CN109988818A - Chinese medicine pre-treating method suitable for PCR identification - Google Patents
Chinese medicine pre-treating method suitable for PCR identification Download PDFInfo
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- 239000003814 drug Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000006228 supernatant Substances 0.000 claims abstract description 42
- 239000007788 liquid Substances 0.000 claims abstract description 38
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000011534 incubation Methods 0.000 claims abstract description 15
- 230000001376 precipitating effect Effects 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- 239000000463 material Substances 0.000 claims description 19
- 102000004882 Lipase Human genes 0.000 claims description 16
- 108090001060 Lipase Proteins 0.000 claims description 16
- 239000004367 Lipase Substances 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 235000019421 lipase Nutrition 0.000 claims description 16
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 239000000284 extract Substances 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract 1
- 239000000843 powder Substances 0.000 description 16
- 210000003056 antler Anatomy 0.000 description 15
- 241000283007 Cervus nippon Species 0.000 description 14
- 241000283026 Cervus elaphus Species 0.000 description 10
- 240000002924 Platycladus orientalis Species 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 210000000582 semen Anatomy 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 244000144725 Amygdalus communis Species 0.000 description 6
- 235000011437 Amygdalus communis Nutrition 0.000 description 6
- 244000144730 Amygdalus persica Species 0.000 description 6
- 235000006040 Prunus persica var persica Nutrition 0.000 description 6
- 241001247821 Ziziphus Species 0.000 description 6
- 235000020224 almond Nutrition 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002798 spectrophotometry method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000003390 Chinese drug Substances 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000218691 Cupressaceae Species 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
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- 239000003960 organic solvent Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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Abstract
The invention discloses a kind of Chinese medicine pre-treating methods suitable for PCR identification, specifically according to being digested after crushing Chinese medicine, it obtains enzymolysis liquid, be incubated for enzymolysis liquid, enzymolysis liquid after incubation is subjected to centrifugal treating, it removes supernatant, DNA extracting solution is added into the enzymolysis liquid of removal supernatant, heat preservation, centrifugation, it collects supernatant, isopropanol is added into the supernatant of step 3 collection, it stands, is then centrifuged for after mixing, remove supernatant, DNA precipitating, the precipitating of the DNA described in ethanol washing are collected, the Chinese medicine DNA solution identified for PCR is obtained.The present invention is suitable for the Chinese medicine pre-treating method of PCR identification, can remove the fat and protein in Chinese medicine, extracts that purity is preferable, the higher Chinese medicine DNA for PCR identification of concentration.
Description
Technical field
The invention belongs to DNA extractive technique fields, are related to a kind of Chinese medicine pre-treating method suitable for PCR identification.
Background technique
Chinese medicine is that the generally acknowledged material with medical function of people's tradition makes since its is high-quality, good effect for a long time
The generally approval of doctor and patient have been obtained in.The pollen standard of Chinese medicine idiomaticity is mainly derived from practical experience, artificially
Factor is larger, also lack of standardization.Vegetable drug such as dried orange peel, Radix Angelicae Sinensis etc. is from a wealth of sources, and yield is high, doctor and patient contact also compared with
More, so the judgement experience to this kind of quality of medicinal material is more sufficient, the quality of this kind of medicinal material is more stable secure, but animal kind medicine
The resources such as material such as pilose antler are very limited, and doctor and patient contact are less, lack the experience of Quality estimation, in addition animal medicinal material
Price is also more expensive, easily there is the phenomenon that adulterating in the market, causes quality of medicinal material seriously irregular.
Since DNA is stable inhereditary material, and the DNA difference of different plant species is larger, so in recent years, often through
Round pcr expands specific gene, and identifies the medicinal material true and false by means such as alignments.The identification of means of this science can be with
The deficiency of artificial micro-judgment is made up, improves resolution, and then guarantee quality of medicinal material.
Although PCR amplification is more mature, it is influenced by many factors such as organic solvent, protein, institute
It is more demanding with purity and concentration of this technology to DNA profiling.However the DNA purity that existing DNA extraction method is extracted is all
It is not high, it needs just to can be carried out PCR after DNA Purification Kit, increases work difficulty.
Summary of the invention
The object of the present invention is to provide a kind of Chinese medicine pre-treating methods suitable for PCR identification, can extract purity
Preferably, the higher Chinese medicine DNA solution of concentration.
