
In some embodiments of the present invention, there is provided a urine preservation solution that is an aqueous solution comprising the following substances in mass percent (wt%): 0.02% -1% of an rnase inhibitor, 2% -10% of a dnase inhibitor, 0.2% -0.8% of a nonionic surfactant, 1% -5% of a cell fixative, 1% -5% of a preservative and 1% -5% of an aldehyde quencher.
In some of its preferred embodiments, the rnase inhibitor comprises at least diethyl pyrocarbonate (DEPC).
In some of its preferred embodiments, the dnase inhibitor comprises at least ethylenediamine tetraacetic acid (EDTA).
In some of its preferred embodiments, the rnase inhibitor comprises at least diethyl pyrocarbonate and the dnase inhibitor comprises at least ethylenediamine tetraacetic acid.
The RNase inhibitor can effectively inhibit the activity of RNase in urine, prevent RNA degradation in urine, and simultaneously can not cause rupture of exfoliated cells in urine. The mass percent of the rnase inhibitor in the urine preservation solution can be 0.02% -1%, for example, any one or two of the following ranges: 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and the like. The interval between any two percentages is, for example, 0.02% -0.1%.
In some embodiments, the rnase inhibitor comprises at least diethyl pyrocarbonate (DEPC). The amount of DEPC may be selected from the amounts of any suitable RNase inhibitor described above.
In some embodiments, the rnase inhibitor is diethyl pyrocarbonate (DEPC). The amount of DEPC may be selected from the amounts of any suitable RNase inhibitor described above.
In some embodiments, the mass percent of diethyl pyrocarbonate in the urine preservation solution is 0.02% -0.1%. In some embodiments, the mass percent of diethyl pyrocarbonate in the urine preservation solution is 0.05% -0.1%.
In some embodiments, the rnase inhibitor comprises diethyl pyrocarbonate and further comprises one or both of ammonium sulfate and guanidine isothiocyanate, when both cell disruption and RNA degradation are prevented. Wherein the concentration of ammonium sulfate in the urine preservation solution may be 0.5% -1%, for example, any one or two of the following percentages: 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, etc. The concentration of guanidine isothiocyanate in the urine preservation solution can be 0.02% -1%, for example, any one or two of the following ranges: 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and the like.
DNase inhibitors are effective in chelating divalent metal ions in urine to inhibit nuclease activity and prevent degradation of free DNA. The dnase inhibitor may be present in the urine preservation solution in an amount of 2% to 10% by mass, for example in an interval consisting of any one or any two of the following percentages: 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.
In some embodiments, the dnase inhibitors include at least ethylenediamine tetraacetic acid (EDTA). The amount of EDTA may be selected from the amounts of any of the suitable dnase inhibitors described above.
In some embodiments, the dnase inhibitor is ethylenediamine tetraacetic acid (EDTA). The amount of EDTA may be selected from the amounts of any of the suitable dnase inhibitors described above.
In some embodiments, the mass percent of ethylenediamine tetraacetic acid in the urine preservation solution is 4% -10%, e.g., may be 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc. In some embodiments, the mass percent of ethylenediamine tetraacetic acid in the urine preservation solution is 6% -10%.
In some embodiments, the dnase inhibitors include ethylenediamine tetraacetic acid, and further include one or both of diethylenetriamine pentaacetic acid and citric acid, when both cell disruption and DNA degradation are prevented. Wherein the concentration of diethyl triamine pentaacetic acid in the urine preservation solution may be 4% -10%, for example, any one or two of the following percentages: 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc. The concentration of citric acid in the urine preservation solution may be 1% -5%, for example, any one or two of the following percentages: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.
Cell fixatives can terminate endogenous and/or exogenous enzymatic reactions, preventing cell rupture or autolysis. In some embodiments, the cell fixative is selected from one or both of paraformaldehyde and imidazolidinyl urea. The mass percentage of the cell fixative in the urine preservation solution can be 1% -5%, for example, any one or two of the following ranges: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc. The interval between any two percentages is, for example, 1.5% -5%, 1.5% -4.5% and the like.
In some embodiments, the concentration of paraformaldehyde in the urine retention solution can be from 1.3% to 1.5% (e.g., 1.3%, 1.4%, 1.5%, etc.). The concentration of imidazolidinyl urea in the urine retention solution may be 1.3% -1.5% (e.g., 1.3%, 1.4%, 1.5%, etc.).
The surfactant used in the invention can be selected from nonionic surfactants, so that the stability of the membrane protein can be improved in the biological environment of the cell fixative, and a certain inhibition effect can be achieved on the RNase. In some embodiments, the nonionic surfactant is selected from one or both of tween 20 and Triton X-100, and the like. Preferably, the nonionic surfactant comprises at least tween 20. The mass percent of the nonionic surfactant in the urine preservation solution can be 0.2-0.8%, such as any one or two of the following ranges: 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%. Examples of the interval between any two percentages are 0.4% to 0.6%.
In some embodiments, the concentration of Triton X-100 in the urine preservation solution may be 0.2% -0.8%.
In some embodiments, the preservative may be present in the urine retention solution in an amount of 1% to 5% by mass, in the interval consisting of either or both of the following percentages: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%. Examples of the interval of any two percentages are 1% to 3%. In some embodiments, the preservative used is selected from one or more of diazolidinyl urea, paraformaldehyde, formalin, hydantoin, trinitromethane, and the like. In some embodiments, the combination of diazolidinyl urea and paraformaldehyde as the preservative has a broad spectrum of antimicrobial effects, and is effective in inhibiting the proliferation of bacteria and fungi in urine.
