CN105961375A - Saliva preserving fluid, preparation method and usage thereof - Google Patents
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- 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
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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Abstract
The invention discloses a saliva preserving fluid which comprises the following components and contents: 5-80mmol/L Tris-HCl, 0.1-4%(w/v) SDS, 1-20mmol/L EDTA and 50-200mmol/L sodium chloride. The pH scope of the system is from 7 to 10. The saliva preserving fluid provided by the invention is simple in formula, is used for storing the human saliva sample and is capable of long-term storing saliva under room temperature condition; DNA in the stored saliva is complete; the stability of the saliva sample in the process of transporting to various gathering places can be ensured; the saliva preserving fluid is fit for gene detection and related scientific research.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly relate to a kind of saliva and preserve liquid and its production and use.
Background technology
DNA sample in molecular biology, medical verification, detect and be widely used in the fields such as treatment, occupy an important position.The DNA being generally used for PCR amplification or detection extracts from anticoagulated blood, but blood sample is restricted to a certain extent as DNA source, and both ways, on the one hand blood preseration the most easily forms coagulation to major embodiment, brings inconvenience extraction；On the other hand gather blood also can cause certain wound and also professional skill to be relied on just can complete to human body.
Saliva is the body fluid that a kind of human body is easier to obtain, and gathers more convenient, and hurtless measure, is preferable body fluid sample, is suitable for sampling on a large scale.But salivary component mainly has the materials such as water, Oral Mucosal Cells, antibacterial, ptyalin, viscosity sugar, complicated component, is difficult to preserve for a long time, therefore, it is to avoid in saliva corruption, holding exfoliative cyte, genomic DNA integrity is applied to most important.
Protective agent complicated component in existing patent, such as Patent publication No CN102440234B and CN102919218B, containing magnesium chloride in CN102919218B, magnesium ion is nuclease activated dose, is unfavorable for the stability of DNA and the preservation of integrity；Containing protein denaturant such as guanidinium isothiocyanate in CN102440234B, the activity of nuclease can be suppressed, but also cracked cell simultaneously so that DNA is in free state, be unfavorable for that the integrity of DNA preserves；CN102919218B and CN105039306A contains sucrose and dextrose components respectively, although this saliva can well be kept to preserve the viscosity of fixative and maintain Premeabilisation of cells pressure, but easily cause growing of the microorganisms such as antibacterial；Magnesium chloride, as the activator of nuclease, easily causes the degraded of DNA；Complicated component in CN105368812A, although the organic substances such as the mucin in saliva and globulin can be carried out degenerative treatments by the composition such as carbamide, ethanol, but also cell can be caused cracking simultaneously, it is unfavorable for that the integrity of DNA preserves, and these compositions provided in this application file are essential component, i.e. the indispensable ability of all the components stably keeps the stability of saliva DNA.
For the deficiencies in the prior art, the present invention provides a kind of simple human saliva to preserve liquid prescription, and described human saliva preserves liquid and is used for preserving saliva, it is possible to long-term keep the integrity of DNA in cell, and it can be avoided that the growing of the microorganisms such as antibacterial.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of saliva and preserves liquid and its production and use.
Embodiments provide a kind of saliva and preserve liquid, including following component and content: Tris-HCl 5-80mmol/L, SDS 0.1-4%(w/v), EDTA 1-20mmol/L, sodium chloride 50-200mmol/L, system pH scope is between 7-10.
Selectively, above-mentioned saliva preserves in liquid, and the content of above-mentioned Tris-HCl is 5-60mmol/L, the content of above-mentioned SDS is 0.2-2.8%(w/v), the content of above-mentioned EDTA is 1-10mmol/L, and the content of above-mentioned sodium chloride is 90-200mmol/L, and above-mentioned system pH scope is between 7-9.
Selectively, above-mentioned saliva preserves in liquid, and the content of above-mentioned Tris-HCl is 15-50mmol/L, the content of above-mentioned SDS is 0.3-1.5%(w/v), the content of above-mentioned EDTA is 1-5mmol/L, and the content of above-mentioned sodium chloride is 100-180mmol/L, and above-mentioned system pH scope is between 7.5-8.5.
