CN106498072B - The guard method and its application of exogenous DNA internal standard compound in a kind of liquid form product - Google Patents
The guard method and its application of exogenous DNA internal standard compound in a kind of liquid form product Download PDFInfo
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Abstract
The present invention relates to a kind of guard methods of exogenous DNA internal standard compound in liquid form product; specific as follows: the ratio by polycationic compounds solution and target DNA solution according to phosphoramidic acid than >=10:1 mixes; 2-20 DEG C placement 0.5-12 hours after mixing; stable DNA complex solution is obtained, can be added directly in liquid form product；The phosphoramidic acid is than the ratio of the phosphate group molal quantity carried for amino molal quantity entrained by polycation and DNA.Exogenous DNA can be improved to the tolerance of the conditions such as nuclease, acid and free radical in polycationic compounds in this method, it is made to can be used as to trace to the source and be stabilized for a long time in liquid form product or commodity with anti-fake internal standard compound.
Description
Technical field
The invention belongs to a kind of protections of exogenous DNA internal standard compound in authentication technical field more particularly to liquid form product
Method and its application.
Background technique
In recent years, it emerges one after another the phenomenon that fraud adulterated due to commodity, consumer is for the anti-fake of commodity and traces to the source especially
Pay attention to.Especially for foodstuff product, since it is concerning national health, even more consumer and state supervision department
Focal point.The anti-fake and measure of tracing to the source of commodity is mainly package anti-fake at present, i.e., various special identifiers is invested outer packing
On, or using special outsourcing assembling structure so as to the identification of consumer and inspection body.However, easy due to package anti-fake
Destructive and easy imitation property, leaves opportunity to criminal.With the foundation of food industries traceability system and perfect, lead to
It crosses and exogenous nucleic acid is added in food as the new technique for marker of tracing to the source gradually by development and application.Such as Chinese patent application
CN 101665825A discloses a kind of method for identifying falsification of distilled spirit using nucleic acid detection technique, by animal, plant, micro- life
Object or artificial synthesized genomic DNA or genetic fragment, which are added in white wine, is used as its internal standard compound, then in round pcr detection
The presence or absence of object is marked to judge the true and false of commodity.
However, since there are many nucleic acid damage factor (such as cores in most of liquid form product especially food liquid
Sour enzyme, free radical and acid etc.), the damage such as depurination, oxidation or fracture can occur after through a long time storage in liquid form product for DNA
Wound, causes the loss of sequence information, eventually leads to the false negative of detection, this will limit significantly it as marker in product false proof
With trace to the source in application.Therefore, a kind of method of raising DNA stability is needed at present to realize that it is long-term in liquid form product
It is stabilized.Have some methods for improving DNA stability in practical applications at present.For example, using Tris-EDTA buffer
Dissolution nucleic acid carrys out the weakly alkaline environment of maintenance system；Reducing agent (such as DTT) is added in the reaction system to prevent nucleic acid to be oxidized；
Or nucleic acid is subjected to structure of modification or modification to improve its tolerance etc. to nuclease.But since these methods need to change
Poisonous and hazardous chemical reagent is added in the physicochemical property of product, or needs transformation or modification step complicated, that cost is high, is difficult
It is applied in practical industry.
Obviously, if can be easy to use, low in cost, nontoxic, and the method for DNA structure and property is not influenced
Improve exogenous DNA stability in the product, will expand significantly application range of the DNA marker in commodity counterfeit prevention and in tracing to the source and
Prospect.
Summary of the invention
In view of the above-mentioned problems, the present invention is intended to provide a kind of simple and convenient, low in cost, nontoxic, and do not influence DNA
The method of structure and property, to protect the exogenous DNA in liquid form product.
The guard method of exogenous DNA internal standard compound in a kind of liquid form product, it is specific as follows: by polycationic compounds solution with
Ratio of the target DNA solution according to phosphoramidic acid than >=10:1 mixes, and 2-20 DEG C placement 0.5-12 hours, are stablized after mixing
DNA complex solution, can be added directly in liquid form product；The phosphoramidic acid is than for amino entrained by polycation
The ratio for the phosphate group molal quantity that molal quantity and DNA are carried.
In solution, the negative electrical charge on the phosphate group of nucleic acid can be with the positive charge phase interaction of polycationic compounds carrying
With, slow down significantly DNA in acid condition depurination reaction, to improve its tolerance to acidic environment.In addition, by
It is centered around around DNA in polycationic compounds, hinders nuclease and the combination of DNA, while also counteracting free radical pair
The attack of DNA, to also improve DNA to the tolerance of nuclease and free radical.
The temperature placed after mixing polycationic compounds solution with target DNA solution in the present invention is controlled at 2-20 DEG C
Between, first ensure that solution is in liquid condition to carry out the interaction between DNA and polycationic compounds, but temperature
Unsuitable excessively high, verified, when temperature is more than 20 DEG C, polycationic compounds are bad to the protecting effect of DNA, and easily lead to micro-
Biological self reproducing.
The time placed after mixing polycationic compounds solution with target DNA solution in the present invention controls in 0.5-12
Hour, it is verified more than 0.5 hour when DNA can form compound with polycationic compounds, and more have as time went on
It is sufficiently acted on conducive to DNA and polycationic compounds, but overlong time then easily leads to microbial reproduction.
Further, the temperature that polycationic compounds solution and target DNA solution are placed after mixing is 2-8 DEG C, the time
It is 6-12 hours, selects low temperature, long-time within the bounds of possibility, can guarantee that DNA is sufficiently acted on polycationic compounds, and
Prevent microbial reproduction.
