CN105695447A - Composition for preserving DNA in saliva - Google Patents
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Abstract
The invention relates to composition for preserving DNA in saliva. The composition is prepared from a denaturing agent, a buffer solution, a chelating agent, sodium chloride and polyethylene glycol. The composition can preserve the DNA in the saliva effectively for a long time under the room temperature condition, reduce degradation of the DNA in the in-vitro saliva effectively, guarantee integrity of the DNA and facilitate subsequent nucleic acid detection and research and is simple to use and convenient to operate, and treatment time for a saliva sample is greatly shortened.
Description
Technical field
The invention belongs to biomedical sector, particularly to a kind of for preserving the compositions of DNA in saliva。
Background technology
At present, gene test is widely used to biology, medical science, the science of law, field of medicaments, it is by being measured and positioning analysis the gene coded sequence of the DNA in person under inspection's blood, serum, blood plasma, saliva, urine, other body fluid, tissue or cell etc., thus knowing person under inspection's gene information。Gene test is significant with prevention, the formulation of personalized medicine scheme, personally identifiable information confirmation, parenthood determination etc. for the control of genetic diseases, such as can pass through examination susceptibility genes of drug-induced deafness type, prevent aminoglycosides antibiotics toxic deafness；By detecting CYP2C19 gene pleiomorphism, thus predicting drug administration condition, the power of toxic and side effects and curative effect of medication；The individual risk that cerebro-vascular diseases and neonate defect occur can be pointed out by individual mthfr gene C677T loci gene type being carried out detection, and the treatment curative effect of the chemotherapeutics such as predicting methotrexate and 5-fluorouracil and toxic and side effects；By extracting the DNA of detection family members, the dead's identity is carried out identification etc.。
Body fluid for extracting DNA is generally the leukocyte in venous blood, but, gathering venous blood needs professional to operate, and adopting intrusive mood mode, human body can cause a degree of wound and discomfort, blood collection there is likely to be the risk of cross infection, in addition, blood is prone to caking, is unfavorable for long-time preservation, brings much inconvenience to extraction。
DNA is extracted relative to conventional blood DNA acquisition mode from saliva, gatherer process is simple and convenient, without through the personnel of professional training and operable, and be non-intrusion type acquisition mode, can avoid person under inspection is caused unnecessary misery, easily accepted, sampling scope can be expanded to a greater extent, be especially suitable for large-scale crowd investigation。At present, extracting DNA from saliva, mainly adopt cotton swab or buccal swab to obtain oral mucosa exfoliative cyte, the amount of DNA that the method gathers is few, and the sample preservation time is short, and needs cryopreservation, additionally, saliva is susceptible to go bad, degrade after departing from human body。Therefore, how effectively complete preservation saliva sample, for significant based on the gene amplification of DNA in saliva and detection。
Prior art CN102440234A discloses a kind of human saliva and preserves fixative, including sucrose, Tris-HCl, MgCl2, guanidinium isothiocyanate and water, it is possible to the activity kill the antibacterial contained in saliva, suppressing ribozyme。But, this fixative component comprises magnesium ion, belongs to nuclease activated dose, be unfavorable for that the stability of DNA preserves；Although guanidinium isothiocyanate can suppress the activity of nuclease, but the destructiveness of cell is relatively strong, it is unfavorable for that the integrity of DNA preserves。
Prior art CN102676501A6 discloses a kind of Preservative agent for DNA of saliva, including trishydroxymethylaminomethane, disodiumedetate, sugar, sodium chloride and surfactant, preserves for a long time under its room temperature being capable of in saliva DNA。But, owing to saliva sample existing deoxyribonuclease and ribonuclease, it is possible to decomposing D NA or RNA, the surfactant of low concentration, it is difficult to fully suppress the activity of nuclease, it is possible to affect the stability of DNA。