Selectively, above-mentioned saliva preserves in liquid, and the content of above-mentioned Tris-HCl is 40mmol/L, and the content of above-mentioned SDS is 0.5%(w/v), the content of above-mentioned EDTA is 3mmol/L, and the content of above-mentioned sodium chloride is 150mmol/L, and above-mentioned system pH scope is 8.
The embodiment of the present invention additionally provides a kind of above-mentioned saliva and preserves the preparation method of liquid, and above-mentioned preparation method comprises the steps:
A. following component and content are weighed: Tris-HCl 5-80mmol/L, SDS 0.1-4%(w/v), EDTA 1-20mmol/L, sodium chloride 50-200mmol/L, add sterilized water and dissolve and stir；Or by each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. between the pH to 7-10 of regulating step a gained solution, and sterilized water constant volume is used；
C. step b gained solution is degerming by filtering with microporous membrane, obtain above-mentioned saliva and preserve liquid.
Selectively, the preparation method of above-mentioned saliva preservation liquid can also comprise the steps:
A. weighing each component: the content of above-mentioned Tris-HCl is 5-60mmol/L, the content of above-mentioned SDS is 0.2-2.8%(w/v), the content of above-mentioned EDTA is 1-10mmol/L, and the content of above-mentioned sodium chloride is 90-200mmol/L, adds sterilized water and dissolves and stir；Or by each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. between the pH to 7-9 of regulating step a gained solution, and sterilized water constant volume is used；
C. step b gained solution is degerming by filtering with microporous membrane, obtain above-mentioned saliva and preserve liquid.
Selectively, the preparation method of above-mentioned saliva preservation liquid can also comprise the steps:
A. each component is weighed: the content of above-mentioned Tris-HCl is 15-50mmol/L, the content of above-mentioned SDS is 0.3-1.5%(w/v), the content of above-mentioned EDTA is 1-5mmol/L, and the content of above-mentioned sodium chloride is 100-180mmol/L, adds sterilized water and dissolves and stir；Or by each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. between the pH to 7.5-8.5 of regulating step a gained solution, and sterilized water constant volume is used；
C. step b gained solution is degerming by filtering with microporous membrane, obtain above-mentioned saliva and preserve liquid.
Selectively, the preparation method of above-mentioned saliva preservation liquid can also comprise the steps:
A. weighing each component: the content of above-mentioned Tris-HCl is 40mmol/L, the content of above-mentioned SDS is 0.5%(w/v), the content of above-mentioned EDTA is 3mmol/L, and the content of above-mentioned sodium chloride is 150mmol/L, adds sterilized water and dissolves and stir；Or by each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. the pH to 8 of regulating step a gained solution, and use sterilized water constant volume；
C. step b gained solution is degerming by filtering with microporous membrane, obtain above-mentioned saliva and preserve liquid.
The embodiment of the present invention additionally provides a kind of above-mentioned saliva and preserves liquid purposes in preserving saliva.
Selectively, above-mentioned saliva is human saliva.
The saliva that the present invention provides preserves in liquid, and wherein EDTA component can chelate the bivalent cations such as Mg, Ca of self containing in saliva, the activity of suppression nuclease, it is ensured that DNA is not degraded；Wherein SDS component may act as denaturant and antibacterial, the organic substances such as the mucin in saliva and globulin can be carried out degenerative treatments, make microorganism be not easy to grow while reducing liquid viscosity, can kill or suppress the microbial growth in the saliva after preserving；SDS can be formed complex with protein as cosolvent and strengthen the solubility of protein again simultaneously；Wherein NaCl component can maintain Premeabilisation of cells pressure, protects saliva cell stability, simultaneously because reducing microbial growth further without the saccharide such as sucrose, glucose.Wherein Tris-HCl component is as buffer, can maintain solution environmental pH in the range of 7-10, preferred 7.5-8.5.It is simple that the saliva of the present invention preserves formula of liquid, for preserving human body saliva sample, saliva can be preserved for a long time, in the saliva preserved, DNA integrity is good, ensure that saliva sample stability between the most locality in transportation, it is adaptable to gene test and related science research.