The technical scheme adopted by the invention is that the Chinese medicine pre-treating method suitable for PCR identification, specifically according to following
Step carries out:
Step 1, it is digested after Chinese medicine being crushed, obtains enzymolysis liquid；
Step 2, enzymolysis liquid is incubated for, the enzymolysis liquid after incubation is subjected to centrifugal treating, removes supernatant；
Step 3, DNA extracting solution is added into the enzymolysis liquid of removal supernatant, keeps the temperature, supernatant is collected in centrifugation；
Step 4, isopropanol is added in the supernatant collected to step 3, stands, is then centrifuged for after mixing, removes supernatant,
Collect DNA precipitating；
Step 5, the DNA described in ethanol washing is precipitated, and obtains the Chinese medicine total DNA identified for PCR.
The features of the present invention also characterized in that:
Specific progress by the following method is digested in step 1:
Into smashed Chinese medicine be added 1mol/l sodium hydroxide solution and lipase, 40 ± 1 DEG C at a temperature of
Water-bath 30min~60min.Wherein, Chinese medicine, sodium hydroxide solution, lipase ratio be 1g:5ml:0.1g, lipase is
Alkaline lipase.
The temperature that enzymolysis liquid is incubated in step 2 is 65 ± 1 DEG C, and the time of incubation is 10~30min, and centrifugation turns in step 2
Speed is 12000r/min, and the time is 10min~15min.
EDTA, 1.5mol/L's of Tris-HCl, 0.1mol/L in step 3 in DNA extracting solution including 0.1mol/L
The KCl of the CTAB and 0.5mol/L of NaCl, 10g/L, the PH of DNA extracting solution are 8.0.
The volume mass of DNA extracting solution and Chinese medicine ratio is 1~2ml:1g.
Water-bath keeps the temperature 20~40min under the conditions of 37 ± 1 DEG C in step 3, and revolving speed when being centrifuged in step 3 is 4000r/min
~5000r/min, centrifugation time are 10min~15min.
Isopropanol is pre-chilled through 4 DEG C in step 4, and the volume ratio of the supernatant of isopropanol and step 3 collection is 0.6:1.
Temperature when step 4 is stood is -20 DEG C, and time of repose is 30~240min.
Revolving speed is 12000r/min, time 10min when being centrifuged in step 4.
The beneficial effects of the invention are as follows
The present invention is suitable for the Chinese medicine pre-treating method of PCR identification, can remove the fat and protein in Chinese medicine,
It is proposed goes purity preferable, the higher Chinese medicine total DNA for PCR identification of concentration.
Specific embodiment
The present invention is described in detail With reference to embodiment.
Suitable for the Chinese medicine pre-treating method of PCR identification, specifically carry out as steps described below:
Step 1, Chinese medicine is dried, crushed into powder, the sodium hydroxide of 1mol/l is then added into Chinese drugs powder
Solution and alkaline lipase, wherein Chinese drugs powder, sodium hydroxide solution, lipase ratio be 1g:5ml:0.1g；
Then in 40 ± 1 DEG C of at a temperature of water-bath 30min~60min, enzymolysis liquid is obtained；
Step 2, by enzymolysis liquid in 65 ± 1 DEG C of incubation 10min~30min, by the enzymolysis liquid after incubation in 12000r/min
Revolving speed under be centrifuged 10min~15min, remove supernatant；
Step 3, DNA extracting solution is added into the enzymolysis liquid of removal supernatant, wherein the volume of DNA extracting solution and Chinese medicine
Mass ratio be 1ml~2ml:1g, under the conditions of 37 ± 1 DEG C water-bath keep the temperature 20min~40min, then 4000r/min~
It is centrifuged 10min~15min under the revolving speed of 5000r/min, collects supernatant；
Wherein in DNA extracting solution including 0.1mol/L Tris-HCl, 0.1mol/L EDTA, 1.5mol/L NaCl,
The KCl of the CTAB and 0.5mol/L of 10g/L, the PH of DNA extracting solution are 8.0
Step 4, the isopropanol being pre-chilled through 4 DEG C of 0.6 times of volume is added in the supernatant collected to step 3, mixes ,-
30~240min is stood at a temperature of 20 DEG C, 10min is then centrifuged with the revolving speed of 12000r/min, removes supernatant, is collected
DNA precipitating；
Step 5, the DNA described in ethanol washing is precipitated, and obtains the Chinese medicine DNA identified for PCR.