In some embodiments, the mass ratio of diazolidinyl urea to paraformaldehyde in the urine retention solution is (0.8-1.2): 1. In some embodiments, the mass ratio of diazolidinyl urea to paraformaldehyde in the urine preservative solution is 1:1.
In some embodiments, the aldehyde quencher is selected from one or more of glycine, arginine, lysine, and the like. In some embodiments, the aldehyde quencher is glycine. The mass percent of the aldehyde quencher in the urine preservation solution can be 1% -5%, for example, the following range is formed by any one or two of the following percentages: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.
The pH value of the urine preservation solution provided by the invention can be selected from 7-8, and examples of the pH value of the system are 7, 7.5, 8 and the like. The inventors of the present application found through a large number of experiments that the above pH conditions are advantageous for achieving good preservation effects of both DNA and RNA.
In some embodiments, the urine preservation solution further comprises a pH buffer. The pH buffer can stabilize the pH of the urine preservation solution within a suitable range, such as between 7 and 8. The pH buffer may be selected from one or more of Tris-HCl, citric acid, trisodium citrate, phosphate, acetate, borate and carbonate. In some embodiments, the pH buffer is a combination of Tris-HCl, citric acid and trisodium citrate, and the pH of the urine sample can be effectively stabilized between 7 and 8 by the combination, so that cell rupture, genomic DNA interference and the like caused by pH fluctuation can be prevented. In some of these embodiments, the mass ratio of Tris-HCl, citric acid, and trisodium citrate is (44% ± 2%): (37% ± 2%): (67% ± 2%). The pH buffer is preferably used in an amount to stabilize the pH of the system within a predetermined range.
In some embodiments, the urine retention solution is an aqueous solution comprising the following mass percent: 0.02% -0.1% of diethyl pyrocarbonate, 0.2% -0.8% of tween 20, 2% -10% of ethylenediamine tetraacetic acid, 1% -5% of glycine, 1% -5% of paraformaldehyde and 1% -5% of diazolidinyl urea, and further, the pH value of the urine preservation solution is selected from 7-8. Further, the pH of the urine preservation solution is adjusted to 7-8 by using a combination of Tris-HCl, citric acid and trisodium citrate.
In some embodiments, the urine retention solution is an aqueous solution comprising the following mass percent: 0.08-0.1% of diethyl pyrocarbonate, 0.4-0.5% of tween 20, 5-6% of ethylenediamine tetraacetic acid, 4-5% of glycine, 1.3-1.5% of paraformaldehyde and 1.3-1.5% of diazolidinyl urea, and further, the pH value of the urine preservation solution is selected from 7.5-8. Further, the pH of the urine preservation solution is adjusted to 7.5-8 by using a combination of Tris-HCl, citric acid and trisodium citrate.
In a second aspect of the invention there is provided a method of preserving urine using the urine preservative fluid of the first aspect of the invention, which is simple to operate, has a long shelf life, and can be stored for up to 7 days or more.
In some embodiments, the method of preserving urine comprises the steps of: the urine to be preserved is mixed with the urine preservation solution of the first aspect of the present invention. Mixing methods are known to those skilled in the art. For example, after mixing at room temperature, shaking and mixing.
In some embodiments, the urine retention solution: the ratio of the amount of urine to be stored can be (15-25): 1, calculated by volume ratio. For example 19:1 by volume.
In some embodiments, the volume of urine is diluted 10-20 times, such as 10-fold, 15-fold, 20-fold, etc., with a urine retention solution.
In some embodiments, the urine retention solution: the dosage ratio of the urine to be stored can be (15-25): 1, calculated by mass ratio. For example 19:1, in mass ratio.
In some embodiments, urine preservation solution is used to dilute the mass of urine by a factor of 10-20, such as, for example, 10-fold, 15-fold, 20-fold, etc.
In some embodiments, after mixing the urine and the urine retention solution, the working concentrations of the components in the resulting mixture may be: 0.004% DEPC, 0.024% Tween 20, 0.291% ethylenediamine tetraacetic acid (EDTA), 0.243% glycine, 0.073% paraformaldehyde, 0.073% diazolidinyl urea, 0.022% Tris-HCl,0.019% citric acid and 0.034% trisodium citrate, and the concentration fluctuation range can be (+ -) (0-0.001%) in terms of mass percent (wt%).
After mixing the urine and the urine preservation solution, the pH (i.e., the working pH) of the resulting mixture may be selected from 7-8.
The mixed solution after even mixing can be stored under the condition of room temperature (such as 4 ℃ to 35 ℃).
In some embodiments, more accurate free DNA and RNA information is provided after 7 days or more of storage, degradation of nucleic acid material is inhibited, and interfering signals, including but not limited to genomic DNA and microbial interference, are suppressed, and the overall detection solution results are still more sensitive and accurate. See the following examples.
In a third aspect of the invention, a urine retention tube is provided that provides a pre-packaged urine sample collection tube that facilitates clinical handling.
The urine retention tube is a sample collection tube containing the urine retention solution of the first aspect of the invention.