Verified (referring to embodiment 1), in 1-100:1, polycationic compounds can play pair phosphoramidic acid ratio
The protective effect of DNA, with the increase of phosphoramidic acid ratio, protective effect is gradually increased.But when phosphoramidic acid ratio is greater than 10:1,
Protective effect is basically unchanged, therefore the optimal selection of phosphoramidic acid ratio is 10:1.Ideal protecting effect can be reached and had
Effect control cost.
Further, the DNA include but is not limited to genomic DNA, mitochondrial DNA, chloroplast DNA, Plasmid DNA,
PCR product, DNA enzymatic are sliced one of section or poly oligonucleotides or a variety of.Preferred plasmid DNA, PCR product are easier to be made,
Advantage of lower cost is suitable for industrial applications.
The genomic DNA includes the zoic genomic DNA of institute, preferably salmon sperm dna, herring sperm dna or λ DNA
One of or it is a variety of because these genomic DNA amounts are easy to get greatly, and be commercialized, be easily commercially available.
The Plasmid DNA includes but is not limited to one in pUC18, pUC19, pUC57, pBR322, pTZ19R or pET28
Kind is a variety of.
Further, the polycationic compounds are the compound that one kind can assemble positive charge in the solution, including
But one of it is not limited to chitosan oligosaccharide, chitosan, spermine, putrescine, cadaverine, spermidine, polypeptide or alkaline protein or a variety of.
Further, the alkaline protein includes protamine and histone etc..
Further, the molecular weight of the chitosan is in 20-250kDa, and the molecular weight of general commercially available chitosan is at this
In range, therefore such chitosan is more easy to get.
Further, the pH=6-8 of the DNA complex solution influences product itself after avoiding being added in product
Physicochemical property.
It is a further object to provide a kind of concrete applications of above-mentioned DNA guard method, that is, are applied to liquid production
It product anti-fake and traces to the source.
A kind of anti-fake and source tracing method of liquid form product, specific steps are as follows:
(1) ratio by polycationic compounds solution and target DNA solution according to phosphoramidic acid than >=10:1 mixes, and mixes
2-20 DEG C placement 0.5-12 hours after even, obtain stable DNA complex solution, and the phosphoramidic acid ratio is polycationic
Close the ratio for the phosphate group molal quantity that amino molal quantity entrained by object and DNA are carried；
(2) DNA complex solution obtained in step (1) is added in liquid form product, additive amount with the meter of DNA,
Make the content 1ng/mL-50 μ g/mL of DNA in liquid form product；Since protecting effect of the method for the present invention to DNA is excellent, DNA exists
Can be stabilized in liquid form product, as long as therefore additional amount reach the detection limit of DNA；The DNA compound of actual interpolation
Volume is far smaller than the volume of liquid form product, can effectively ensure that quality and appearance of product etc.；
(3) it detects in liquid form product and whether contains added DNA.
Further, in step (3) method of detection DNA include agarose gel electrophoresis, polyacrylamide gel electrophoresis,
One of the methods of denaturing polyacrylamide gel electrophoresis, Standard PCR, real-time fluorescence quantitative PCR or constant temperature nucleic acid amplification or
It is a variety of.
Compared with the prior art the shortcomings that and deficiency, the invention has the following advantages:
(1) present invention carries out exogenous DNA using nontoxic polycationic compounds (such as chitosan oligosaccharide and chitosan)
Protection improves exogenous DNA to the tolerance of the conditions such as nuclease, acid and free radical, DNA internal standard compound is enable steadily to be present in
In the liquid form product of complicated component, be applied to liquid form product especially food liquid trace to the source with it is anti-fake in.
(2) simple and convenient in such a way that DNA to be mixed to placement with polycationic compounds, technical level requires low, side
Just it produces in batches.
(3) type of polycationic compounds is more, and raw material is easy to get, and cost is relatively low.
(4) since nucleic acid has the fundamental characteristics of polyanion, in the solution, the negative electrical charge on the phosphate group of nucleic acid can
It interacts with the positive charge that polycationic compounds carry, forms metastable compound.Due to polycationic compounds
It is and the Non-covalent binding through positive and negative charge interaction in conjunction with DNA, on the structure of DNA and property all almost without influence, because
This does not influence the detection that electrophoresis, PCR or other modes are carried out to DNA.