The Chinese medicine pre-treating method for being suitable for PCR identification of the invention is suitable for animal horn class medicinal material or fat content is high
Plant medicinal material, wherein animal horn class medicinal material such as sika deer velvet antler, cervus elaphus linnaeus, cornu bubali etc.；Plant medicinal material such as almond, peach
Benevolence, the seed of Oriental arborvitae, SEMEN PINI KORAIENSIS or jujube kernel etc..
The present invention is suitable for the Chinese medicine pre-treating method that PCR is identified, due to animal class and high fat kind Chinese medicine sample
Containing compared with fattiness and protein in product, easily PCR is had an impact, so these objects should be removed as far as possible when extracting DNA
Matter.The present invention degraded using sodium hydroxide and lipase fat and protein, in addition sodium hydroxide also has broken wall and cracking
The effect of cell；The degradation of DNA in extraction process is prevented using KCI.
Embodiment 1
Step 1, Chinese medicine sika deer velvet antler is taken, by sika deer velvet antler in 60 DEG C of at a temperature of drying 2h, is ground into powder.Take 1g
Then 5ml, the sodium hydroxide solution and 0.1g alkaline lipase of 1mol/l is added in sika deer velvet antler powder into sika deer velvet antler powder
In (Ao Weike biotechnology), then in 39 DEG C of at a temperature of water-bath 30min, test tube 1 time for during which shaking installed reagents obtains enzyme
Solve liquid；
Step 2, by enzymolysis liquid 64 DEG C at a temperature of be incubated for 10min, by the enzymolysis liquid after incubation 12000r/min's
It is centrifuged 10min under revolving speed, removes supernatant；
Step 3,1mlDNA extracting solution is added into the enzymolysis liquid of removal supernatant, water-bath is kept the temperature under the conditions of 36 DEG C
Then 20min is centrifuged 10min under the revolving speed of 4000r/min, collect supernatant；
Wherein in DNA extracting solution including 0.1mol/L Tris-HCl, 0.1mol/L EDTA, 1.5mol/L NaCl,
The KCl of the CTAB and 0.5mol/L of 10g/L, the PH of DNA extracting solution are 8.0
Step 4, the isopropanol being pre-chilled through 4 DEG C of 0.6 times of volume is added in the supernatant collected to step 3, mixes ,-
30min is stood at a temperature of 20 DEG C, then to be centrifuged 10min under the revolving speed of 12000r/min, removes supernatant, it is heavy to collect DNA
It forms sediment；
Step 5, DNA described in the ethanol washing for the volume fraction 70% being pre-chilled with 4 DEG C is precipitated, and is air-dried, is then used ddH2O is molten
The air-dried DNA precipitating of solution, obtains the sika deer velvet antler DNA solution identified for PCR.
The sika deer velvet antler DNA solution that the present embodiment obtains takes 2 μ l sika deer velvet antler DNA solutions, scannable in faint yellow
Absorption value at ultraviolet specrophotometer measurement detection 260nm, 280nm, with Eppendorf Biophotomete type nucleic acid egg
White matter analyzes the concentration of DNA, and the purity of DNA is judged according to 280 value of OD 260/OD, the results show that sika deer velvet antler DNA is molten
The OD260/OD280 value of liquid is 1.83, and in the DNA purity range that PCR allows, concentration is 453.1ng/ μ l.
Embodiment 2
Take cervus elaphus linnaeus as traditional Chinese medicinal material samples, remaining treatment process is identical with the technique of embodiment 1, obtains in yellowish
Color obtains cervus elaphus linnaeus DNA solution；Take 2 μ l cervus elaphus linnaeus DNA solutions, scannable ultraviolet specrophotometer measure detection 260nm,
Absorption value at 280nm analyzes the concentration of DNA with Eppendorf Biophotomete type nucleic acid protein, and according to OD
280 value of 260/OD judges the purity of DNA, the results show that the OD260/OD280 value of cervus elaphus linnaeus DNA solution is 1.82, in PCR
In the DNA purity range allowed, concentration is 467.1ng/ μ l.