In some embodiments, the urine retention tube comprises a container and a urine retention solution contained in the container, the urine retention solution being the urine retention solution of the first aspect of the invention or a concentrate thereof. In some of these embodiments, the urine retention solution or concentrate thereof is hermetically preserved. In some embodiments, the volume of the container is 11mL and the graduation line is 10mL. In some embodiments, the volume of urine retention solution in each urine retention tube is 0.5mL.
In some embodiments, the package is ready for use without additional weighing, dispensing, and dilution. The urine sample with preset quantity can be directly added into the urine preservation tube, and the urine sample is preserved after being uniformly mixed and used as a sample to be detected.
In some embodiments, the concentrate of urine retention solution may be diluted for use after unpacking. The dilution may be performed with water or an aqueous solution of the buffer of the first aspect of the invention.
In some embodiments, the container is provided with graduations that may be used to indicate the amount of substance added.
The following are some specific examples.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy and/or operational accuracy.
In the following examples, DEPC is diethyl pyrocarbonate and EDTA is ethylenediamine tetraacetic acid.
In the following examples, the procedure not limited to the reaction temperature was carried out at an ordinary temperature of 4℃to 35 ℃.
In the examples below, the concentration percentages are referred to as mass percentages wt% unless indicated.
EXAMPLE 1 DNA preservation Capacity
The preparation method of the urine preservation solution comprises the following steps: 0.1wt% diethyl pyrocarbonate (DEPC) was prepared in a fume hood using ultrapure water, and solution 1 was prepared using the DEPC solution as a vehicle, solution 1 containing the following final concentration reagents in mass percent (wt%): 0.5% Tween 20,6% ethylenediamine tetraacetic acid (EDTA), 5% glycine, 1.5% paraformaldehyde and 1.5% diazolidinyl urea, and the pH of the solution was adjusted to 8 with hydrochloric acid and sodium hydroxide. The concentration of DEPC in solution 1 was estimated to be about 0.09wt%. The ultrapure water is used for preparing solutions 2, 3 and 4, and the solutions 2, 3 and 4 respectively contain final concentration reagents with mass percent: solutions 2, 3 and 4 were prepared from 44% Tris-HCl, 37% citric acid and 67% trisodium citrate, and the pH of the solutions was adjusted to 8 with hydrochloric acid and/or sodium hydroxide, respectively. Sterilizing the prepared solution at high temperature for 30 minutes, and cooling the solution for later use. And (3) preparing the preservation solution according to the volume of the preservation solution to be prepared, if the volume of the preservation solution to be prepared is V, mixing the solutions 1, 2, 3 and 4 with the volumes of 0.97V, 0.01V and 0.01V respectively, using ultrapure water to fix the volume to the volume V, and measuring the pH value to be 8.
In the example, morning urine of a healthy person (sample 1) and a haematuria patient (sample 2 and sample 3) is adopted respectively, each sample is divided into 16 parts after being uniformly mixed, 10mL of each part is filled into a sterile centrifuge tube, and 8 tubes in the sample are randomly picked according to urine and preservation solution 19:1 (the operation is completed within 1 hour of urine separation), the control group does not treat the rest 8 tubes of urine, and the implementation group (the implementation case) and the control group (the control case) are different in that no preservation solution is added in the control case, and the two groups of specimens are preserved at normal temperature. Taking 1 tube for total urine nucleic acid extraction at the following 4 time points, taking 1 tube for nucleic acid extraction of urine supernatant and urine precipitation cells, and taking 4mL of total urine to extract strictly according to the specification, wherein the total urine extraction kit is a common commercial serum/plasma circulating DNA kit within 2 hours of the day of urine separation, 3 days of normal temperature preservation, 5 days of normal temperature preservation and 7 days of normal temperature preservation. The urine supernatant extraction kit is a common commercial serum/plasma circulating DNA kit, and 4mL of urine supernatant is extracted strictly according to the specification by centrifuging a centrifuge tube at 3500rpm for 10 minutes. The urine sediment cell extraction kit is a blood/cell/tissue genome DNA extraction kit, a centrifuge tube is centrifuged at 3500rpm for 10 minutes, supernatant is removed, 500 mu L of PBS is used for resuspension sediment, and 500 mu L of resuspension is taken for extraction according to the specification. The results of the concentration of the extracted nucleic acid can be seen in Table 1, and the results can be seen in FIGS. 1 and 2.
According to the experimental results (see table 1, fig. 1 and fig. 2), after urine is preserved for 7 days at normal temperature under the protection of preservation solution, nucleic acid of whole urine is not obviously changed, and nucleic acid of whole urine without preservation solution is obviously increased, because microorganisms in urine can be greatly propagated under the condition without preservation solution, and the urine preservation solution can inhibit the propagation of microorganisms under the condition of normal temperature. The urine can be preserved for at least 7 days at normal temperature under the protection of the preservation solution, and free nucleic acid in the urine supernatant has no obvious change, which indicates that the free nucleic acid has no obvious degradation, and also indicates that urine abscisic cells have no obvious rupture and no genomic DNA pollution free nucleic acid is released. In contrast, in the control group, the cfDNA of the urine supernatant was monitored, and it was found that one sample nucleic acid was continuously increased, wherein the two sample nucleic acids were increased and then decreased, and it was likely that the urine supernatant nucleic acid was increased due to the rupture of the urine abscisic cells, and the nucleic acid was slowly degraded due to the release of nuclease at the same time. Urine sediment genomic DNA is monitored in a control group, one sample is found to be obviously and continuously increased, two samples are raised and then lowered, and the result of combining with whole urine nucleic acid shows that urine without preservation solution is likely to have mass microbial reproduction under normal temperature conditions, so that the total amount of the sediment nucleic acid is increased, and nuclease released by cell rupture can lead to slow degradation of the nucleic acid. It was found that when the urine retention solution was not added, the information of the free nucleic acid changed greatly at the room temperature storage condition. In contrast, after the preservation solution of example 1 was added, urine was preserved for at least 7 days at normal temperature, and neither the urine supernatant cfDNA nor urine sediment genomic DNA was significantly changed, and no significant cell rupture and microbial proliferation were observed during preservation.
