CN103122380A - Penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as establishment method and application - Google Patents
Penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as establishment method and application Download PDFInfo
- Publication number
- CN103122380A CN103122380A CN 201210593298 CN201210593298A CN103122380A CN 103122380 A CN103122380 A CN 103122380A CN 201210593298 CN201210593298 CN 201210593298 CN 201210593298 A CN201210593298 A CN 201210593298A CN 103122380 A CN103122380 A CN 103122380A
- Authority
- CN
- China
- Prior art keywords
- dna
- deer
- deer whip
- whip
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of traditional Chinese medicine identification and particularly relates to a penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as an establishment method and an application thereof. The penis cervi capillary electrophoresis DNA fingerprint spectrum comprises extraction of the penis cervi DNA, PCR (Polymerase Chain Reaction) amplification of the penis cervi DNA, analysis of a PCR amplified product, and final establishment of the penis cervi capillary electrophoresis DNA fingerprint spectrum. An identification method of a penis cervi DNA fingerprint comprises the steps of: establishment of the penis cervi capillary electrophoresis DNA fingerprint spectrum, establishment of a sample DNA fingerprint spectrum and result identification by utilizing similarity software. The penis cervi capillary electrophoresis DNA fingerprint spectrum has the advantages of high resolution ratio, high sensitivity, simplicity and convenience, rapidness and safety in operation, and accurate result, and can be used for identifying a penis cervi sample.
Description
Technical field
The present invention relates to the Materia Medica Identification technology, specifically a kind of deer flagellum cons electrophoresis DNA fingerprinting and establishment method and application.
Background technology
The deer whip has another name called the deer kidney, is rare traditional Chinese medicine, is the male penis of animal in deer family spotted deer Cervus nippon Temminck and red deer Cervuselaphus Linnaeus and the dry product of testis.Have kidney tonifying, establishing-Yang, benefit essence, control soreness of waist and knee joint, nephrasthenia deafness, impotence, the cold infertile effect in palace.Due to expensive, demand is large, and on market, rate is seen with reinder whip, penis and testlicle of Bos grunniens and donkey whip etc. and filled pseudo-phenomenon.
At present, the discriminating Main Basis appearance character of deer whip, microscopical identification and physico-chemical property etc. is differentiated.These methods can be to a certain extent provide foundation to discriminating and the quality evalution of deer whip, but need to rely on rich experience, and subjectivity is strong.In addition, deer whip proterties plasticity-in drying and the course of processing is very strong, causes the character identification result inaccurate, is difficult to satisfy differentiate fast and accurately requirement.Therefore, to set up a kind of easy, accurate, practical method particularly important for the authenticity of deer whip.
Along with the development of Modern Molecular Biotechnology, the DNA analysis technology constantly is penetrated into the identification and assessment of Chinese medicines field, has promoted the development of identification and assessment of Chinese medicines research.DNA molecular is not subjected to external environment factor and ontogenetic the impact as the direct carrier of genetic information, has genetic stability and chemical stability.Make a general survey of the achievement in research in this field in recent years, find that DNA fingerprinting is highly suitable for easily mixed cultivar identification of Chinese medicine (especially rare traditional Chinese medicine).Such technology has the germplasm specificity, can set up the electrophoretogram for the identification of the biont difference.At present, the molecule marking method that is widely used in the constructed dna finger printing is broadly divided into 3 classes: the molecular marking technique take electrophoretic technique and molecular hybridization as core at first, and the technology that wherein represents is restriction fragment length polymorphism (RFLP) analysis; The 2nd class is the evaluation of markers technology take electrophoretic technique and round pcr as core, actually uses the more technology that represents to be: random amplification DNA polymorphism (RAPD) is analyzed, simple sequence is distinguished labeling technique (ISSR) and amplified fragment length polymorphism (SFLP) analysis between repeating.The 3rd class is the evaluation of markers technology take DNA sequence dna as core, and it represents that technology is cytochrome b (Cyt b) sequencing technologies.Because RFLP and DNA sequencing technological operation are complicated, experimental period is long, ISSR need to understand the genetic information of experiment material, and AFLP is applied patent protection, so all there is certain limitation in these technology in practical application.
And because the RAPD technology has following advantage:
1. need not Specialty Design RAPD amplimer, do not need to understand in advance composition of genome, but primer random synthesis and selected.
2. RAPD primer amount is large, and the binding site in whole genome is many, can carry out the blanket type polymorphism analysis to genome, detect small gap between genome.
