CN108642042A - A kind of preservative agent and device of urine nucleic acid - Google Patents
A kind of preservative agent and device of urine nucleic acid Download PDFInfo
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- CN108642042A CN108642042A CN201810333596.5A CN201810333596A CN108642042A CN 108642042 A CN108642042 A CN 108642042A CN 201810333596 A CN201810333596 A CN 201810333596A CN 108642042 A CN108642042 A CN 108642042A
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- nucleic acid
- urine
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- preservative agent
- preservative
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- 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
Abstract
The invention discloses a kind of preservative agents of urine nucleic acid, by mass percentage, including 50~80 parts of buffer solution, 10~30 parts of Antiseptic and fixation agent, 0.5~5 part of nucleic acid inhibitor, 1~5 part of membrane protective agent, 1~5 part of metabolic poison；The present invention further discloses a kind of save sets of urine nucleic acid, including rubber plug, safety cap, collection container and above-mentioned preservative agent.The urine nucleic acid preservation agent that the present invention program provides is a kind of effective room temperature urine preservative agent, urine nucleic acid can be preserved with room temperature 7 days even for more time, also allow for room temperature transport.
Description
Technical field
The present invention relates to nucleic acid preservation fields, particularly relate to a kind of preservative agent and device of urine nucleic acid.
Background technology
Urine is a kind of common biological sample, is generated by human kidney, and body is discharged by ureter, bladder and urethra
Outside.Contain nucleic acid in urine, for human body, the inspection of urine nucleic acid has more hurtless measure compared with the inspection of blood nucleic acid
Property；Urine will not be infected by inhibition of HIV, be not easy to be infected by other pathogenic microorganisms；It is carried from blood plasma and urine
DNA with the RNA methods taken are similar, but the materials of urine will not be limited by measuring；Simultaneously as the albumen in urine
Matter content is less compared with blood, and for PCR processes, the interfering substance in test process is also than few in blood plasma.
It requires to preserve and transport under the conditions of 4 DEG C after urine collecting, centrifuges urine supernatant urinary precipitation as early as possible, if
Long-term preservation need to be transferred to -80 DEG C of ultra low temperature freezers storages, to ensure the original shape of dissociative DNA in urine down to greatest extent
State.Nonetheless, low temperature is relied solely on, it still cannot the effectively dissociative DNA in preservation urine sample within a certain period of time.
Therefore, a growth that can effectively inhibit bacterium, fungi etc. in nuclease and sample is developed, it can be steady under room temperature
Surely preserving the preservative agent of the nucleic acid in urine is necessary.
Invention content
In view of this, it is an object of the invention to propose a kind of preservation that can stablize preservation urine amplifying nucleic acid at room temperature
Agent and device.
A kind of preservative agent of urine nucleic acid, by mass percentage, including 50~80 parts of buffer solution, Antiseptic and fixation agent 10~
30 parts, 0.5~5 part of nucleic acid inhibitor, 1~5 part of membrane protective agent, 1~5 part of metabolic poison.
The buffer solution is sodium citrate buffer solution, and mass concentration is 2%~6%.
The Antiseptic and fixation agent is selected from formaldehyde and its derivative, sodium hydroxymethylglycinate, dimethylol urea, diazonium alkyl
One or more in urea, imidazolidinyl urea, mass concentration is 200~700g/L.
The nucleic acid inhibitor is selected from heparin, aurin tricarboxyli acid (ATA), formamide, ethylenediamine tetra-acetic acid, DTT, two sulphur threoses
Alcohol is one or more.
The membrane protective agent includes glycine, aspartic acid, half Guang amide, glutamine, glyceraldehyde-3-phosphate one
Kind is a variety of.
The metabolic poison includes dihydroxyacetone phosphate, mannose, glyceraldehyde, sodium fluoride, pyruvic acid, glycerine.
In addition, the present invention also provides a kind of save set of urine nucleic acid, including rubber plug, safety cap, collection container and
The preservative agent of urine nucleic acid described above.
The collection container is glass or plastic production, rubber plug are that rubber makes, safety cap is plastic production.
Further, the collection container is polyethylene naphthalate or polyethylene terephthalate making, institute
Rubber plug is stated to make for butyl rubber.