Be currently, there are develop a kind of efficiently, long-time for preserving the demand of the compositions of DNA in saliva under stable, room temperature。
Summary of the invention
It is an object of the invention to the technological deficiency existed for prior art, it is provided that a kind of efficiently, long-time for preserving the compositions of DNA in saliva under stable, room temperature。
In order to realize the purpose of the present invention, the present invention adopts the following technical scheme that
A kind of for preserving the compositions of DNA in saliva, including denaturant, buffer, chelating agen, sodium chloride and Polyethylene Glycol。
In the embodiment of the invention, described denaturant is one or more in guanidine hydrochloride, phenol and surfactant。Wherein, described surfactant includes but not limited to: sodium lauryl sulphate (Sodiumdodecylsulfate, SDS), sarcosyl (SodiumN-lauroylsarcosine, NLS), Triton X-100 (TritonX-100), polysorbas20 (polyoxyethylene 20 sorbitan monolaurate), polysorbate40 (polyoxyethylene 20 sorbitan monopalmitate), polysorbate60 (polyoxyethylene 20 sorbitan monostearate), polysorbate65 (polyoxyethylene 20 sorbitan tristearate), one or more in Tween 80 (polyoxyethylene 20 sorbitan monooleate)。
In the embodiment of the invention, described denaturant concentration in the composition presses quality volume percentage more than 30%, it is preferred to 30～60%, more preferably 40～60%。
In embodiment of the present invention, suppressed deoxyribonuclease (DNA enzymatic) and the activity of ribonuclease (RNase) by denaturant, and be not result in the fracture of DNA, it is ensured that the integrity of DNA in saliva。
In the embodiment of the invention, described denaturant includes guanidine hydrochloride。
In embodiment of the present invention, guanidine hydrochloride can suppress the activity of DNA enzymatic and RNase preferably, it is prevented that the DNA degradation in saliva, makes DNA in saliva keep long-time integrity and stability；Additionally, the protein that can pass through in guanidine hydrochloride dissolution saliva, albumen is disintegrated down from nucleic acid, reduce in subsequent detection DNA extraction and purification time in saliva sample。
In other embodiment of the present invention, described denaturant is the combination of guanidine hydrochloride and surfactant。
In the embodiment of the invention, described guanidine hydrochloride concentration in the composition presses quality volume percentage more than 20%, it is preferred to 20～60%, more preferably 30～60%, it is most preferred that be 40～60%。
In the embodiment of the invention, described buffer is the one in trishydroxymethylaminomethane, Tris-HCl buffer, phosphate buffer, barbitol buffer solution, it is preferred to Tris-HCl buffer。
In embodiment of the present invention, by buffer for the compositions suitable pH value of offer and buffer environment, it is prevented that nucleic acid is destroyed。
In the embodiment of the invention, described chelating agen is one or more in ethylenediaminetetraacetic acid, edetate or its hydrate (such as disodiumedetate, disodiumedetate two water, tetrasodium ethylenediamine tetraacetate two water etc.), citric acid, citrate or its hydrate (such as sodium citrate, sodium citrate two water, sodium citrate three water, potassium citrate, potassium citrate one water, potassium citrate three water etc.), 1,2-diaminocyclohexane tetraacetic acid。
In embodiment of the present invention, by the Mg in chelant ties saliva2+、Ca2+Or Mn2+Ion, it is prevented that metal ion combines with the phosphate backbones of DNA, additionally, may also suppress the activity of DNA enzymatic。
In the embodiment of the invention, described Polyethylene Glycol mean molecule quantity is 4000-8000, for instance PEG4000, PEG6000, PEG8000 etc., it is preferred to PEG6000。In the present invention, PEG6000 refers to the Polyethylene Glycol that mean molecule quantity is about 6000。
In the embodiment of the invention, described include the component of following concentration for preserving the compositions of DNA in saliva: the denaturant of 30～60%, the Tris-HCl buffer (pH6.8-8) of 5～20mM, the EDTA of 1.5～2.5%, the sodium citrate of 100～300mM or its hydrate, the sodium chloride of 600～900mM, 0.2～1% PEG6000, solvent is water；
Wherein, described denaturant, EDTA, PEG6000 concentration by quality volume percentage；