Accompanying drawing explanation
The saliva of the preparation that Fig. 1 provides for the embodiment of the present invention 1 preserves the saliva sample of the same person that liquid room temperature condition preserves agarose gel electrophoresis figure of the genomic DNA of extraction after preserving different time, the i.e. DNA electrophoretogram of embodiment 3, in figure: M represents DNA Marker (D2000
plus DNA Ladder)；1-3 be saliva sample room temperature preservation 1 week, 2 weeks, extract, after 1 month, the DNA obtained；
The saliva of the preparation that Fig. 2 provides for the embodiment of the present invention 4 preserves same person saliva sample agarose gel electrophoresis figure of the genomic DNA of extraction after preserving 1 week that liquid room temperature condition preserves, in figure: M represents DNA Marker (D2000
plus DNA Ladder)；1-10 is that saliva sample uses formula 1-10 to extract, after processing, the DNA obtained；
The saliva sample of the collection that Fig. 3 provides for the embodiment of the present invention 2 use the saliva of embodiment 1 preserve liquid preserve 1 week, 2 weeks, after 1 month, the genomic DNA of extraction is template, carries out the agarose gel electrophoresis figure that PCR amplification obtains, in figure: M represents DNA
Marker；NA is blank, 1-3 be saliva sample room temperature preservation 1 week, 2 weeks, to extract the DNA obtained after 1 month be that template carries out PCR and expands the PCR of the result that obtains, i.e. embodiment 5 and identify figure.
Detailed description of the invention
In order to make those skilled in the art be more fully understood that, technical scheme can be practiced, and the invention will be further described with specific embodiment below in conjunction with the accompanying drawings, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1 saliva preserves liquid and prepares
The saliva of the present embodiment preserves liquid and includes following component and content: Tris-HCl
40mmol/L, SDS0.5% (w/v), EDTA3mmol/L, NaCl150mmol/L, system pH is 8.
The preparation method that the saliva of the present embodiment preserves liquid is as follows:
Measure 2mL Tris-HCl (1mol/L), 2.5mL SDS (10%), 0.3mL EDTA (0.5mol/L) and 7.5mLNaCl(1mol/L) the most respectively, it is added sequentially in 50ml beaker, by magnetic stirring apparatus mix homogeneously;
B. solution ph is adjusted to 8 with dilute hydrochloric acid;
C. mixed liquor in beaker is transferred in 50ml volumetric flask, and is settled to scale with ultra-pure water;
D. with disposable filter membrane device, above-mentioned preservation liquid is filtered, be stored in liquid containing bottle.
Or optional, wherein Tris-HCl stores the concentration of liquid can be configured to 1mol/L standby, and SDS stores the concentration of liquid, and can be configured to 10% (w/v) standby, and EDTA stores liquid, and to can be configured to 0.5mol/L standby；It is standby that NaCl storage liquid can be configured to 1mol/L；During preparation, correct amount takes each component storage liquid respectively, with the pH of hydrochloric acid regulation gained mixed solution to purpose scope after mixing, then uses sterilized water constant volume；Gained solution is operated by step (d) with degerming；Step (d) microporous filter membrane can select less than 0.45 micron of aperture, the microporous filter membrane of preferably less than 0.22 micron.
Embodiment 2 saliva sample collection and preservation
In the present embodiment, saliva sample collection and store method are as follows:
(1) sample first 30 minutes and gargle to remove food debris and residual microorganism, do not take food in 30 minutes after gargling, drink water, brush teeth, smoking or chew gum;
(2) start before ptysis, loosen cheek, and rub the 15-30 second gently to produce saliva, by saliva collection to aseptic 2mL EP pipe;
(3) the saliva 2mL of a people is gathered according to the method described above;
(4) take saliva prepared by the present embodiment isopyknic with saliva to preserve liquid and join in saliva sample, and reverse mixing 10-20 time;
(5) after mixing, sample can place room temperature preservation, and respectively at 1 week, 2 weeks, 1 month time extraction preservation sample in DNA.