Embodiment 3
Step 1, water intaking ox horn is ground into powder, then by cornu bubali in 45 DEG C of at a temperature of drying 4h as Chinese medicine
2g powder is added the sodium hydroxide solution and 0.2g alkaline lipase (Ao Weike biotechnology) of 10ml 1mol/l, then exists
Water-bath 60min at a temperature of 41 DEG C test tube 3 times of shake installed reagents, obtains enzymolysis liquid；
Step 2, by enzymolysis liquid in 66 DEG C of incubation 30min, by the enzymolysis liquid after incubation under the revolving speed of 12000r/min from
Heart 15min removes supernatant；
Step 3, the DNA extracting solution of 2ml is added into the enzymolysis liquid of removal supernatant, water-bath is kept the temperature under the conditions of 38 DEG C
Then 40min is centrifuged 15min under the revolving speed of 5000r/min, collect supernatant；
Wherein in DNA extracting solution including 0.1mol/L Tris-HCl, 0.1mol/L EDTA, 1.5mol/L NaCl,
The KCl of the CTAB and 0.5mol/L of 10g/L, the PH of DNA extracting solution are 8.0
Step 4, the isopropanol being pre-chilled through 4 DEG C of 0.6 times of volume is added in the supernatant collected to step 3, mixes ,-
240min is stood at a temperature of 20 DEG C, then to be centrifuged 10min under the revolving speed of 12000r/min, removes supernatant, it is heavy to collect DNA
It forms sediment；
Step 5, the ethanol washing DNA for the volume fraction 70% being pre-chilled with 4 DEG C is precipitated, and is air-dried, is then used ddH2O dissolves wind
Dry DNA precipitating, obtains the cornu bubali DNA solution identified for PCR.
The present embodiment obtains cornu bubali DNA solution and is transparent and colorless, and 2 μ l cornu bubali DNA solutions is taken, scannable ultraviolet
Spectrophotometric determination detects the absorption value at 260nm, 280nm, with Eppendorf Biophotomete type nucleic acid protein
The concentration of DNA is analyzed, and judges the purity of DNA according to 280 value of OD 260/OD, the results show that cornu bubali DNA solution
OD260/OD280 value is 1.80, and in the DNA purity range that PCR allows, concentration is 462.4ng/ μ l.
Embodiment 4
Suitable for the Chinese medicine pre-treating method of PCR identification, specifically carry out as steps described below:
Step 1, take almond as Chinese medicine, 42 DEG C of dry 2h are ground into powder, take 1g powder, and 5ml 1mol/l is added
Sodium hydroxide solution and 0.1g alkaline lipase (Ao Weike biotechnology) during which shake dress in 40 ± 1 DEG C of water-bath 45min
The test tube of reagent 2 times, obtains enzymolysis liquid；
Step 2, by enzymolysis liquid in 65 DEG C of incubation 20min, by the enzymolysis liquid after incubation under the rate of 12000r/min from
Heart 13min removes supernatant；
Step 3,1ml DNA extracting solution is added into the enzymolysis liquid of removal supernatant, water-bath is kept the temperature under the conditions of 37 DEG C
Then 30min is centrifuged 13min under the revolving speed of 4500r/min, collect supernatant；
Wherein in DNA extracting solution including 0.1mol/L Tris-HCl, 0.1mol/L EDTA, 1.5mol/L NaCl,
The KCl of the CTAB and 0.5mol/L of 10g/L, the PH of DNA extracting solution are 8.0
Step 4, the isopropanol being pre-chilled through 4 DEG C of 0.6 times of volume is added in the supernatant collected to step 3, mixes ,-
180min is stood at a temperature of 20 DEG C, then to be centrifuged 10min under the revolving speed of 12000r/min, removes supernatant, it is heavy to collect DNA
It forms sediment；
Step 5, DNA described in 70% ethanol washing of volume fraction being pre-chilled with 4 DEG C is precipitated, and is air-dried, and is then dissolved with 1 × TE
Air-dried DNA precipitating, obtains the almond DNA solution identified for PCR.