TABLE 1 comparison of total urine, urine supernatant and urine sediment nucleic acid extraction concentration with or without preservative fluid for different days of storage
As can be seen from the comparison result of FIG. 2, after urine is preserved for 7 days at normal temperature under the protection of the preservative, the urine sediment genomic DNA has no obvious change, which indicates that urine abscisic cells have no obvious rupture; from the results of the control group, it can be inferred that the urine sediment nucleic acid is increased without adding urine preservation solution, and that the nucleic acid is gradually degraded by nuclease while a large amount of microorganisms are propagated in urine to increase the nucleic acid. Under the condition that urine is free of urine preservation solution, the molecular signals of microorganisms, the free nucleic acid signals of people in urine supernatant and the genome signals of people in urine sediment can interfere with each other, so that the test result is inaccurate. The urine preservation solution can play a very good role in protecting urine at normal temperature, can inhibit the propagation of microorganisms, can prevent cell rupture and degradation of free nucleic acid, and effectively maintains the stability of free nucleic acid and exfoliated cells.
EXAMPLE 2 RNA preservation Capacity
In this example, morning urine of a patient with hematuria (sample 4) is adopted, each sample is divided into 8 parts after being uniformly mixed, 10mL of each part is filled into a centrifuge tube without nuclease, 4 tubes of the sample are randomly picked, and the preservation solution 19 is prepared according to urine and embodiment 1: 1 (the operation is completed within 0.5 hour of urine separation), the control group does not treat the rest 4 tubes of urine, and the implementation group (the implementation case) and the control group (the control case) are different in that no urine preservation solution is added in the control group, and the two groups of specimens are preserved at normal temperature. The RNA extraction and QPCR (real-time fluorescence quantitative PCR) detection of RNA were performed by taking 1 tube from each of the two groups at the following 4 time points, and 300. Mu.L of whole urine was directly extracted according to the specification requirements within 1 hour of the day of urine isolation, 3 days at room temperature, 5 days at room temperature, and 7 days at room temperature, with the whole urine extraction kit being a nucleic acid extraction or purification reagent (Santa Clara S10015, santa Clara Biotechnology Co., ltd.). The urine sediment cell extraction kit is a nucleic acid extraction or purification reagent (St. Hunan S10015), a centrifuge tube is centrifuged at 3500rpm for 10 minutes, the supernatant is removed, 300 mu L of PBS is used for resuspension sediment, and 300 mu L of resuspension is taken for extraction according to the specification. The extracted eluent adopts a novel coronavirus 2019-nCoV nucleic acid detection kit (Santa Hunan biotechnology Co., ltd.) to detect RNA, and the internal standard of the kit is set for a housekeeping gene GAPDH of a human, so that the content of the human RNA can be detected, and the kit can be used for comparing and analyzing the preservation condition of the RNA of an implementation group and a comparison group. The amplification CT values of the human internal standard genes of the two groups can be seen in Table 2 and FIG. 3. The CT value represents the cycle number experienced by the fluorescent signal in each reaction tube when reaching a set threshold value, the numerical value of CT can reflect the concentration of a detection sample, the lower the CT value is, the higher the concentration of the nucleic acid of the detection sample is, and the higher the CT value is, the lower the concentration of the nucleic acid in the detection sample is; sample nucleic acid concentrations that differ by 1 CT value are twice different, by 2 CT values are four times different, and so on.
According to the experimental results (see Table 2), the control group without urine preservative is placed at normal temperature from day 3 to day 7, the RNA content is obviously reduced (the CT value is higher than that of the implementation case group), and after the control group is preserved for 7 days, the RNA content of whole urine and urine sediment is reduced by more than 10 times, which indicates that the RNA is severely degraded under the condition without urine preservative. However, after urine is preserved for 7 days at normal temperature under the protection of preservation solution, the RNA content of whole urine and urine sediment is not obviously changed, which indicates that RNA is not obviously degraded. Therefore, the urine preservation solution provided by the application has a very strong protection effect on RNA in urine.