Detailed description of the invention
Fig. 1: the route schematic diagram of 1-7 implementation method of the embodiment of the present invention；
Fig. 2: the nucleic acid agarose gel electrophoresis results figure of the embodiment of the present invention 1；Wherein, digital ratio represents polycation
The phosphoramidic acid of compound and DNA ratio；
Fig. 3: the nucleic acid agarose gel electrophoresis results figure of the embodiment of the present invention 1；
Fig. 4: the nucleic acid denaturation polyacrylamide gel electrophoresis result figure of the embodiment of the present invention 2；
Fig. 5: the real-time fluorescence quantitative PCR testing result of the embodiment of the present invention 3；
Fig. 6: real-time fluorescence quantitative PCR of the DNA after 20 DEG C of storage different times in the embodiment of the present invention 3, in white wine
Testing result；
Fig. 7: the real-time fluorescence quantitative PCR testing result of the embodiment of the present invention 4；
Fig. 8: the real-time fluorescence quantitative PCR testing result of the embodiment of the present invention 5；
Fig. 9: the real-time fluorescence quantitative PCR testing result of the embodiment of the present invention 6；
Figure 10: the real-time fluorescence quantitative PCR testing result of the embodiment of the present invention 7.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, right below in conjunction with drawings and examples
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
1 chitosan oligosaccharide of embodiment and spermine improve λ DNA to the tolerance of DNase I
1, the preparation of chitosan oligosaccharide and solution of spermine
The preparation of chitosan oligosaccharide solution: it accurately weighs 150mg chitosan oligosaccharide and (it is limited to be purchased from Laizhou City, Shandong Province sea power biological products
Company；Lot number: HL130317G；Molecular weight: 6500Da；Deacetylation: 91.1%), the hydrochloric acid solution that 8mL pH 2.0 is added makes
Chitosan oligosaccharide is completely dissolved, and then adjusts solution ph to 7.0 with the sodium hydroxide solution of 0.1M.Finally, fixed with sterilizing ultrapure water
Hold to 10mL, in 4 DEG C of preservations.The concentration of required working solution is diluted to when use with sterilizing ultrapure water.
The preparation of solution of spermine: it accurately weighs 100mg spermine and (is purchased from Sigma-Aldrich；Article No.: S3256), use 8mL
It sterilizes after ultrapure water dissolution, 10mL is settled to, in 4 DEG C of preservations.The dense of required working solution is diluted to sterilizing ultrapure water when use
Degree.
2, the preparation of λ DNA- chitosan oligosaccharide compound and λ DNA- spermine compound
After λ DNA and chitosan oligosaccharide or spermine are mixed according to the ratio of different phosphoramidic acid ratios (1:1-100:1), make λ
The concentration of DNA is 300ng/ μ L, and mixture is placed 12 hours in 4 DEG C, λ DNA is acted on sufficiently with polycationic compounds, leads to
It crosses electrostatic interaction and forms compound.
3, tolerance test of the compound to DNase I
λ DNA- chitosan oligosaccharide compound or λ DNA- spermine compound are added in the system containing DNase I, 20 μ L bodies
The content of the 600ng of DNA containing λ in system, DNase I are 0.02U and 1 × DNase I buffer.After 37 DEG C of reaction 10min, 65
Warm bath 10min inactivates DNase I at DEG C.Then it is examined by integrity degree of the method for agarose gel electrophoresis to λ DNA
It surveys.
Fig. 2 the results show that with phosphoramidic acid ratio increase, polycationic compounds gradually increase the protective effect of λ DNA
By force.As phosphoramidic acid ratio >=10:1, chitosan oligosaccharide and spermine are good to the protecting effect of λ DNA.
4, the influence of DNA and polycationic compounds composite construction to protecting effect
Influence for validating DNA compound to protecting effect, setting do not form the control group of compound in advance, specifically:
λ DNA, chitosan oligosaccharide or spermine are added sequentially in the system containing DNase I respectively, make the phosphoramidic acid ratio in system
10:1 (the same experimental group of ratio).The content of DNA containing λ 600ng, DNase I are 0.02U and 1 × DNase I in 20 μ L systems
buffer.After 37 DEG C of reaction 10min, warm bath 10min inactivates DNase I at 65 DEG C.Pass through the side of agarose gel electrophoresis
Method detects the integrity degree of λ DNA in experimental group and control group.
Fig. 3 is the results show that in control group: DNA and polycationic compounds are directly sequentially added the body containing DNase I
After in system, although polycationic compounds have certain protective effect, the ratio that DNA still degrades is more serious.And in experimental group:
By DNA and polycationic compounds premix and 4 DEG C are placed 12 hours, add meeting in reaction system after so that the two is formed compound
DNA is significantly improved to the tolerance of DNase I.
Protection of the chitosan to poly oligonucleotides in 2 lactic acid drink of embodiment
1, the preparation of external source single-stranded polynucleotides oligonucleotides and chitosan complexes
The preparation of chitosan solution: it accurately weighs 250mg chitosan and (is purchased from ZHEJIANG AOXING BIOTECHNOLOGY CO., LTD；Batch
Number: 20140220；Molecular weight: 50-100kDa), 20mL sterilizing ultrapure water is added, magnetic agitation 2h, makes chitosan powder at room temperature
Last sufficiently hydration.The hydrochloric acid solution of 20mL pH 2 is gradually added dropwise to the chitosan solution of hydration, magnetic agitation makes it sufficiently overnight
Dissolution.Then, its pH value is adjusted to 7.0 using the sodium hydroxide solution of 0.1M.Finally it is settled to sterilizing ultrapure water
100mL, in 4 DEG C of preservations.The concentration of required working solution is diluted to when use with sterilizing ultrapure water.
By single-stranded polynucleotides oligonucleotides (36mer, sequence GTCTTAAATAGGCCACTAAGTCGTTAATTCGAGACT)
It is mixed with chitosan according to the ratio of phosphoramidic acid ratio 10:1, so that the final concentration of DNA is reached 100 μM, and place 8 hours in 8 DEG C,
It acts on DNA sufficiently with chitosan, compound is formed by electrostatic interaction.