The almond DNA solution clear, colorless that this method obtains, takes 2 μ l almond DNA solutions, in scannable ultraviolet spectrometry light
Degree measurement regular inspection surveys the absorption value at 260nm, 280nm, analyzes DNA with Eppendorf Biophotomete type nucleic acid protein
Concentration, and the purity of DNA is judged according to 280 value of OD 260/OD, the results show that the OD260/OD280 of almond DNA solution
Value is 1.83, and in the DNA purity range that PCR allows, concentration is 446.2ng/ μ l.
Embodiment 5
Take peach kernel as Chinese medicine, remaining extraction process is identical with embodiment 5, obtains peach kernel DNA solution, the party
Method obtains peach kernel DNA solution clear, colorless, takes 2 μ l peach kernel DNA solutions, measures and detects in scannable ultraviolet specrophotometer
Absorption value at 260nm, 280nm analyzes the concentration of DNA, and root with Eppendorf Biophotomete type nucleic acid protein
The purity of DNA is judged according to 280 value of OD 260/OD, the OD260/OD280 value of peach kernel sample is 1.81 as the result is shown, in PCR
In the DNA purity range allowed, concentration is 489.3ng/ μ l.
Embodiment 6
Suitable for the Chinese medicine pre-treating method of PCR identification, specifically carry out as steps described below:
Step 1, it takes the seed of Oriental arborvitae as Chinese medicine, then by 60 DEG C of the seed of Oriental arborvitae dry 2h, is ground into powder, takes 1g powder,
The sodium hydroxide solution and 0.1g neutral lipase (Ao Weike biotechnology) of 5ml 1mol/l is added, in 40 ± 1 DEG C of water-baths
30min, test tube 1 time for during which shaking installed reagents, obtains enzymolysis liquid；
Step 2, by enzymolysis liquid in 64 DEG C of incubation 15min, by the enzymolysis liquid after incubation under the rotational speed rate of 12000r/min
It is centrifuged 12min, removes supernatant；
Step 3,1ml DNA extracting solution is added into the enzymolysis liquid of removal supernatant, water-bath is kept the temperature under the conditions of 37 DEG C
Then 25min is centrifuged 14min under the revolving speed of 4200r/min, collect supernatant；
Wherein in DNA extracting solution including 0.1mol/L Tris-HCl, 0.1mol/L EDTA, 1.5mol/L NaCl,
The KCl of the CTAB and 0.5mol/L of 10g/L, the PH of DNA extracting solution are 8.0
Step 4, the isopropanol being pre-chilled through 4 DEG C of 0.6 times of volume is added in the supernatant collected to step 3, mixes ,-
200min is stood at a temperature of 20 DEG C, then to be centrifuged 10min under the revolving speed of 12000r/min, removes supernatant, it is heavy to collect DNA
It forms sediment；
Step 5, the ethanol solution for the volume fraction 70% being pre-chilled with 4 DEG C cleans DNA precipitating, air-dries, then molten with 1 × TE
The air-dried DNA precipitating of solution, obtains the seed of Oriental arborvitae DNA solution identified for PCR.
The seed of Oriental arborvitae DNA solution that the present embodiment obtains takes 2 μ l seed of Oriental arborvitae DNA solutions, scannable ultraviolet in faint yellow
Spectrophotometric determination detects the absorption value at 260nm, 280nm, with Eppendorf Biophotomete type nucleic acid protein
The concentration of DNA is analyzed, and judges the purity of DNA according to 280 value of OD 260/OD, the results show that seed of Oriental arborvitae sample
OD260/OD280 value is 1.82, and in the DNA purity range that PCR allows, concentration is 513.0ng/ μ l.
Embodiment 7
Take SEMEN PINI KORAIENSIS as Chinese medicine, remaining extraction process is identical with embodiment 7, obtains the cypress identified for PCR
Sub- benevolence DNA solution.
The seed of Oriental arborvitae DNA solution that the present embodiment obtains takes 2 μ l seed of Oriental arborvitae DNA solutions, scannable ultraviolet in faint yellow
Spectrophotometric determination detects the absorption value at 260nm, 280nm, with Eppendorf Biophotomete type nucleic acid protein
The concentration of DNA is analyzed, and judges the purity of DNA according to 280 value of OD 260/OD, as the result is shown the OD260/ of SEMEN PINI KORAIENSIS sample
OD280 value is 1.82, and in the DNA purity range that PCR allows, concentration is 498.0ng/ μ l.