TABLE 2 comparison of CT values of RNA amplification of human housekeeping Gene in Whole urine and urine sediment with preservation solution
Example 3 ability to preserve DNA and RNA simultaneously
In this example, morning urine of patients with hematuria (sample 5 and sample 6) is used, each sample is divided into 8 parts after being uniformly mixed, 10mL of each part is put into a sterile centrifuge tube, and 4 tubes of the sample are randomly picked according to urine and preservation solution 19 prepared in example 1: 1 (the operation is completed within 1 hour of urine separation), the comparison group uniformly mixes the rest 4 tubes of urine with the preservation solution of the urine preservation tube A (the comparison reagent, commodity number: CW 2657) according to the requirement of the urine preservation tube A (the operation is completed within 1 hour of urine separation), and the implementation group (the example group) and the comparison group are different in preservation solution and the two groups of specimens are preserved at normal temperature. At the following 4 time points, 1 tube is taken for extracting free nucleic acid of urine supernatant, and urine is stored for 3 days at normal temperature, 5 days at normal temperature and 7 days at normal temperature within 2 hours of the day of separating from the body. The extraction kit of the free DNA of the urine supernatant is a common commercial serum/plasma circulating DNA kit, a centrifuge tube is centrifuged at 3500rpm for 10 minutes, and 4mL of urine supernatant is extracted strictly according to the specification. The results of the extracted free DNA can be seen in Table 3 and the comparison of the results can be seen in FIG. 4. The whole urine extraction kit is a nucleic acid extraction or purification reagent (St. Hunan S10015), and 300 mu L of whole urine is directly extracted according to the specification. The extracted eluent is subjected to RNA detection by using a novel coronavirus 2019-nCoV nucleic acid detection kit (St. Hunan) with an internal standard set for a housekeeping gene GAPDH of a human, so that the content of human RNA can be detected, and the extracted free DNA result can be seen in Table 4.
According to the experimental results (see fig. 4 and table 3), the preservation solution of the example group had a more stable protective effect than the comparative reagent, and the free DNA of the example was not significantly degraded for 0 to 7 days, but there was less degradation in the comparative group. Comparing the example group with the control group, it can be seen that the urine preservation solution of the invention has more stable protection effect on urine free DNA.
TABLE 3 comparison of the free DNA extraction concentration of urine supernatant from different preservation days for different preservation solutions
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TABLE 4 comparison of RNA amplification CT values of human housekeeping genes of whole urine for different preservation days in different preservation solutions
According to the experimental results (see table 4), the preservation solution of the example group has a more stable protective effect than the comparative reagent, and the CT value of the example group has no obvious change from 0 to 7 days, which indicates that the RNA content has no obvious change, the visible RNA has no obvious degradation, and the CT value of the comparative group has obviously increased, which indicates that the RNA content gradually decreases, and the visible RNA in the urine supernatant has obvious degradation. The urine preservation solution of the example group has more stable protection effect on RNA than the control group.
EXAMPLE 4 presence or absence of added RNase inhibitor
Implementation group (implementation case) the method of preparing urine preservation solution is identical to the implementation case in example 1. The control (control) was formulated substantially similar to the stock solutions of the examples, but no diethyl dicarbonate was added to the formulation, and the remaining steps remained the same.
Morning urine of a patient with hematuria (sample 7) is adopted, the sample is evenly mixed and divided into 8 parts, 10mL of each part is filled into a nuclease-free centrifuge tube, 4 tubes of the sample are randomly picked by a comparison group, and the preservation solution 19 is prepared according to urine and comparison cases: 1, mixing evenly; implementation group remaining 4 tubes were prepared with urine and implementation case as preservative fluid 19:1 (the operation is completed within 0.5 hour of urine separation), and the two groups of specimens are stored at normal temperature. Taking 1 tube for total urine RNA extraction and QPCR detection of RNA at the following 4 time points, wherein the total urine extraction kit is a nucleic acid extraction or purification reagent (Saint Hunan S10015), and 300 mu L of total urine is directly extracted according to the strict requirements of the specification within 1 hour of urine in vitro, 3 days at normal temperature, 5 days at normal temperature and 7 days at normal temperature. The extracted eluent adopts a novel coronavirus 2019-nCoV nucleic acid detection kit (Saint Hunan) to detect RNA, and the internal standard of the kit is set aiming at a housekeeping gene GAPDH of a human, so that the content of the human RNA can be detected, and the kit can be used for comparing and analyzing the preservation condition of the RNA of an implementation group and a comparison group. The amplification CT values of the human internal standard genes of the two groups can be seen in Table 5.
TABLE 5 comparison of RNA amplification CT values of human housekeeping genes of whole urine with different preservation days in different preservation solutions
According to the experimental results (see table 5), the preservation solution of the implementation group (implementation case) has a more stable protective effect than the preservation solution of the comparison group (comparison case, no DEPC added), the CT value of the comparison group is obviously increased from 0 to 7 days, which indicates that the RNA content is gradually reduced, and the RNA in the urine supernatant is obviously degraded, but the implementation group is not obviously degraded. Therefore, the urine preservation solution added with diethyl pyrocarbonate has a strong protection effect on RNA.