2, long-term storage of the DNA- chitosan complexes in sour milk beverage
Taking the compound prepared in 4 μ L steps 1 to be added to 396 μ L sour milk beverages, (name of an article: the every benefit of Erie adds low sugar activity
Sour milk beverage；Specification: 350mL；Manufacturer: Inner Mongolia Yili Industry Group Co., Ltd) in, make poly few nucleosides
The final concentration of acid reaches 1 μM (~10ng/ μ L), after mixing, by EP ferrule and is respectively placed in 20 DEG C and 37 DEG C with sealed membrane
Lower storage takes out sample after a period of time and detects the content of DNA.It is provided with the control group for not adding chitosan simultaneously, i.e., will
Poly oligonucleotides is added directly into above-mentioned sour milk beverage, makes the final concentration phase of the final concentration of DNA with DNA in compound
Together, other holding conditions are identical.
3, detection-denaturing polyacrylamide gel electrophoresis of poly oligonucleotides stability
It is chitosan in detecting step 2 to the protecting effect of poly oligonucleotides, uses denaturing polyacrylamide gel electricity
The content of poly oligonucleotides after swimming detection is stored for a long time in sour milk beverage.To avoid the ingredient shadow in sour milk beverage
Electrophoresis is rung, loading after 10 times being diluted containing the sour milk beverage of poly oligonucleotides.The concentration for being denaturalized PAGE gel is 15%,
Wherein urea containing 8M.Analysis result is shown in Fig. 4.
The result shows that stability of the single-stranded polynucleotides oligonucleotides in sour milk beverage is poor, stored 30 days when at 20 DEG C
Or after 37 DEG C are stored 5 days, most of single-stranded polynucleotides oligonucleotides is all degraded.The compound of oligonucleotides and chitosan is added
The stability for entering to greatly improve oligonucleotides in sour milk beverage, DNA content is still higher after 37 DEG C are stored 20 days.
The protection of chitosan, chitosan oligosaccharide and spermine to pUC18 Plasmid DNA in 3 white wine of embodiment
1, the preparation of exogenous plasmid dna and chitosan, chitosan oligosaccharide and spermine compound
The preparation of chitosan solution: it accurately weighs 250mg chitosan and (is purchased from Sigma-Aldrich；Article No.: 740500；Point
Son amount: 110-150kDa), 20mL sterilizing ultrapure water is added, magnetic agitation 2h, is hydrated Chitosan powder sufficiently at room temperature.To
The hydrochloric acid solution of 20mL pH 2 is gradually added dropwise in the chitosan solution of hydration, and magnetic agitation dissolves it sufficiently overnight.Then, make
PH value is adjusted to 7.0 with the sodium hydroxide solution of 0.1M.It finally is settled to 100mL with sterilizing ultrapure water, in 4 DEG C of preservations.Make
Used time is diluted to the concentration of required working solution with sterilizing ultrapure water.
The preparation of chitosan oligosaccharide solution: it accurately weighs 150mg chitosan oligosaccharide and (it is limited to be purchased from Laizhou City, Shandong Province sea power biological products
Company；Lot number: HL130317G；Molecular weight: 6500Da；Deacetylation: 91.1%), the hydrochloric acid solution that 8mL pH 2.0 is added makes
Chitosan oligosaccharide is completely dissolved, and then adjusts solution ph to 7.0 with the sodium hydroxide solution of 0.1M.Finally, fixed with sterilizing ultrapure water
Hold to 10mL, in 4 DEG C of preservations.The concentration of required working solution is diluted to when use with sterilizing ultrapure water.
The preparation of solution of spermine: it accurately weighs 100mg spermine and (is purchased from Sigma-Aldrich；Article No.: S3256), use 8mL
It sterilizes after ultrapure water dissolution, 10mL is settled to, in 4 DEG C of preservations.The dense of required working solution is diluted to sterilizing ultrapure water when use
Degree.
PUC18 Plasmid DNA is mixed with chitosan, chitosan oligosaccharide and solution of spermine according to the ratio of phosphoramidic acid ratio 10:1 respectively
It closes, so that the final concentration of DNA is reached 29ng/ μ L, and place 12 hours in 4 DEG C, act on DNA sufficiently with polycationic compounds,
Compound is formed by electrostatic interaction.
2, Plasmid DNA-chitosan, Plasmid DNA-chitosan oligosaccharide, Plasmid DNA-long-term storage of the spermine compound in white wine
The compound prepared in 4 μ L steps 1 is taken to be added to the 396 μ L white wine (name of an article: three well pocket knives；Alcoholic strength: 45%vol；
Specification: 248mL；Manufacturer: Hebei San Jing the wine industry limited liability company) in, so that the final concentration of Plasmid DNA is reached 290pg/ μ L
(~108Copy/μ L), after mixing, EP ferrule and being respectively placed at 4 DEG C, 20 DEG C and 37 DEG C is stored with sealed membrane, one section
Sample is taken out after time and detects the content of Plasmid DNA.It is provided with the control group for not adding polycationic compounds simultaneously, i.e., will
Plasmid DNA is added directly into above-mentioned white wine, keeps the final concentration of Plasmid DNA identical as the final concentration of Plasmid DNA in compound,
His holding conditions are identical.
3, detection-fluorescence quantitative PCR method of Plasmid DNA stability
It is chitosan, chitosan oligosaccharide and spermine in detecting step 2 to the protecting effect of Plasmid DNA, uses fluorescence quantitative PCR method
(RT-qPCR) content of Plasmid DNA in white wine after storing for a long time is detected.To avoid the composition influence RT-qPCR in white wine anti-
It answers, the template after the white wine containing Plasmid DNA is diluted 100 times as RT-qPCR reaction.For the design of pUC18 Plasmid DNA
Specific PCR primers are shown in Table 1, and the purpose product length of amplification is 170bp.Each system contains 52 × SYBR of μ L in reaction
Select Master Mix (it is purchased from Invitrogen, article No.: 4472908), and 0.3 μM of specific primer, after the 1 above-mentioned dilution of μ L
The white wine containing Plasmid DNA, use ddH2O is mended to 10 μ L.Response parameter are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation 20s, 59 DEG C
Anneal 20s, 72 DEG C of extension 20s, 40 circulations.Analysis result is shown in Fig. 5 and Fig. 6.