Embodiment 8
Take jujube kernel as Chinese medicine, remaining extraction process is identical with embodiment 7, obtains the jujube kernel identified for PCR
DNA solution.
The jujube kernel DNA solution that the present embodiment obtains takes 2 μ l jujube kernel DNA solutions, in scannable ultraviolet spectrometry in faint yellow
Photometric determination detects the absorption value at 260nm, 280nm, is analyzed with Eppendorf Biophotomete type nucleic acid protein
The concentration of DNA, and judge according to 280 value of OD 260/OD the purity of DNA, the as the result is shown OD260/OD280 of jujube kernel sample
Value is 1.84, and in the DNA purity range that PCR allows, concentration is 476.5ng/ μ l.
In order to verify the effect of enzymolysis processing step and DNA extract recipe of the present invention, We conducted following comparison is real
It tests, and compared with conventional CTAB method, respectively using sika deer velvet antler, cervus elaphus linnaeus, cornu bubali and SEMEN PINI KORAIENSIS as traditional Chinese medicinal material samples, if
Count following comparative example.
Comparative example 1
A kind of sample-pretreating method suitable for Chinese medicine PCR identification, method and step and embodiment 1 are essentially identical, area
It is not to be changed to step 1 " to take traditional Chinese medicinal material samples, 60 DEG C of dry 2h are ground into powder, take 1g powder, and 5ml 1mol/l is added
Sodium hydroxide solution, in 40 ± 1 DEG C of water-bath 30min, test tube 1 time for during which shaking installed reagents obtains enzymolysis liquid ".
This method acquisition DNA solution is brown, and the purity of DNA solution and the measurement result of concentration are shown, sika deer velvet antler sample
The OD260/OD280 value of product is 1.67, and concentration is 347.8ng/ μ l；The OD260/OD280 value of cervus elaphus linnaeus sample is 1.66, concentration
For 321.4ng/ μ l；The OD260/OD280 value of SEMEN PINI KORAIENSIS sample is 1.67, and concentration is 344.5ng/ μ l；Cornu bubali sample
OD260/OD280 value is 1.65, and concentration is 321.4ng/ μ l；Above-mentioned sample is not in 1.8~2.0 ranges that PCR allows.
Comparative example 2
A kind of sample-pretreating method suitable for Chinese medicine PCR identification, method and step and embodiment 1 are essentially identical, area
Be not for step 1 to be changed to " take traditional Chinese medicinal material samples, 60 DEG C of dry 2h are ground into powder, take 1g powder, be added 5ml distilled water and
0.1g alkaline lipase (Ao Weike biotechnology), in 40 ± 1 DEG C of water-bath 30min, test tube 1 time for during which shaking installed reagents is obtained
To enzymolysis liquid ".
It is in yellowish-brown that this method, which obtains DNA solution, and the purity of DNA solution and the measurement result of concentration are shown, sika deer velvet antler
The OD260/OD280 value of sample is 1.71, and concentration is 322.4ng/ μ l；The OD260/OD280 value of cervus elaphus linnaeus sample is 1.66, dense
Degree is 368.1ng/ μ l；The OD260/OD280 value of SEMEN PINI KORAIENSIS sample is 1.70, and concentration is 354.2ng/ μ l；Cornu bubali sample
OD260/OD280 value is 1.69, and concentration is 304.8ng/ μ l；Above-mentioned sample is not in 1.8~2.0 ranges that PCR allows.
Comparative example 3
A kind of sample-pretreating method suitable for Chinese medicine PCR identification, method and step and embodiment 1 are essentially identical, area
It is not to be changed to every liter of DNA extracting solution to prepare " 0.1mol Tris-HCl, 0.1mol EDTA, 1.5mol in accordance with the following methods
NaCl, 10g CTAB, solvent are distilled water, pH 8.0 ".
It is in yellow that this method, which obtains DNA solution, and the purity of DNA is judged according to 280 value of OD 260/OD, the results show that
The OD260/OD280 value of sika deer velvet antler sample is 2.14, and concentration is 356.1ng/ μ l；The OD260/OD280 value of cervus elaphus linnaeus sample
It is 2.04, concentration is 391.0ng/ μ l；The OD260/OD280 value of SEMEN PINI KORAIENSIS sample is 2.07, and concentration is 335.2ng/ μ l；Buffalo
The OD260/OD280 value of angle sample is 2.11, and concentration is 336.1ng/ μ l；Above-mentioned sample not PCR allow 1.8~
In 2.0 ranges.