Embodiment 3 saliva sample DNA extraction
In the present embodiment, the extracting method of saliva sample DNA is as follows:
(1) take the saliva during 500ul preserves, add 1ml buffer (100mmol/L Tri-HCl, pH8.0,0.5%SDS, 10mmol/L
EDTA) and the E.C. 3.4.21.64 of 6ul 20mg/ul, concussion mixing on vortex instrument;
(2) saliva after above-mentioned steps in the present embodiment (1) being processed carries out 55 DEG C of water bath processing 20min；
(3) adding 600ul phenol: chloroform: isoamyl alcohol (25:24:1), reverse mixing, 10000rpm is centrifuged 5min, takes supernatant；
(4) supernatant of above-mentioned steps (3) adds in the present embodiment the chloroform of 600ul: isoamyl alcohol (24:1), after reverse mixing, 10000rpm is centrifuged 5min, takes supernatant；
(5) supernatant fluid of above-mentioned steps (4) adds in the present embodiment the isopropanol of isopyknic-20 DEG C of pre-coolings, mixes postprecipitation 1h；
(6) aforesaid liquid 14000rpm is centrifuged 10min, abandons supernatant；
(7) by the above-mentioned precipitation of 500ul70% washing with alcohol once, 14000rpm is centrifuged 10min, abandons supernatant；
(8) room temperature dries DNA, uses 30ulEB buffer solution；
(9) DNA taking dissolving carries out agarose gel electrophoresis detection, and result is as shown in Figure 1.
Result shows: saliva sample room temperature preservation 1 week, 2 weeks, to extract the DNA electrophoretic band obtained after 1 month the sharpest keen, does not has diffusing phenomenon.Illustrating that the saliva using the present embodiment to prepare preserves liquid and is able to maintain that saliva sample preserves at least 1 month at ambient temperature, and integrity is good, therefore the saliva preservation liquid of the present invention can be used for the long-term room-temperature preservation of saliva sample.
The different saliva of embodiment 4 preserves the saliva sample DNA extraction that liquid component formula preserves
The present embodiment preserves liquid according to following table formula preparation saliva, and preserves liquid with the saliva for preparing and preserve the saliva sample of the method collection according to embodiment 2, after preserving 1 week, extract the saliva sample DNA of same person respectively according to the method for embodiment 3:
| Formula | Tris-HCl(mmol/L) | EDTA(mmol/L) | SDS(w/v) | NaCl(mmol/L) |
| 1 | 80 | 3 | 2.8 | 90 |
| 2 | 60 | 20 | 0.2 | 100 |
| 3 | 45 | 4 | 0.3 | 120 |
| 4 | 5 | 1 | 0.1 | 50 |
| 5 | 40 | 5 | 0.5 | 100 |
| 6 | 50 | 8 | 1.5 | 150 |
| 7 | 15 | 2 | 4 | 200 |
| 8 | 60 | 10 | 2.8 | 180 |
| 9 | 40 | 3 | 0.5 | 150 |
| 10 | 45 | 5 | 1.2 | 100 |
The DNA taking dissolving carries out agarose gel electrophoresis detection, and result is as shown in Figure 2.
Result shows: it is the sharpest keen that the formula obtained according to the concentration range combination of each component fixed given in form extracts the DNA electrophoretic band obtained, and does not has diffusing phenomenon.Illustrate that the formula that the saliva preservation liquid each component concentration ranges random combine using the present embodiment to prepare is formed can effectively extract saliva sample DNA, and integrity is good.
Embodiment 5 PCR identifies
The present embodiment is that the genomic DNA extracted from saliva sample is carried out gene PCR amplification, use the saliva sample room temperature preservation that gathers in embodiment 31 week, 2 weeks, extract the DNA obtained after 1 month and carry out PCR amplification as template, institute's amplification gene fragment length is 494bp.