Fig. 5 the result shows that, stability of the pure plasmid DNA in white wine is poor, after 37 DEG C, the storage of 10d, control group
The DNA content of middle white wine is only initial 0.07%.It is containing organic acids, total acidities such as lactic acid, acetic acid, caproic acid and butyric acid in white wine
0.25-0.45g/L, therefore exogenous DNA can degrade in white wine because of depurination.After reducing reserve temperature, the stability of DNA is bright
It is aobvious to improve, but after storing for a long time DNA to be destroyed degree still larger.By Plasmid DNA-chitosan, DNA- chitosan oligosaccharide and
The stability that can greatly improve DNA is added in white wine after DNA- spermine composition compound.Wherein protection of the chitosan to DNA
Effect is best, by 4 DEG C 30 days, after 20 DEG C of 20 days and 37 DEG C of storages in 10 days, DNA content does not have significant changes.Due to spermine
Smaller with the molecular weight of chitosan oligosaccharide, the compound formed with DNA is more unstable, therefore spermine and chitosan oligosaccharide are for Plasmid DNA
Protecting effect is weaker than chitosan.
The changes of contents that the Plasmid DNA in white wine is stored in 30 days at 20 DEG C is illustrated in Fig. 6.The results show that pure DNA
Stability in white wine is poor, and DNA- chitosan complexes and DNA- chitosan oligosaccharide compound stable for a long time in white wine can be deposited
?.
Primer sequence used in 1. embodiment 3 of table
The protection of spermidine and chitosan to pUC18 Plasmid DNA in 4 grape wine of embodiment
1, the preparation of exogenous plasmid dna and spermidine and chitosan complexes
The preparation of spermidine solution: it accurately weighs 100mg spermidine and (is purchased from Sigma-Aldrich；Article No.: S2626), it uses
8mL sterilizes after ultrapure water dissolution, 10mL is settled to, in 4 DEG C of preservations.Required working solution is diluted to sterilizing ultrapure water when use
Concentration.
The preparation of chitosan solution: it accurately weighs 250mg chitosan and (is purchased from Sigma-Aldrich；Article No.: 740179；Point
Son amount: 140-220kDa), 20mL sterilizing ultrapure water is added, magnetic agitation 2h, is hydrated Chitosan powder sufficiently at room temperature.To
The hydrochloric acid solution of 20mL pH 2 is gradually added dropwise in the chitosan solution of hydration, and magnetic agitation dissolves it sufficiently overnight.Then, make
PH value is adjusted to 7.0 with the sodium hydroxide solution of 0.1M.It finally is settled to 100mL with sterilizing ultrapure water, in 4 DEG C of preservations.Make
Used time is diluted to the concentration of required working solution with sterilizing ultrapure water.
PUC18 Plasmid DNA is mixed with spermidine, chitosan according to the ratio of phosphoramidic acid ratio 10:1 respectively, makes DNA's
Final concentration reaches 2.9ng/ μ L, and places 10 hours in 2 DEG C, acts on DNA sufficiently with chitosan, passes through electrostatic interaction shape
At compound.
2, the long-term storage of Plasmid DNA-spermidine, Plasmid DNA-chitosan complexes in grape wine
The compound prepared in 4 μ L steps 1 is taken to be added to the 396 μ L grape wine (name of an article: Qingdao East China claret；Wine
Precision: 13%vol；Specification: 750mL；Manufacturer: Qingdao Huadong Grape Brewing Co., Ltd.) in, keep the end of Plasmid DNA dense
Degree reaches 29pg/ μ L (~107Copy/μ L), after mixing, by EP ferrule and 4 DEG C, 20 DEG C and 37 are respectively placed in sealed membrane
It is stored at DEG C, take out sample after a period of time and detects the content of Plasmid DNA.It is provided with simultaneously and does not add polycationic compounds
Control group, i.e., Plasmid DNA is added directly into above-mentioned grape wine, makes the final concentration of DNA in the final concentration and compound of DNA
Identical, other holding conditions are identical.
3, detection-fluorescence quantitative PCR method of Plasmid DNA stability
It is spermidine in detecting step 2 and chitosan to the protecting effect of Plasmid DNA, uses quantitative fluorescent PCR (RT-
QPCR the content of Plasmid DNA in grape wine after storing for a long time) is detected.To avoid the composition influence RT-qPCR in grape wine anti-
It answers, the template after the grape wine containing Plasmid DNA is diluted 100 times as RT-qPCR reaction.It is designed for pUC18 Plasmid DNA
Specific PCR primers be shown in Table 2, the purpose product length of amplification is 138bp.In reaction each system contain 5 μ L 2 ×
(be purchased from Invitrogen, article No.: 4472908), 0.3 μM of specific primer, 1 μ L is above-mentioned dilute by SYBR Select Master Mix
The grape wine containing Plasmid DNA after releasing, uses ddH2O is mended to 10 μ L.Response parameter are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation
20s, 59 DEG C of annealing 20s, 72 DEG C of extension 20s, 40 recycle.Analysis result is shown in Fig. 7.