Described EDTA is one or more in ethylenediaminetetraacetic acid, disodiumedetate, disodiumedetate two water, tetrasodium ethylenediamine tetraacetate two water；
Described sodium citrate or its hydrate include one or more in sodium citrate, sodium citrate two water, sodium citrate three water。
In the embodiment of the invention, described include the component of following concentration for preserving the compositions of DNA in saliva: the denaturant of 30～60%, the Tris-HCl buffer (pH6.8-8) of 5～20mM, 2% the sodium citrate of EDTA, 200mM or its hydrate, the sodium chloride of 800mM, 0.5% PEG6000, solvent is water；
Wherein, described denaturant, EDTA, PEG6000 concentration by quality volume percentage；
Described EDTA is one or more in ethylenediaminetetraacetic acid, disodiumedetate, disodiumedetate two water, tetrasodium ethylenediamine tetraacetate two water；
Described sodium citrate or its hydrate include one or more in sodium citrate, sodium citrate two water, sodium citrate three water。
In the embodiment of the invention, described include the component of following concentration for preserving the compositions of DNA in saliva: the guanidine hydrochloride of 40-60%, 0.1～1% surfactant, the Tris-HCl buffer (pH6.8-8) of 5～20mM, 2% disodiumedetate, sodium citrate two water of 200mM, the sodium chloride of 800mM, 0.5% PEG6000, solvent is water；
Wherein, described denaturant, EDTA, PEG6000 concentration by quality volume percentage。
In the embodiment of the invention, the present composition can test kit form exist。
The present composition can be used for preserving saliva, especially human saliva。Said composition can keep the completeness and efficiency of DNA in saliva at normal temperatures for a long time。
The method have the benefit that
The present composition can long-time (more than 15 months) DNA effectively preserved in saliva at ambient temperature, and can effectively reduce the degraded of DNA in vitro saliva, it is prone to follow-up detection of nucleic acids research, and use simple, easy to operate, it is greatly saved the process time of saliva sample。
Accompanying drawing explanation
In Fig. 1 embodiment of the present invention 1 in agarose gel electrophoresis testing result figure, the figure of saliva sample 1-sample 10, sample 1 to sample 10 is by arraying from left to right。
In Fig. 2 embodiment of the present invention 2 in result figure, the figure of the real-time fluorescence quantitative PCR detection of saliva sample 11 DNA in different time sections, different lines represents 0th month, the 3rd month, the 6th month, the 9th month, the 12nd month and 15th month respectively。
In Fig. 3 embodiment of the present invention 2 in result figure, the figure of the real-time fluorescence quantitative PCR detection of saliva sample 12 DNA in different time sections, different lines represents 0th month, the 3rd month, the 6th month, the 9th month, the 12nd month and 15th month respectively。
The result figure of the real-time fluorescence quantitative PCR detection of saliva sample 13 (wild type) in Fig. 4 embodiment of the present invention 3。
The result figure of the real-time fluorescence quantitative PCR detection of saliva sample 14 (wild type) in Fig. 5 embodiment of the present invention 3。
The result figure of the real-time fluorescence quantitative PCR detection of saliva sample 15 (1555A > G sudden change) in Fig. 6 embodiment of the present invention 3。
The result figure of the real-time fluorescence quantitative PCR detection of whole blood sample 16 (wild type) in Fig. 7 embodiment of the present invention 3。
The result figure of the real-time fluorescence quantitative PCR detection of whole blood sample 17 (wild type) in Fig. 8 embodiment of the present invention 3。
The result figure of the real-time fluorescence quantitative PCR detection of whole blood sample 18 (1555A > G sudden change) in Fig. 9 embodiment of the present invention 3。
In the present invention in Fig. 4-9, abscissa is temperature, and unit is DEG C；Vertical coordinate is-d (Rn)/dT, Rn is Raw fluorescence value。
Detailed description of the invention
By further illustrating the technology contents of the present invention, structural feature, being realized purpose and effect, below in conjunction with specific examples below (but unrestricted) and coordinate accompanying drawing to illustrate, described embodiment sets forth the several preferred embodiment of the present invention, and other embodiments of the invention are predicted by those skilled in the art under the premise without departing substantially from the core of the present invention。