There is helicitic texture, rubber plug to be built in safety cap, internal threading arrangement increases safety inside the safety cap
Frictional resistance between cap and rubber plug is not in rubber plug and peace while operating personnel pull open safety cap from vacuum blood collection tube
The situation of full cap separation, it is easy to operation.
The urine nucleic acid preservation agent that the present invention program provides is a kind of effective room temperature urine preservative agent, wherein containing quality
The membrane protective agent and 1~5 part of metabolic poison that number is 1~5 part, the two collective effect can ensure to fall off in urine thin
The stabilization of born of the same parents, the nucleic acid inhibitor that 0.5~5 part of mass fraction can ensure that the DNA to dissociate in urine is non-degradable, while at it
Under the synergistic effect of his additive, the urine nucleic acid preservation agent can room temperature preserve urine nucleic acid 7 days even longer time.
Description of the drawings
Fig. 1 is the schematic diagram of urine save set of the present invention；
Fig. 2 is that the embodiment of the present invention 6 is not added with preservative agent, the analysis chart of 0 day extraction urine dissociative DNA；
Fig. 3 is that the embodiment of the present invention 6 is not added with preservative agent, the analysis chart of 7 days extraction urine dissociative DNAs；
Fig. 4 is the embodiment of the present invention 6 plus preservative agent, the analysis chart of 0 day extraction urine dissociative DNA；
Fig. 5 is the embodiment of the present invention 6 plus preservative agent, the analysis chart of 7 days extraction urine dissociative DNAs；
Fig. 6 be 7 room temperature of the embodiment of the present invention, 4 DEG C, under 37 DEG C of storage, preservative agent is added and is not added with preservative agent and is externally added
Urine internal reference (NCS) DNA stability influences；
Fig. 7 a are the amplification curve and solubility curve of internal reference (NCS) DNA when embodiment of the present invention 7 is not added with preservative agent；
The amplification curve and solubility curve of internal reference (NCS) DNA when Fig. 7 b are the embodiment of the present invention 7 plus preservative agent；
Fig. 8 be 8 room temperature of the embodiment of the present invention, 4 DEG C, 37 DEG C storage under, be added preservative agent and be not added with preservative agent to urine from
Body internal reference (RPL13a) DNA stability influences；
Fig. 9 a be the embodiment of the present invention 8 when being not added with preservative agent the amplification curve of urine itself internal reference (RPL13a) DNA with it is molten
Solution curve；
The amplification curve of urine itself internal reference (RPL13a) DNA and dissolving when Fig. 9 b are the embodiment of the present invention 8 plus preservative agent
Curve；
Figure 10 be 9 room temperature of the embodiment of the present invention, 4 DEG C, 37 DEG C storage under, be added preservative agent and be not added with preservative agent to urine from
Body internal reference (GAPDH) DNA stability influences；
Figure 11 a are the amplification curve of urine itself internal reference (GAPDH) DNA when embodiment of the present invention 9 is not added with preservative agent；
The amplification curve of urine itself internal reference (GAPDH) DNA when Figure 11 b are the embodiment of the present invention 9 plus preservative agent.
Specific implementation mode
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.
Embodiment 1
Urine nucleic acid preservation agent prescription 1
Buffer solution is that sodium citrate delays liquid, and mass fraction 65%, Antiseptic and fixation agent are imidazolidinyl urea, and mass fraction is
30%, nucleic acid inhibitor is ethylenediamine tetra-acetic acid, and mass fraction 2%, membrane protective agent are glycine, and mass fraction is
1%, metabolic poison is glyceraldehyde, mass fraction 2%.
Embodiment 2
Urine nucleic acid preservation agent prescription 2
Buffer solution is that sodium citrate delays liquid, and mass fraction 70%, Antiseptic and fixation agent are attached most importance to azanyl urea, and mass fraction is
20%, nucleic acid inhibitor is heparin, and mass fraction 5%, membrane protective agent are aspartic acid, mass fraction 3%, generation
It is dihydroxyacetone phosphate, mass fraction 2% to thank to inhibitor.