Example 5 different RNase inhibitor concentrations
This example is substantially similar to the preservative fluid formulation of example 1, but the formulation provides 3 different concentration gradients of diethyl pyrocarbonate (rnase inhibitor), 0.01wt% in group a, 0.1wt% in group B, and 1wt% in group C. In this example, morning urine from a patient with hematuria (sample 8) was used, the sample was homogenized and divided into 12 portions, 10mL portions each were placed into a nuclease-free centrifuge tube, and 4 tubes of the sample were randomly picked up and mixed with the preservative fluid 19 prepared in example 5-A: 1, mixing evenly; 4 tubes of the stock solution 19 prepared from urine and example 5-B were randomly picked: 1, mixing evenly; remaining 4 tubes were prepared with urine and preservation solution 19 from example 5-C: 1, and preserving the three groups of specimens at normal temperature. Taking 1 tube for total urine RNA extraction and QPCR detection of RNA at the following 4 time points, wherein the total urine extraction kit is a nucleic acid extraction or purification reagent (Saint Hunan S10015), and 300 mu L of total urine is directly extracted according to the strict requirements of the specification within 1 hour of urine in vitro, 3 days at normal temperature, 5 days at normal temperature and 7 days at normal temperature. The extracted eluent adopts a novel coronavirus 2019-nCoV nucleic acid detection kit (Saint Hunan) to detect RNA, and the internal standard of the kit is set aiming at a housekeeping gene GAPDH of a human, so that the content of the human RNA can be detected, and the kit can be used for comparing and analyzing the preservation condition of the RNA of an implementation group and a comparison group. The amplification CT values of the human internal standard genes of the two groups can be summarized in Table 6.
TABLE 6 comparison of RNA amplified CT values of human housekeeping genes of whole urine with different preservation days in different preservation solutions
According to the experimental results, referring to table 6, the protection effect of RNA is better when the concentration of diethyl pyrocarbonate is 0.1% and 1%, the CT value is not significantly changed, which indicates that the RNA content is not significantly changed, and the RNA in the urine supernatant is not significantly degraded, and considering that the cost of diethyl pyrocarbonate is higher, 0.1% is more beneficial to saving the cost. However, when the concentration of diethyl pyrocarbonate is 0.01%, the CT value is obviously increased, which shows that the RNA content is gradually reduced, and the RNA in the urine supernatant has obvious RNA degradation and poor preservation effect.
Example 6 with or without addition of aldehyde quencher
Implementation group (implementation case) the method of preparing urine preservation solution is identical to the implementation case in example 1. The control (control) was formulated substantially similar to the preservative solution of the embodiment, but no aldehyde quencher was added to the formulation, and the remaining steps remained identical.
Morning urine of a haematuria patient (sample 9), a diabetes patient (sample 10) and a healthy patient (sample 11) is adopted, each sample is divided into 8 parts after being uniformly mixed, 10mL of each part is filled into a nuclease-free centrifuge tube, 4 tubes of the preservative solution 19 prepared according to urine and a comparison case are randomly selected by a comparison group: 1, mixing evenly; implementation group remaining 4 tubes and implementation case formulated preserved urine and real solution 19:1 (the operation is completed within 0.5 hour of urine separation), and the two groups of specimens are stored at normal temperature. Taking 1 tube for total urine RNA extraction and QPCR detection of RNA at the following 4 time points, wherein the total urine extraction kit is a nucleic acid extraction or purification reagent (Saint Hunan S10015), and 300 mu L of total urine is directly extracted according to the strict requirements of the specification within 1 hour of urine in vitro, 3 days at normal temperature, 5 days at normal temperature and 7 days at normal temperature. The extracted eluent adopts a novel coronavirus 2019-nCoV nucleic acid detection kit (Saint Hunan) to detect RNA, and the internal standard of the kit is set aiming at a housekeeping gene GAPDH of a human, so that the content of the human RNA can be detected, and the kit can be used for comparing and analyzing the preservation condition of the RNA of an implementation group and a comparison group. The amplification CT values of the human internal standard genes of the two groups can be seen in Table 7.
According to the experimental results, see table 7, there was no significant difference between the control group and the control group for the normal human samples. However, aiming at the blood urine sample and the urine sample of the diabetic patient, the implementation group (adding the aldehyde quencher) has a more stable protection effect than the preservation solution of the comparison group (without the aldehyde quencher), the CT value of the RNA detection of the comparison group is obviously increased from 0 to 7 days, which proves that the RNA content is gradually reduced, the preservation solution of the comparison group has obvious degradation in the blood urine sample and the urine sample of the diabetic patient, the CT value of the RNA detection of the implementation group has no obvious change, which proves that the RNA content has no obvious change, and the RNA of the implementation group has no obvious degradation. Therefore, for special samples, especially blood urine samples and urine samples of diabetics, the urine preservation solution added with the aldehyde quencher provided by the invention has a strong protection effect on RNA.
TABLE 7 comparison of RNA amplification CT values of human housekeeping genes of whole urine for different preservation days in different preservation solutions
Example 7 investigation of pH Effect
Implementation group (implementation case) the method of preparing urine preservation solution is identical to the implementation case in example 1. The comparative group (comparative case) was formulated substantially in accordance with the preservative solution of the embodiment, but when the pH was adjusted, the pH of solutions 1, 2, 3, and 4 was adjusted to 6 with hydrochloric acid and/or sodium hydroxide solution, the remaining steps remained in accordance, and the pH of the urine preservative solution formulated in the comparative case was about 6. Morning urine of a patient with hematuria (sample 12) is adopted, each sample is divided into 8 parts after being uniformly mixed, 10mL of each part is filled into a nuclease-free centrifuge tube, 4 tubes of the preservative solution 19 prepared according to urine and a comparison case are randomly picked by a comparison group: 1, mixing evenly; control group the remaining 4 tubes of the preserved urine formulated in case 1 with real solution 19:1 (the operation is completed within 0.5 hour of urine separation), and the two groups of specimens are stored at normal temperature. Taking 1 tube for total urine RNA extraction and QPCR detection of RNA at the following 4 time points, wherein the total urine extraction kit is a nucleic acid extraction or purification reagent (Saint Hunan S10015), and 300 mu L of total urine is directly extracted according to the strict requirements of the specification within 1 hour of urine in vitro, 3 days at normal temperature, 5 days at normal temperature and 7 days at normal temperature. The extracted eluent adopts a novel coronavirus 2019-nCoV nucleic acid detection kit (Saint Hunan) to detect RNA, and the internal standard of the kit is set aiming at a housekeeping gene GAPDH of a human, so that the content of the human RNA can be detected, and the kit can be used for comparing and analyzing the preservation condition of the RNA of an implementation group and a comparison group. The amplification CT values of the human internal standard genes of the two groups can be seen in Table 8.