Embodiment 1 is for preserving the compositions of DNA in saliva
A kind of for preserving the compositions of DNA in saliva, its component and content are as shown in table 1 below:
Table 1
In upper table, % representation quality percent by volume。
Said composition compound method is as follows:
Weigh each component by the proportioning in table 1, and disodiumedetate is completely dissolved makes mother solution；After adding other components in deionized water and fully dissolving, add above-mentioned mother solution and be sufficiently mixed, add deionized water and be settled to final volume。
The compositions prepared by above-mentioned formula is sufficiently mixed uniformly with 10 saliva samples gathered by the volume ratio of 1:1 respectively, the each 500 μ L nucleic acid extraction of above-mentioned mixed liquor or purification kit (Chong Ding Bioisystech Co., Ltd of Ningbo City is taken after preserving a period of time at normal temperatures, production code member: ZD-XY-Mini) extract DNA, and it is dissolved separately in the eluent of 100 μ L, obtain 10 samples, it is numbered at 10,4 DEG C of sample 1 to sample respectively to save backup。
Adopting ultra-violet absorption spectrum to detect the amount of DNA in above-mentioned 10 samples, testing result is as shown in table 2 below:
Table 2
| Numbering | Abs260 | Abs280 | A260/A280 | Concentration (ng/ μ L) |
| 1 | 1.429 | 0.887 | 1.612 | 71.5 |
| 2 | 1.17 | 5.418 | 0.216 | 58.5 |
| 3 | 0.998 | 0.669 | 1.49 | 49.9 |
| 4 | 1.588 | 1.654 | 0.96 | 79.4 |
| 5 | 1.343 | 0.829 | 1.62 | 67.2 |
| 6 | 0.998 | 0.669 | 1.49 | 49.9 |
| 7 | 1.092 | 0.708 | 1.542 | 54.6 |
| 8 | 1.164 | 0.748 | 1.557 | 58.2 |
| 9 | 0.47 | 0.382 | 1.229 | 23.5 |
| 10 | 0.428 | 0.374 | 1.146 | 21.4 |
Additionally, above-mentioned 10 samples are carried out agarose gel electrophoresis detection, testing result is as shown in Figure 1, the numeral marked in Fig. 1 is counter sample numbering respectively, as it is shown in figure 1, after the saliva gathered preserves a period of time at normal temperatures, the DNA in 10 samples all remains stable for。
The embodiment 2 different holding time is on preserving the impact of DNA in saliva
It is sufficiently mixed uniformly with 2 saliva samples gathered by the volume ratio of 1:1 respectively by the compositions of embodiment 1 formula preparation, numbered samples 11 and 12 respectively, these two samples take each 500 μ L nucleic acid extraction of above-mentioned mixed liquor or purification kit (Chong Ding Bioisystech Co., Ltd of Ningbo City after preserving certain time (preserving 0 month, 3 months, 6 months, 9 months, 12 months, 15 months respectively) at ambient temperature, production code member: ZD-XY-Mini) extract DNA, and be dissolved separately in the eluent of 100 μ L, obtain corresponding 2 samples。DNA concentration in sample is detected, and result is as shown in table 3 below:
Table 3 (concentration is ng/ μ L)
| Sample | Initial | March | June | JIUYUE | December | 15th month |
| 11 | 43.5 | 43.1 | 42.9 | 42.7 | 42.6 | 42.1 |
| 12 | 47.6 | 47.3 | 47 | 46.9 | 46.7 | 46 |
By testing result it can be seen that sample 11 and sample 12 are after preservation was more than 1 year, its DNA content is basically unchanged, and loss amount is only small。
In addition, the DNA extracted after at ambient temperature sample 11 and sample 12 are preserved certain time carries out real-time fluorescence quantitative PCR detection, testing result is respectively as Figure 2-3, from testing result, preserve the DNA extracted after more than a year under room temperature and still can carry out pcr amplification reaction, show that DNA is not destroyed, still keep complete。
Embodiment 3
The saliva impact on DNA cloning and detection is preserved for characterizing the employing present composition further, adopting DNA in the drug induced deafness gene mutation detection kit saliva to extracting to carry out gene test, DNA sample respectively carries the human gene group DNA of mtDNA 12S rRNA gene 1555A > G saltant type and wild type。