The result shows that stability of the pure plasmid DNA in grape wine is poor, after 37 DEG C, the storage of 10d, control group
The Plasmid DNA content of middle grape wine is only 0.08%.The total acidity of grape wine is 1.66-4.52g/L, wherein containing tartaric acid, apple
Lactic acid, succinic acid and the acetic acid etc. that tartaric acid and fermentation generate, therefore exogenous DNA can degrade in grape wine because of depurination.It will
The stability that can greatly improve DNA is added in grape wine after Plasmid DNA-chitosan and DNA- spermidine composition compound.
Wherein chitosan is better than spermidine to the protecting effect of DNA, by 4 DEG C 30 days, after 20 DEG C of 20 days and 37 DEG C of storages in 10 days,
DNA content in DNA- chitosan complexes does not have significant changes.
Primer sequence used in 2. embodiment 4 of table
Protection of the nucleoprotamine to pUC19 Plasmid DNA in 5 milk of embodiment
1, the preparation of exogenous plasmid dna and nucleoprotamine compound
The preparation of nucleoprotamine solution: it accurately weighs 10mg nucleoprotamine and (is purchased from Sigma-Aldrich；Article No.:
P4005), 8mL sterilizing ultrapure water and 50 μ L 0.5M EDTA (pH 8.0) are added, it is ultrapure with sterilizing after nucleoprotamine dissolution
Water is settled to 10mL, in 4 DEG C of preservations.The concentration of required working solution is diluted to when use with sterilizing ultrapure water.
PUC19 Plasmid DNA and nucleoprotamine are mixed according to the ratio of phosphoramidic acid ratio 10:1, reach the final concentration of DNA
It is placed 10 hours to 290pg/ μ L, and in 6 DEG C, acts on DNA sufficiently with nucleoprotamine, formed by electrostatic interaction compound
Object.
2, long-term storage of the DNA- nucleoprotamine compound in milk
The compound prepared in 4 μ L steps 1 is taken to be added to the 396 μ L milk (name of an article: the full-cream pure cow's milk of sterilizing of Erie；Specification:
250mL is box-packed；Manufacturer: Inner Mongolia Yili Industry Group Co., Ltd) in, reach the final concentration of Plasmid DNA
2.9pg/ μ L (~106Copy/μ L), after mixing, by EP ferrule and it is respectively placed at 4 DEG C, 20 DEG C and 37 DEG C with sealed membrane
Storage takes out sample after a period of time and detects the content of Plasmid DNA.It is provided with the control group for not adding nucleoprotamine simultaneously,
Plasmid DNA is added directly into above-mentioned milk, keeps the final concentration of DNA identical as the final concentration of DNA in compound, other storages
Hiding condition is identical.
3, detection-fluorescence quantitative PCR method of Plasmid DNA stability
It is nucleoprotamine in detecting step 2 to the protecting effect of Plasmid DNA, is detected using quantitative fluorescent PCR (RT-qPCR)
After storing for a long time in milk Plasmid DNA content.To avoid the composition influence RT-qPCR in milk from reacting, plasmid will be contained
The template that the milk of DNA reacts after diluting 100 times as RT-qPCR.For the Specific PCR primers of pUC19 Plasmid DNA design
3 are shown in Table, the purpose product length of amplification is 154bp.Each system contains 5 μ L 2 × SYBR Select Master in reaction
Mix (it is purchased from Invitrogen, article No.: 4472908), 0.3 μM of specific primer, the ox containing Plasmid DNA after the 1 above-mentioned dilution of μ L
Milk uses ddH2O is mended to 10 μ L.Response parameter are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation 20s, 59 DEG C of annealing 20s, 72 DEG C extend
20s, 40 circulations.Analysis result is shown in Fig. 8.
The result shows that stability of the pure plasmid DNA in milk is lower, and content is only initial after 10d is stored at 37 DEG C
1%.After DNA- nucleoprotamine compound is added, the stability of DNA is significantly improved, by 4 DEG C 30 days, 20 DEG C
DNA content does not have significant changes after 20 days and 37 DEG C of storages in 10 days.
Primer sequence used in 3. embodiment 5 of table
Protection of the chitosan oligosaccharide to pUC19 Plasmid DNA in 6 lemon juice beverage of embodiment
1, the preparation of exogenous plasmid dna and chitosan oligosaccharide compound
The preparation of chitosan oligosaccharide solution: it accurately weighs 150mg chitosan oligosaccharide and (it is limited to be purchased from Laizhou City, Shandong Province sea power biological products
Company；Lot number: HL130317G；Molecular weight: 6500Da；Deacetylation: 91.1%), the hydrochloric acid solution that 5mL pH 2 is added makes shell
Oligosaccharides is completely dissolved, and then adjusts solution ph to 7.0 with the sodium hydroxide solution of 0.1M.Finally, with sterilizing ultrapure water constant volume
To 10mL, in 4 DEG C of preservations.The concentration of required working solution is diluted to when use with sterilizing ultrapure water.
PUC19 plasmid and chitosan oligosaccharide are mixed according to the ratio of phosphoramidic acid ratio 10:1, reach the final concentration of DNA
290ng/ μ L, and placed 10 hours in 4 DEG C, it acts on DNA sufficiently with chitosan oligosaccharide, compound is formed by electrostatic interaction.