Embodiment 3
Urine nucleic acid preservation agent prescription 3
Buffer solution is that sodium citrate delays liquid, and mass fraction 75%, Antiseptic and fixation agent are dimethylol urea, and mass fraction is
15%, nucleic acid inhibitor is edetate, and mass fraction 3%, membrane protective agent are half Guang amide, mass parts
Number is 5%, metabolic poison is mannose, mass fraction 2%.
Embodiment 4
Urine nucleic acid preservation agent prescription 4
Buffer solution is that sodium citrate delays liquid, and mass fraction 80%, Antiseptic and fixation agent are sodium hydroxymethylglycinate, mass parts
Number is 10%, nucleic acid inhibitor ATA, and mass fraction 5%, membrane protective agent are glyceraldehyde-3-phosphate, mass fraction
It is pyruvic acid, mass fraction 2% for 3%, metabolic poison.
Embodiment 5
Plasma DNA (cfDNA) in urine when fluorimeter measures addition urine nucleic acid preservation agent and is not added with preservative agent
Concentration.
(1) working solution is prepared：With fluorescent dye：Sodium citrate buffer=1:200 volume ratio prepares working solution.
(2) working solution of 190uL is added in the cfDNA standard items S1 of a concentration of 0ng/uL of 10uL, obtains standard curve
1；The working solution of 190uL is added in the cfDNA standard items S2 of a concentration of 10ng/uL of 10uL, obtains standard curve 2.
(3) sample test：3uL samples are added in 197uL working solutions, mixing 5S is protected from light 2min, uses Life
Technologies's2.0 fluorimeters measure the cfDNA concentration in urine.
The content that table one is cfDNA in urine under different storage conditions.
Embodiment 6
CfDNA in urine when 2100 biological analysers of Agilent measure addition urine nucleic acid preservation agent and are not added with preservative agent
Concentration.
Required reagent is placed into equilibrium at room temperature 20min, 15uL high sensitivity DNA dyestuffs (indigo plant is managed) are added highly sensitive
Glue in DNA gel matrix bottle (red pipe), and draw 9uL gels-dye mixture and be added in chip, chip is placed into moulding
On device, syringe position is located at 1mL, locks chip, pushes syringe until chip blocks, 60s gently pushes syringe
It is placed at 1mL, takes out chip pick-up 9uL gels-dye mixture.It draws 5uL marker (green pipe) and sets chip all samples slot
In, 1uL ladder (Huang pipe) are drawn to the holes chip marker, draw 1uL samples to chip sample hole, chip is placed and is vibrated
Chip is placed into Agilent 2100 after 2400rpm concussions 1min on device and runs program, and is analyzed, analysis result such as Fig. 2-5 institutes
Show.
Embodiment 7
Test room temperature, 4 DEG C, 37 DEG C of storages externally addition urine internal reference (NCS) DNA influences.
(1) morning awake rear first bubble urina sanguinis (centrifuge tube sterilized 50ml) beaker mixing is collected, after 15mL packing respectively
The preservative agent mixing that 1ml is prepared is added；
(2) standard curve of NCS fragment concentrations is made；
(3) it when fluorescent quantitation CT values are 20 or so, takes NCS solution to dilute 100,000 times and urine is added in 1/20 ratio,
The NCS internal reference segments that 750ul dilutes 100,000 times are added in 15ml urines；
(5) use certain company's urine free nucleic acid extracts kit, respectively extract room temperature, 4 DEG C, 37 DEG C place 0 day, 4 days,
7 days urine dissociative DNAs；
(6) reference gene NCS primers are used to carry out fluorogenic quantitative detection；
(7) test result is as shown in fig. 6-7.
Embodiment 8
Test room temperature, 4 DEG C, 37 DEG C storage on urine itself internal reference (RPL13a) DNA influence.
(1) morning awake rear first bubble urina sanguinis (centrifuge tube sterilized 50ml) beaker mixing is collected, after 15ml packing respectively
The preservative agent mixing that 1ml is prepared is added；
(2) use certain company's urine free nucleic acid extracts kit, respectively extract room temperature, 4 DEG C, 37 DEG C place 0 day, 4 days,
7 days urine dissociative DNAs；
(3) reference gene RPL13a primers are used to carry out fluorogenic quantitative detection；
(4) test result is as Figure 8-9.