TABLE 8 comparison of RNA amplification CT values of human housekeeping genes of whole urine for different days of preservation in different pH preservation solutions
According to the experimental results, see Table 8, the preservation solution has good RNA protection effect and no obvious degradation when the pH value is 8. And when the pH value of the preservation solution is 6, the CT value of RNA detection is obviously increased, which indicates that the content of RNA is gradually reduced, and indicates that more obvious RNA degradation exists and the preservation effect is poor.
Example 8.
The preparation method of the urine preservation solution comprises the following steps: the solutions of the respective groups were prepared as solvents for the respective groups of solutions 1 based on the respective components and the respective concentrations of the corresponding RNase inhibitors in Table 9 using ultrapure water in a fume hood, and the respective groups of solutions 1 were prepared based on the respective components and the respective concentrations of the corresponding DNase inhibitors, nonionic surfactants, cell fixatives, preservatives, aldehyde quenchers in Table 9, and the respective pH values of the respective groups of solutions were adjusted to the pH values of the respective groups of solutions 1 in Table 9 with hydrochloric acid and/or sodium hydroxide, respectively. Buffer solutions 2, 3, 4 of each group were prepared using ultrapure water, and solutions 2, 3, 4 of each group were prepared according to the respective components and the respective concentrations of the buffer solutions 2, 3, 4 in table 9, and the pH of each group of buffer solutions was adjusted to the pH values of the respective groups of solutions 2, 3, 4 in table 9 with hydrochloric acid and/or sodium hydroxide, respectively. All the prepared solutions are sterilized at high temperature for 30 minutes, and the solutions are cooled for later use. The stock solutions of each group were prepared in volumes corresponding to solutions 1, 2, 3, and 4 in the stock solutions of Table 9, and each was independently prepared using water to determine 100mL, and the pH of each group was the pH corresponding to each group in Table 9.
In the example, mixed morning urine (sample 13) of a haematuria patient is adopted, the sample is divided into 48 parts after being uniformly mixed, 10mL of each part is filled into a sterile centrifuge tube, and 8 tubes in each implementation group (8-1, 8-2, 8-3, 8-4 and 8-5) and a control group (D8-1) are randomly picked according to urine and preservation solution 19:1 (the operation is completed within 1 hour of urine separation), and each group is different in that different preservation solutions are added, and each group of specimens is preserved at normal temperature. At 4 time points (0 day, 3 day, 5 day, 7 day), 1 tube was taken for total urine RNA extraction and QPCR detection of RNA, 1 tube was taken for nucleic acid extraction of urine supernatant and urine precipitation cells, and urine was stored for 3 days at room temperature, 5 days at room temperature, and 7 days at room temperature within 2 hours of the day of ex vivo. The RNA extraction kit of the whole urine is a nucleic acid extraction or purification reagent (Saint Hunan S10015), and 300 mu L of the whole urine is directly extracted according to the specification. The extracted eluent adopts a novel coronavirus 2019-nCoV nucleic acid detection kit (Saint Hunan) to detect RNA, and the internal standard of the kit is set aiming at a housekeeping gene GAPDH of a human, so that the content of the human RNA can be detected, and the kit can be used for comparing and analyzing the preservation condition of each group of human RNA. The amplification CT values of the human internal standard genes of each group can be seen in Table 10.
The nucleic acid extraction kit of the urine supernatant is a common commercial DNA extraction kit, and 4mL of urine supernatant is extracted strictly according to the specification by centrifuging a centrifuge tube at 3500rpm for 10 minutes. The urine sediment cell extraction kit is a common commercial blood/cell/tissue genome DNA extraction kit, a centrifuge tube is centrifuged at 3500rpm for 10 minutes, supernatant is removed, 500 mu L of PBS is used for resuspension sediment, and 500 mu L of heavy suspension is taken for extraction according to the specification. The results of the concentration of the nucleic acid after extraction in each group can be seen in Table 11.
Table 9. Urine preservation solution preparation Table (percent concentration "%" unit is wt%)
TABLE 10 comparison of RNA amplification CT values of human housekeeping genes of whole urine for different preservation days in different preservation solutions
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As can be seen from Table 10, the CT values detected by the implementation groups 8-1, 8-2, 8-3, 8-4 and 8-5 at 4 time points are not changed obviously, which indicates that the RNA content is not changed obviously, the degradation of the visible RNA is not caused, and the preservation solutions of the implementation groups can play a good role in protecting the RNA. And the CT value of the control group D8-1 is obviously increased along with the increase of the preservation time, which shows that the RNA content is obviously reduced, the visible RNA is obviously degraded, and the protection effect on the RNA of the urine sample is poor.