Comparative example 4
Conventional CTAB method extracts Chinese medicine total DNA, bibliography " Wang Peixun, botanical herbs material Total DNA extraction method
Comparison new Chinese medicine and clinical pharmacology, the 1st phase of volume 10 in January, 1999 " in CTAB method described in 1.4.3.
It is in yellow that this method, which obtains DNA solution, and the purity of DNA is judged according to 280 value of OD 260/OD, the results show that
The OD260/OD280 value of sika deer velvet antler sample is 1.77, and concentration is 364.5ng/ μ l；The OD260/OD280 value of cervus elaphus linnaeus sample
It is 1.66, concentration is 340.7ng/ μ l；The OD260/OD280 value of SEMEN PINI KORAIENSIS sample is 1.77, and concentration is 354.6ng/ μ l；Buffalo
The OD260/OD280 value of angle sample is 2.03, and concentration is 367.4ng/ μ l；Above-mentioned sample not PCR allow 1.8~
In 2.0 ranges.
1-9 of the embodiment of the present invention and comparative example 1-4 are compared, show that the present invention is suitable for the Chinese medicine of PCR identification
The DNA solution that pre-treating method extracts all has preferable purity and higher concentration, does not need DNA purification kit and additionally mentions
It is pure, it can be directly used for PCR amplification, and the purity and concentration of the DNA solution of comparative example 1~4 are poor, need to purify through DNA and try
The purifying of agent box, could effectively carry out PCR amplification.
Claims (10)
1. being suitable for the Chinese medicine pre-treating method of PCR identification, which is characterized in that specifically carry out as steps described below:
Step 1, it is digested after Chinese medicine being crushed, obtains enzymolysis liquid；
Step 2, the enzymolysis liquid is incubated for, the enzymolysis liquid after incubation is subjected to centrifugal treating, removes supernatant；
Step 3, DNA extracting solution is added into the enzymolysis liquid of removal supernatant, keeps the temperature, supernatant is collected in centrifugation；
Step 4, isopropanol is added in the supernatant collected to step 3, stands, is then centrifuged for after mixing, removes supernatant, collects
DNA precipitating；
Step 5, the DNA described in ethanol washing is precipitated, and obtains the Chinese medicine DNA solution identified for PCR.
2. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that institute in step 1
State the specific progress by the following method of enzymatic hydrolysis:
The sodium hydroxide solution and lipase of 1mol/l are added into smashed Chinese medicine, in 40 ± 1 DEG C of at a temperature of water-bath
30min~60min.
3. the Chinese medicine pre-treating method according to claim 2 suitable for PCR identification, which is characterized in that the Chinese medicine
Material, sodium hydroxide solution, lipase ratio be 1g:5ml:0.1g, the lipase be alkaline lipase.
4. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that institute in step 2
The temperature for stating enzymolysis liquid incubation is 65 ± 1 DEG C, and the time of incubation is 10~30min, and the rate being centrifuged in step 2 is 12000r/
Min, time are 10min~15min.
5. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that institute in step 3
State the CTAB of NaCl, 10g/L of EDTA, 1.5mol/L of Tris-HCl, 0.1mol/L in DNA extracting solution including 0.1mol/L
With the KCl of 0.5mol/L, the PH of the DNA extracting solution is 8.0.
6. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that the DNA is mentioned
Taking the volume mass of liquid and Chinese medicine ratio is 1~2ml:1g.
7. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that the step 3
In under the conditions of 37 ± 1 DEG C water-bath keep the temperature 20~40min, revolving speed when being centrifuged in step 3 is 4000r/min~5000r/min, from
The heart time is 10min~15min.
8. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that institute in step 4
It states isopropanol to be pre-chilled through 4 DEG C, the volume ratio of the supernatant of the isopropanol and step 3 collection is 0.6:1.
9. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that step 4 is stood
When temperature be -20 DEG C, time of standing is 30~240min.
10. the Chinese medicine pre-treating method according to claim 1 suitable for PCR identification, which is characterized in that in step 4
The speed of the centrifugation is 12000r/min, and the time of centrifugation is 10min.
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