Amplimer is as follows:
F: 5’-GGGGTCAGAAGCATATCAGTC-3’
R: 5’-GGGAAGAACTCAGCGAACT-3’
PCR response procedures is as follows:
95℃4min；95 DEG C of 40s, 60 DEG C of 30s, 72 DEG C of 50s, 36 circulations；72℃2min；4℃∝.
Result shows: the genes of interest fragment amplified is consistent with expection size, and band is clear, illustrates that saliva sample uses the saliva of embodiment 1 to preserve the genomic DNA extracted after liquid preserves 1 month complete.
Embodiment described above is only the preferred embodiment lifted by absolutely proving the present invention, and protection scope of the present invention is not limited to this.The equivalent that those skilled in the art are made on the basis of the present invention substitutes or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (10)
1. a saliva preserves liquid, it is characterised in that include following component and content: Tris-HCl
5-80mmol/L, SDS 0.1-4%(w/v), EDTA 1-20mmol/L, sodium chloride 50-200mmol/L, system pH scope is between 7-10.
Saliva the most according to claim 1 preserves liquid, the content that it is characterized in that described Tris-HCl is 5-60mmol/L, the content of described SDS is 0.2-2.8%(w/v), the content of described EDTA is 1-10mmol/L, the content of described sodium chloride is 90-200mmol/L, and described system pH scope is between 7-9.
Saliva the most according to claim 2 preserves liquid, the content that it is characterized in that described Tris-HCl is 15-50mmol/L, the content of described SDS is 0.3-1.5%(w/v), the content of described EDTA is 1-5mmol/L, the content of described sodium chloride is 100-180mmol/L, and described system pH scope is between 7.5-8.5.
Saliva the most according to claim 3 preserves liquid, it is characterised in that the content of described Tris-HCl is 40mmol/L, and the content of described SDS is 0.5%(w/v),
The content of described EDTA is 3mmol/L, and the content of described sodium chloride is 150mmol/L, and described system pH scope is 8.
5. the preparation method of a saliva according to claim 1 preservation liquid, it is characterised in that described preparation method comprises the steps:
Weigh the saliva described in claim 1 and preserve each component of liquid, add sterilized water and dissolve and stir；Or by described in claim 1 saliva preserve liquid each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
Between the pH to 7-10 of regulating step a gained solution, and use sterilized water constant volume；
Step b gained solution is degerming by filtering with microporous membrane, obtain described saliva and preserve liquid.
6. the preparation method of a saliva according to claim 2 preservation liquid, it is characterised in that described preparation method comprises the steps:
A. weigh the saliva described in claim 2 and preserve each component of liquid, add sterilized water and dissolve and stir；Or by described in claim 2 saliva preserve liquid each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. between the pH to 7-9 of regulating step a gained solution, and sterilized water constant volume is used；
C. step b gained solution is degerming by filtering with microporous membrane, obtain described saliva and preserve liquid.
7. the preparation method of a saliva according to claim 3 preservation liquid, it is characterised in that described preparation method comprises the steps:
A. weigh the saliva described in claim 3 and preserve each component of liquid, add sterilized water and dissolve and stir；Or by described in claim 3 saliva preserve liquid each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. between the pH to 7.5-8.5 of regulating step a gained solution, and sterilized water constant volume is used；
C. step b gained solution is degerming by filtering with microporous membrane, obtain described saliva and preserve liquid.
8. the preparation method of a saliva according to claim 4 preservation liquid, it is characterised in that described preparation method comprises the steps:
A. weigh the saliva described in claim 4 and preserve each component of liquid, add sterilized water and dissolve and stir；Or by described in claim 4 saliva preserve liquid each component respectively be prepared as store liquid form standby, measure the volume of each component during preparation respectively, mixing；
B. the pH to 8 of regulating step a gained solution, and use sterilized water constant volume；
C. step b gained solution is degerming by filtering with microporous membrane, obtain described saliva and preserve liquid.
9. preserve liquid purposes in preserving saliva according to the arbitrary described saliva of Claims 1-4.
Purposes the most according to claim 9, it is characterised in that described saliva is human saliva.
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