2, long-term storage of the DNA- chitosan oligosaccharide compound in lemon juice beverage
The compound prepared in 4 μ L steps 1 is taken to be added to the 396 μ L lemon juice beverages (name of an article: U.S. juice source ice tangerine lemon；Rule
Lattice: 480mL；Manufacturer: Shanghai Shen-Mei Beverage and Food Co., Ltd.) in, so that the final concentration of Plasmid DNA is reached 2.9ng/ μ L
(~109Copy/μ L), after mixing, EP ferrule and being respectively placed at 4 DEG C, 20 DEG C and 37 DEG C is stored with sealed membrane, one section
Sample is taken out after time and detects the content of DNA.Be provided with the control group for not adding chitosan oligosaccharide simultaneously, i.e., it is Plasmid DNA is direct
It is added in above-mentioned lemon juice beverage, keeps the final concentration of DNA identical as the final concentration of DNA in compound, other holding conditions are complete
It is exactly the same.
3, detection-fluorescence quantitative PCR method of Plasmid DNA stability
It is chitosan oligosaccharide in detecting step 2 to the protecting effect of Plasmid DNA, is detected and grown using quantitative fluorescent PCR (RT-qPCR)
Plasmid DNA content after time storage in lemon juice beverage.To avoid the composition influence RT-qPCR in lemon juice beverage from reacting,
Template after lemon beverage containing Plasmid DNA is diluted 100 times as RT-qPCR reaction.It is designed for pUC19 Plasmid DNA
Specific PCR primers be shown in Table 4, the purpose product length of amplification is 208bp.In reaction each system contain 5 μ L 2 ×
(be purchased from Invitrogen, article No.: 4472908), 0.3 μM of specific primer, 1 μ L is above-mentioned dilute by SYBR Select Master Mix
The lemon juice beverage containing Plasmid DNA after releasing, uses ddH2O is mended to 10 μ L.Response parameter are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of changes
Property 20s, 59 DEG C of annealing 20s, 72 DEG C of extension 20s, 40 circulation.Analysis result is shown in Fig. 9.
At 37 DEG C, stability of the pure plasmid DNA in lemon juice beverage is poor, the DNA after storing 10d, in lemon juice
Content is only initial 0.3%.This is because the pH value of lemon juice is lower, depurination reaction easily occurs wherein for DNA, due to
Chitosan oligosaccharide can significantly inhibit the depurination of DNA in acid condition, therefore show after DNA- chitosan oligosaccharide compound is added in system
Work improves stability of the DNA in lemon juice beverage.
Primer sequence used in 4. embodiment 6 of table
Protection of the chitosan to PCR product in 7 light-coloured vinegar of embodiment
1, the preparation of PCR product and chitosan complexes
The preparation of chitosan solution: it accurately weighs 250mg chitosan and (is purchased from Sigma-Aldrich；Article No.: 740063；Point
Son amount: 60-120kDa), 20mL sterilizing ultrapure water is added, magnetic agitation 2h, is hydrated Chitosan powder sufficiently at room temperature.Xiang Shui
The hydrochloric acid solution of 20mL pH 2 is gradually added dropwise in the chitosan solution of conjunction, and magnetic agitation dissolves it sufficiently overnight.Then, it uses
PH value is adjusted to 7.0 by the sodium hydroxide solution of 0.1M.It finally is settled to 100mL with sterilizing ultrapure water, in 4 DEG C of preservations.It uses
When the concentration of required working solution is diluted to sterilizing ultrapure water.
The preparation of PCR product: using pUC18 plasmid as template (1010Copy/μ L), primer pUC19-364a/s be primer into
Row common PCR reaction, primer sequence are shown in Table 5, and the purpose product length of amplification is 364bp.In 50 μ L systems containing 5 μ L 10 ×
Pfu buffer, 0.4 μM of primer pUC19-364a/s, 0.2mM dNTPs, 2U pfu archaeal dna polymerase (are purchased from Thermo
Scientific).PCR parameter are as follows: 72 DEG C of primer extend 5min, 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 59 DEG C are annealed
30s, 72 DEG C of extension 90s, 72 DEG C of heat preservation 5min after 35 circulations.Obtained PCR product is subjected to alcohol precipitation purifying and uses nano
The concentration of drop measurement PCR product.
PCR product and chitosan are mixed according to the ratio of phosphoramidic acid ratio 10:1, the final concentration of DNA is made to reach 4ng/ μ
L, and placed 10 hours in 2 DEG C, it acts on DNA sufficiently with chitosan, compound is formed by electrostatic interaction.
2, long-term storage of the DNA- chitosan complexes in light-coloured vinegar
The compound prepared in 4 μ L steps 1 is taken to be added to the 396 μ L light-coloured vinegars (name of an article: extra large day white rice vinegar；Specification: 450mL；System
Make unit: Foshan Haitian Seasoning Co., Ltd.) in, so that the final concentration of PCR product is reached 40pg/ μ L (~108It copies
Shellfish/μ L), after mixing, EP ferrule and being respectively placed at 4 DEG C, 20 DEG C and 37 DEG C is stored with sealed membrane, is taken after a period of time
Sample and detect the content of PCR product out.It is provided with the control group for not adding chitosan simultaneously, i.e., is directly added into PCR product
Into above-mentioned light-coloured vinegar, keep the final concentration of DNA identical as the final concentration of DNA in compound, other holding conditions are identical.