Embodiment 9
Test room temperature, 4 DEG C, 37 DEG C storage on urine itself internal reference (GAPDH) DNA influence.
(1) morning awake rear first bubble urina sanguinis (centrifuge tube sterilized 50ml) beaker mixing is collected, after 15ml packing respectively
The preservative agent mixing that 1ml is prepared is added；
(2) use certain company's urine free nucleic acid extracts kit, respectively extract room temperature, 4 DEG C, 37 DEG C place 0 day, 4 days,
7 days urine dissociative DNAs；
(3) reference gene GAPDH primers are used to carry out fluorogenic quantitative detection；
(4) test result is as shown in figs. 10-11.
Comparative example
Urine preserves reagent, is quenched by nucleic acid buffer solution, metal ion chelation agent, preservative, cell fixative and formaldehyde
Agent forms, wherein nucleic acid buffer solution is the Tris-HCl of final concentration 2mmol/L, and work pH is 8；Metal ion chelation agent is eventually
The EDTA of concentration 10mmol/L；Preservative by final concentration 1.25mmol/L citric acid and final concentration 3.75mmol/L citric acid
Sodium forms；Cell fixative is made of the paraformaldehyde, diazonium ureine and imidazolidinyl urea of equivalent, the dosage of cell fixative
It is the 0.1% of total weight；Formaldehyde quencher is made of the glycine, lysine and ethylenediamine of equivalent, and total formaldehyde of mixing is quenched
The final concentration of 40mmol/L of agent is (see patent：Application publication number：CN107881213A).
As described above, as it can be seen from table 1 in the case where room temperature is not added with preservative agent, DNA content prolongs at any time in urine
Length is continuously increased, this is because there is the cell to fall off in urine, cast-off cells are unstable, is easy cracking release gDNA, simultaneously
By Fig. 2-5 it can be seen that extending the cfDNA degradations to dissociate in urine at any time, when causing to measure cfDNA, molecular weight is continuously increased
Situation；And after urine preservative agent is added, the molecular weight of cfDNA extends at any time to be kept approximately constant, this is because preservative agent
In containing mass fraction be 1~5 part membrane protective agent and 1~5 part of metabolic poison, the two collective effect can ensure to urinate
The nucleic acid inhibitor of the stabilization of cast-off cells in liquid, 0.5~5 part of the mass fraction contained simultaneously can ensure that cfDNA does not drop
The collective effect of solution and other reagents, enables to urine nucleic acid to be stabilized at normal temperatures at least 7 days.
In addition, compared with the patent that application publication number is CN107881213A, urine nucleic acid preservation provided by the invention
Agent can preserve 7 days even for more time at normal temperatures, and the urine nucleic acid preservation agent in comparative example is merely able at 4 DEG C
It preserves, contrastingly, the urine nucleic acid preservation agent that patent of the present invention provides more can adapt to the market demand, especially be convenient for room temperature
Therefore lower transport has more economy.
It is externally added for urine internal reference (NCS) DNA, after being stored at room temperature 1 day, compared with original time point, is not added with urine
When liquid nucleic acid preservation agent, the extension of amplification curve (Fig. 7) at any time, appearance time is postponed, and the concentration of internal reference (NCS) DNA is bright
It is aobvious to decline, it is continued to decline after 7 days, by solubility curve (Fig. 8) it can be seen that it is same gene, specific amplification is not present；And
After urine preservative agent is added, the concentration of internal reference (NCS) DNA was still remained unchanged at the 7th day, illustrated it under urine preservative agent state
It is stabilized at least 7 days (as shown in Figure 6), by its amplification curve and solubility curve it is also seen that (as shown in Figure 8)；To urine
For itself internal reference (RPL13a) DNA (such as Fig. 9-11) and itself internal reference (GAPDH) DNA also so.
Therefore, the urine nucleic acid preservation agent that the present invention program provides is a kind of effective room temperature urine preservative agent, wherein containing
There is mass fraction that can ensure in urine for 1~5 part of membrane protective agent and 1~5 part of metabolic poison, the two collective effect
The stabilization of cast-off cells, the nucleic acid inhibitor that 0.5~5 part of mass fraction can ensure that the DNA to dissociate in urine is non-degradable,
Simultaneously under the synergistic effect of other additives, the urine nucleic acid preservation agent can room temperature to preserve urine nucleic acid 7 days even longer
Time.