It is sufficiently mixed uniformly with 3 saliva samples gathered by the volume ratio of 1:1 respectively by the compositions of embodiment 1 formula preparation, difference numbered samples 13 (wild type), 14 (wild types), 15 (1555A > G sudden changes), preserve one week at ambient temperature。Take each 10 μ L Drug deaf gene mutation detection kit (fluorescent PCR method) (intelligence marine growth engineering (Beijing) company limited) of above-mentioned mixed liquor and extract DNA, obtain corresponding 3 samples according to the sample treatment of said medicine deaf gene mutation detection kit (fluorescent PCR method)。
Choose 3 whole blood samples, difference numbered samples 16 (wild type), 17 (wild types), 18 (1555A > G sudden changes), take each 10 μ L Drug deaf gene mutation detection kit (fluorescent PCR method) (intelligence marine growth engineering (Beijing) company limited) of above-mentioned whole blood sample and extract DNA, obtain corresponding 3 samples according to the sample treatment of said medicine deaf gene mutation detection kit (fluorescent PCR method)。
The saliva sample DNA (sample 13,14,15) of said extracted and whole blood sample DNA (sample 16,17,18) is carried out real-time fluorescence quantitative PCR detection according to the method for inspection of drug induced deafness gene mutation detection kit (fluorescent PCR method), its detection method and step can refer to Chinese patent application CN201510397875.4, and therefore not to repeat here。Testing result is respectively as shown in figures 4-9。
By Fig. 4 and Fig. 5 result it can be seen that in saliva sample, carrying and only occur in the sample of mtDNA 12S rRNA gene wild type DNA that melts a peak, it is the melting peak of amplified production of Quality Control primer；By the result of Fig. 6 it can be seen that carry and occur in the sample of mtDNA 12S rRNA gene 1555A > G sudden change that two melt peaks, the amplified production of its respectively 1555G melts the melting peak of the amplified production of peak and Quality Control primer。
By Fig. 7 and Fig. 8 result it can be seen that in whole blood sample, carrying and only occur in the sample of mtDNA 12S rRNA gene wild type DNA that melts a peak, it is the melting peak of amplified production of Quality Control primer；By the result of Fig. 9 it can be seen that carry and occur in the sample of mtDNA 12S rRNA gene 1555A > G sudden change that two melt peaks, the amplified production of its respectively 1555G melts the melting peak of the amplified production of peak and Quality Control primer。
By above testing result it can be seen that saliva sample is consistent with the testing result of whole blood sample, after adopting the application compositions to preserve saliva sample at ambient temperature, in saliva there is not degeneration in DNA, and DNA integrity is good, does not affect DNA detection result。
Embodiment 4 is for preserving the compositions of DNA in saliva
A kind of for preserving the compositions of DNA in saliva, compound method is with embodiment 1, and its component and content are as shown in table 4 below:
Table 4
In upper table, % representation quality percent by volume。
Embodiment 5 is for preserving the compositions of DNA in saliva
A kind of for preserving the compositions of DNA in saliva, compound method is with embodiment 1, and its component and content are as shown in table 5 below:
Table 5
In upper table, % representation quality percent by volume。
Embodiment 6
After the compositions prepared by embodiment 4 and embodiment 5 formula is sufficiently mixed uniformly with the saliva sample gathered by the volume ratio of 1:1 and preserves a period of time under room temperature respectively, take each 500 μ L nucleic acid extraction of above-mentioned mixed liquor or purification kit (Chong Ding Bioisystech Co., Ltd of Ningbo City, production code member: ZD-XY-Mini) extract DNA, and it is dissolved separately in the eluent of 100 μ L, it is designated as at sample 19 and 20,4 DEG C respectively to save backup。Adopting ultra-violet absorption spectrum to detect the amount of DNA in above-mentioned 2 samples, testing result is as shown in table 6 below:
Table 6
| Sample number into spectrum | A260/A280 | Concentration (ng/ μ L) |
| 19 | 1.821 | 39.3 |
| 20 | 1.909 | 45.2 |
Claims (10)
1. for preserving a compositions of DNA in saliva, including denaturant, buffer, chelating agen, sodium chloride and Polyethylene Glycol。
2. compositions as claimed in claim 1, it is characterised in that: described denaturant is one or more in guanidine hydrochloride, phenol and surfactant；Described surfactant is selected from: one or more in sodium lauryl sulphate, sarcosyl, Triton X-100, polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene 20 sorbitan monopalmitate, polyoxyethylene 20 sorbitan monostearate, polyoxyethylene 20 sorbitan tristearate, polyoxyethylene 20 sorbitan monooleate。