TABLE 11 comparison of urine supernatant and urine sediment nucleic acid extraction concentration for different days of preservation for different preservation solutions
As can be seen from Table 11, the samples of the respective samples 8-1, 8-2, 8-3, 8-4 and 8-5 were not significantly changed in both the concentration of cfDNA extracted from urine supernatant and the concentration of genomic DNA extracted from urine sediment, and the preservation solutions of the samples were found to have a good protective effect on free DNA degradation and breakage of urine sediment cells within 7 days. The phenomenon that the extraction concentration of precipitated genomic DNA is low and the extraction concentration of cfDNA of urine supernatant is high occurs in the control group D8-1 on the day 0, because the preservation solution of the control group causes cell rupture in urine and genomic DNA in the cells enters the urine supernatant. In the control group D8-1, no matter the urine supernatant cfDNA or urine sediment genomic DNA increases along with the preservation time, the extraction concentration is obviously reduced, obvious degradation of DNA is visible, and the preservation solution of the control group has insufficient protection effect on urine sample DNA.
The technical features of the foregoing embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the foregoing embodiments and examples are not described, however, they should be considered to be within the scope of the description provided in this specification as long as there is no contradiction between the combinations of the technical features.
The above examples merely represent several embodiments of the present invention, which facilitate specific and detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present invention. It should also be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
Claims (10)
1. The urine preservation solution is characterized by being an aqueous solution of the following substances in percentage by mass: 0.05% -1% of an RNase inhibitor, 2% -10% of a DNase inhibitor, 0.2% -0.8% of a nonionic surfactant, 1% -5% of a cell fixative, 1% -5% of a preservative, 1% -5% of an aldehyde quencher and a pH buffer, wherein the pH value of the urine preservation solution is selected from 7-8;
Wherein the rnase inhibitor comprises at least diethyl pyrocarbonate and the dnase inhibitor comprises at least ethylenediamine tetraacetic acid; the mass percentage of the diethyl pyrocarbonate in the urine preservation solution is 0.05-0.15%, and the mass percentage of the ethylenediamine tetraacetic acid in the urine preservation solution is 2-10%;
the nonionic surfactant is selected from one or two of Tween 20 and Triton X-100;
the cell fixative is one or two selected from paraformaldehyde and imidazolidinyl urea;
the preservative is selected from one or more of diazolidinyl urea, paraformaldehyde, formalin, hydantoin and trinitromethane;
the aldehyde quencher is selected from one or more of glycine, arginine, and lysine.
2. The urine retention solution according to claim 1, wherein the mass percentage of the diethyl pyrocarbonate in the urine retention solution is 0.05% -0.1%.
3. The urine retention solution according to claim 1, wherein the mass percentage of ethylene diamine tetraacetic acid in the urine retention solution is 4% -10%.
4. The urine retention solution of claim 1, wherein the rnase inhibitor further comprises one or both of ammonium sulfate and guanidine isothiocyanate; and/or the number of the groups of groups,
The dnase inhibitor further comprises one or both of diethyl triamine pentaacetic acid and citric acid.
5. The urine retention solution according to any one of claims 1 to 4,
the cell fixative is paraformaldehyde;
the preservative is a combination of diazolidinyl urea and paraformaldehyde;
the mass ratio of diazoalkyl urea to paraformaldehyde in the urine preservation solution is (0.8-1.2) 1;
the aldehyde quencher is glycine;
the urine preservation solution is an aqueous solution of the following substances in percentage by mass: 0.05 to 0.1 percent of diethyl pyrocarbonate, 0.2 to 0.8 percent of tween 20, 2 to 10 percent of ethylenediamine tetraacetic acid, 1 to 5 percent of glycine, 1 to 5 percent of paraformaldehyde and 1 to 5 percent of diazolidinyl urea;
the pH buffer is selected from one or more of Tris-HCl, citric acid, trisodium citrate, phosphate, acetate, borate and carbonate.
6. The urine retention solution according to claim 1, wherein the urine retention solution is an aqueous solution of the following substances in mass percent: 0.08% -0.1% of diethyl pyrocarbonate, 0.4% -0.5% of tween 20, 5% -6% of ethylenediamine tetraacetic acid, 4% -5% of glycine, 1.3% -1.5% of paraformaldehyde, 1.3% -1.5% of diazolidinyl urea and a pH buffer, wherein the pH value of the urine preservation solution is selected from 7.5-8.
7. The urine preservation solution according to any one of claims 1 to 4 and 6, wherein the pH buffer is a combination of Tris-HCl, citric acid and trisodium citrate, wherein the mass ratio of Tris-HCl, citric acid and trisodium citrate is (44% ± 2%): (37% ± 2%): (67% ± 2%).
8. The urine preservation method is characterized by comprising the following steps: mixing urine to be preserved with the urine preservation solution according to any one of claims 1 to 7.
9. The preservation method of claim 8, wherein the urine preservation solution: the dosage ratio of the urine to be stored is (15-25) 1, calculated by volume ratio or calculated by mass ratio;
the urine to be preserved is from urine of patients with haematuria and diabetics.
10. A urine retention tube comprising the urine retention solution according to any one of claims 1 to 7.
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