3, detection-fluorescence quantitative PCR method of PCR product stability
It is chitosan in detecting step 2 to the protecting effect of PCR product, is detected and grown using quantitative fluorescent PCR (RT-qPCR)
After time storage in light-coloured vinegar PCR product content.To avoid the composition influence RT-qPCR in light-coloured vinegar from reacting, PCR product will be contained
Light-coloured vinegar dilute 100 times after as RT-qPCR react template.For the Specific PCR primers pUC18- of PCR product design
The sequence of 156a/s is shown in Table 5, and the purpose product length of amplification is 156bp.Each system contains 52 × SYBR of μ L in reaction
Select Master Mix (it is purchased from Invitrogen, article No.: 4472908), and 0.3 μM of specific primer, after the 1 above-mentioned dilution of μ L
The light-coloured vinegar containing PCR product, use ddH2O is mended to 10 μ L.Response parameter are as follows: 95 DEG C of initial denaturation 2min, 95 DEG C of denaturation 20s, 59 DEG C
Anneal 20s, 72 DEG C of extension 20s, 40 circulations.Analyze the result is shown in Figure 10.
Stability of the pure PCR product in light-coloured vinegar is poor, and after 37 DEG C of storage 10d, the DNA content in light-coloured vinegar is only initial
0.1%.This is because the pH value of light-coloured vinegar is lower (~pH 2.2), depurination reaction easily occurs wherein for DNA, since shell is poly-
Sugar can significantly inhibit the depurination of DNA in acid condition, therefore significantly improve after DNA- chitosan complexes are added in system
Stability of the DNA in light-coloured vinegar.
Primer sequence used in 5. embodiment 7 of table
The above embodiments the result shows that method of the invention is greatly improved stability of the DNA in liquid form product,
After high temperature, storage for a long time, the content of exogenous DNA is almost unchanged in product, therefore can be internal by molecular biology method
The presence or absence of mark DNA even content is detected, so that it is determined that the true and false and whereabouts of product, to expand DNA as commodity counterfeit prevention
With the application range for internal standard compound of tracing to the source.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
SEQUENCE LISTING
<110>the tall and erect molecular biosciences in Qingdao thousand Science and Technology Ltd.
<120>in a kind of liquid form product exogenous DNA internal standard compound guard method and its application
<130> 2016
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 36
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<212> DNA
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ccgcctccat ccagtctatt aattgt 26
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Claims (8)
1. the guard method of exogenous DNA internal standard compound in a kind of liquid form product, which is characterized in that specific as follows: by polycationic
The ratio of polymer solution and target DNA solution according to phosphoramidic acid than >=10:1 mixes, and 2-20 DEG C of placement 0.5-12 is small after mixing
When, stable DNA complex solution is obtained, can be added directly in liquid form product；The phosphoramidic acid ratio is polycation
The ratio for the phosphate group molal quantity that amino molal quantity entrained by compound and DNA are carried, the polycationic compounds
For one of chitosan oligosaccharide, chitosan, spermine, spermidine or nucleoprotamine or a variety of, the molecular weight of the chitosan is in 20-
250k Da, the molecular weight of the chitosan oligosaccharide are 6500Da.
2. the guard method of exogenous DNA internal standard compound in liquid form product according to claim 1, which is characterized in that poly- sun from
The temperature that sub- compound solution and target DNA solution are placed after mixing is 2-8 DEG C, and the time is 6-12 hours.
3. the guard method of exogenous DNA internal standard compound in liquid form product according to claim 1 or 2, which is characterized in that described
DNA include that genomic DNA, Plasmid DNA, PCR product, DNA enzymatic slice are one of disconnected or a variety of.
4. the guard method of exogenous DNA internal standard compound in liquid form product according to claim 3, which is characterized in that described
Genomic DNA includes one of salmon sperm dna, herring sperm dna, mitochondrial DNA, chloroplast DNA or λ DNA or a variety of.
5. the guard method of exogenous DNA internal standard compound in liquid form product according to claim 3, which is characterized in that described
Plasmid DNA includes one of pUC18, pUC19, pUC57, pBR322, pTZ19R or pET28 or a variety of.
6. the guard method of exogenous DNA internal standard compound in liquid form product according to claim 1, which is characterized in that the DNA
The pH=6-8 of complex solution.
7. a kind of anti-fake and source tracing method of liquid form product, which is characterized in that specific steps are as follows:
(1) ratio by polycationic compounds solution and target DNA solution according to phosphoramidic acid than >=10:1 mixes, after mixing
2-20 DEG C placement 0.5-12 hours, obtain stable DNA complex solution, the phosphoramidic acid ratio is polycationic compounds
The ratio for the phosphate group molal quantity that entrained amino molal quantity and DNA carries, the polycationic compounds are that shell is few
One of sugar, chitosan, spermine, spermidine or nucleoprotamine are a variety of, the molecular weight of the chitosan in 20-250k Da,
The molecular weight of the chitosan oligosaccharide is 6500Da；
(2) DNA complex solution obtained in step (1) is added in liquid form product, additive amount makes liquid with the meter of DNA
The content of DNA is 1ng/mL-50 μ g/mL in state product；
(3) it detects in liquid form product and whether contains added DNA.
8. the anti-fake and source tracing method of liquid form product according to claim 7, which is characterized in that detect DNA in step (3)
Method include agarose gel electrophoresis, polyacrylamide gel electrophoresis, Standard PCR, real-time fluorescence quantitative PCR or constant temperature nucleic acid
One of amplification is a variety of.
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