3. compositions as claimed in claim 2, it is characterised in that: described denaturant includes guanidine hydrochloride；Preferably, described denaturant is the combination of guanidine hydrochloride and surfactant。
4. compositions as claimed in claim 3, it is characterised in that: described guanidine hydrochloride concentration in the composition presses quality volume percentage more than 20%；It is preferably 20～60%；It is more preferably 30～60%；It most preferably is 40～60%。
5. the compositions as described in any one of claim 1-4, it is characterised in that: described buffer is the one in trishydroxymethylaminomethane, Tris-HCl buffer, phosphate buffer, barbitol buffer solution；It is preferably Tris-HCl buffer。
6. the compositions as described in any one of Claims 1-4, it is characterised in that: described chelating agen is one or more in ethylenediaminetetraacetic acid, edetate or its hydrate, citric acid, citrate or its hydrate, 1,2-diaminocyclohexane tetraacetic acid。
7. the compositions as described in any one of Claims 1-4, it is characterised in that: described Polyethylene Glycol mean molecule quantity is 4000-8000。
8. compositions as claimed in claim 1, it is characterized in that: described compositions includes the component of following concentration: the denaturant of 30～60%, the Tris-HCl buffer of 5～20mM, the EDTA of 1.5～2.5%, the sodium citrate of 100～300mM or its hydrate, the sodium chloride of 600～900mM, 0.2～1% PEG6000, solvent is water；
Wherein, described denaturant, EDTA, PEG6000 concentration by quality volume percentage；
Described EDTA is one or more in ethylenediaminetetraacetic acid, disodiumedetate, disodiumedetate two water, tetrasodium ethylenediamine tetraacetate two water；
Described sodium citrate or its hydrate include one or more in sodium citrate, sodium citrate two water, sodium citrate three water。
9. compositions as claimed in claim 8, it is characterized in that: described compositions includes the component of following concentration: the denaturant of 30～60%, the Tris-HCl buffer of 5～20mM, 2% the sodium citrate of EDTA, 200mM or its hydrate, the sodium chloride of 800mM, 0.5% PEG6000, solvent is water；
Wherein, described denaturant, EDTA, PEG6000 concentration by quality volume percentage；
Described EDTA is one or more in ethylenediaminetetraacetic acid, disodiumedetate, disodiumedetate two water, tetrasodium ethylenediamine tetraacetate two water；
Described sodium citrate or its hydrate include one or more in sodium citrate, sodium citrate two water, sodium citrate three water。
10. compositions as claimed in claim 9, it is characterized in that: described compositions includes the component of following concentration: the guanidine hydrochloride of 40-60%, 0.1～1% surfactant, the Tris-HCl buffer of 5～20mM, 2% disodiumedetate, sodium citrate two water of 200mM, the sodium chloride of 800mM, 0.5% PEG6000, solvent is water；
Wherein, described denaturant, EDTA, PEG6000 concentration by quality volume percentage。
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| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
| CN107586774A (en) * | 2017-11-01 | 2018-01-16 | 上海涛济医药科技有限公司 | Long-time buccal swab DNA preservations and extracting method under a kind of normal temperature |
| CN108546646A (en) * | 2018-04-18 | 2018-09-18 | 成都知己基因生物科技有限公司 | Micro-biological samples for genetic test preserve liquid and preparation method thereof, kit and application |
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| CN109363728A (en) * | 2018-09-27 | 2019-02-22 | 广州维帝医疗技术有限公司 | Buccal swab and its preparation method and application, acquisition and the method for saving saliva DNA |
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| CN111449056A (en) * | 2020-05-25 | 2020-07-28 | 山西汉济堂医疗科技有限公司 | Novel cell preservation solution and preparation method thereof |
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| CN111778242A (en) * | 2020-07-23 | 2020-10-16 | 德州学院 | Oral cavity liquid virus DNA protective agent and preparation method and application thereof |
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| CN112760318A (en) * | 2020-12-30 | 2021-05-07 | 苏州白垩纪生物科技有限公司 | Reagent composition for stabilizing nucleic acid molecules and application thereof |
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