Those of ordinary skills in the art should understand that：The discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under the thinking of the present invention, above example
Or can also be combined between the technical characteristic in different embodiments, step can be realized with random order, and be existed such as
Many other variations of the different aspect of the upper present invention, for simplicity, they are not provided in details.
The embodiment of the present invention be intended to cover fall within the broad range of appended claims it is all it is such replace,
Modifications and variations.Therefore, all within the spirits and principles of the present invention, any omission, modification, equivalent replacement, the improvement made
Deng should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of preservative agent of urine nucleic acid, which is characterized in that by mass percentage, including 50~80 parts of buffer solution, anti-corrosion
10~30 parts of fixative, 0.5~5 part of nucleic acid inhibitor, 1~5 part of membrane protective agent, 1~5 part of metabolic poison.
2. a kind of preservative agent of urine nucleic acid according to claim 1, which is characterized in that the buffer solution is sodium citrate
Buffer solution.
3. buffer solution according to claim 2, mass concentration is 2%~6%.
4. a kind of preservative agent of urine nucleic acid according to claim 1, which is characterized in that the Antiseptic and fixation agent is selected from first
It is one or more in aldehyde and its derivative, sodium hydroxymethylglycinate, dimethylol urea, diazonium ureine, imidazolidinyl urea.
5. Antiseptic and fixation agent according to claim 4, mass concentration is 200~700g/L.
6. a kind of preservative agent of urine nucleic acid according to claim 1, which is characterized in that the nucleic acid inhibitor is selected from
Heparin, aurin tricarboxyli acid (ATA), formamide, ethylenediamine tetra-acetic acid, edetate, DTT, ATA are one or more.
7. a kind of preservative agent of urine nucleic acid according to claim 1, which is characterized in that the membrane protective agent includes
Glycine, aspartic acid, half Guang amide, glutamine, glyceraldehyde-3-phosphate are one or more.
8. a kind of preservative agent of urine nucleic acid according to claim 1, which is characterized in that the metabolic poison includes phosphorus
Sour dihydroxyacetone, mannose, glyceraldehyde, sodium fluoride, pyruvic acid, glycerine.
9. a kind of urine nucleic acid preservation device of the preservative agent including the urine nucleic acid described in the claims 1~8.
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| CN201810333596.5A CN108642042A (en) | 2018-04-13 | 2018-04-13 | A kind of preservative agent and device of urine nucleic acid |
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| CN201810333596.5A CN108642042A (en) | 2018-04-13 | 2018-04-13 | A kind of preservative agent and device of urine nucleic acid |
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Cited By (7)
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| CN109757466A (en) * | 2018-10-29 | 2019-05-17 | 中国医学科学院阜外医院 | Urine saves liquid, urine capture container, method and kit |
| WO2020127815A1 (en) * | 2018-12-19 | 2020-06-25 | Charité-Universitätsmedizin Berlin | Method for preserving urinary cells |
| CN111334560A (en) * | 2018-12-19 | 2020-06-26 | 合肥铼科生物科技有限公司 | Composition and method for preserving nucleic acid in urine |
| CN111334503A (en) * | 2020-04-07 | 2020-06-26 | 江苏康为世纪生物科技有限公司 | Nucleic acid preserving fluid |
| CN111944805A (en) * | 2020-08-26 | 2020-11-17 | 合肥铼科生物科技有限公司 | Urine free DNA normal temperature preservative |
| CN113265443A (en) * | 2021-05-21 | 2021-08-17 | 深圳逗点医疗科技有限公司 | Blood free DNA protection reagent, protection method and blood collection tube |
| WO2023011162A1 (en) * | 2021-08-04 | 2023-02-09 | 江苏臻石生物科技有限公司 | Biological sample nucleic acid release preservative |
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| WO2023011162A1 (en) * | 2021-08-04 | 2023-02-09 | 江苏臻石生物科技有限公司 | Biological sample nucleic acid release preservative |
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