3. the RAPD technology is simple, and is time saving and energy saving, and RAPD is easy to sequencing, but the Import computer system.
4. RAPD specimen in use amount is few, and not high to the DNA purity requirement, is suitable for differentiating dry medicinal material.
Above advantage has guaranteed that the RAPD technology is widely used in the evaluation of each herbal species.But still there are several point defects at present in the RAPD technology:
1. because RAPD is very sensitive to reaction conditions, so experiment condition need to ask strictly, to guarantee experimental repeatability.
2. the RAPD product analysis mainly adopts the classic flat-plate gel electrophoresis technology at present, although method is ripe, but consuming time, effort, non-automaticization and resolving power and sensitivity are all lower, cover to a great extent the polymorphism of RAPD product, seriously restricted the application of RAPD technology aspect structure species DNA fingerprinting.
When 3. utilizing at present the RAPD technology to carry out real and fake discrimination to Chinese medicine, often artificial several representative amplified bands of selection in gel pattern, bring according to these " standard " bars and carry out qualitative identification.Interpretation of result is oversimplified, but due to artificial amplified band is accepted or rejected, reduced the polymorphism of amplified production, reduced simultaneously the objectivity of analytical results.
HPCE (CE) is the Novel liquid-phase isolation technique of a class take kapillary as split tunnel, take high-voltage dc as motivating force, because it has quick, inexpensive, the advantages such as separation efficiency is high, amount of samples is few, antipollution, be widely used in the biological sample analysises such as protein, nucleic acid, DNA, amino acid.CE can satisfy the RAPD amplified production to the requirement of high separation, embodies the polymorphism of amplification; CE can obviously shorten RAPD amplified production analysis time, and is time saving and energy saving; The CE analytical results can adopt statistical method to process, and is more suitable for the accurate analysis of this complicated result of RAPD amplification.Therefore, CE is highly suitable for the analysis of RAPD amplified production as a kind of high efficiency separation detection technique, both in conjunction with the defective (lacking suitable detection means, repeatability and less stable) that can effectively solve the RAPD technology.The present invention combines with High Performance Capillary Electrophoresis with random amplification DNA polymorphism analysis (RAPD) technology, and is used for the evaluation of Chinese medicinal materials deer whip, has set up deer flagellum cons electrophoresis DNA fingerprinting.Present method resolving power is high, highly sensitive, easy and simple to handle, quick, safety, result are accurate, can be used in the evaluation of deer whip sample.
Summary of the invention
The purpose of this invention is to provide a kind of deer flagellum cons electrophoresis DNA fingerprinting and establishment method and application that can be used for the medicinal material real and fake discrimination, to address the deficiencies of the prior art.Present method resolving power is high, highly sensitive, easy and simple to handle, quick, safety, result are accurate, can be used in the evaluation of deer whip sample.
At first the present invention provides deer flagellum cons electrophoresis DNA fingerprinting (comprising spotted deer flagellum cons electrophoresis DNA fingerprinting and red deer flagellum cons electrophoresis DNA fingerprinting), this finger printing can be used for the deer whip real and fake discrimination of (comprising spotted deer whip and red deer whip), and deer flagellum cons electrophoresis DNA fingerprinting is seen accompanying drawing 1, accompanying drawing 2.
The present invention also provides the construction process of above-mentioned deer flagellum cons electrophoresis DNA fingerprinting, and the method comprises the steps: the extraction of deer whip DNA; The pcr amplification of deer whip DNA; The analysis of pcr amplification product; The foundation of deer flagellum cons electrophoresis DNA fingerprinting.
Concrete steps are:
1) deer whip DNA extraction: the deer whip is pulverized, by its Mitochondrial DNA of alkaline lysis method of extracting;
2) pcr amplification of deer whip DNA: carry out RAPD take high purity deer whip (comprising spotted deer whip and the red deer whip) DNA that obtains as template and increase, the primer that adopts is primer 1 (GAGAGCCAAC), primer 2 (CTACGGAGGA), primer 3 (ACGGATCCTG).
3) analysis of pcr amplification product: adopt capillary electrophoresis technique to carry out polymorphism analysis to amplified production, record electrophoretogram;
4) foundation of deer flagellum cons electrophoresis DNA fingerprinting: spotted deer whip, red deer whip and other amplification for the examination material in the comparative electrophoresis collection of illustrative plates, find that spotted deer whip and red deer whip all have its special DNA fingerprint, can distinguish for the examination material with other.This electrophoretogram is deer flagellum cons electrophoresis DNA fingerprinting.
The present invention also provides deer whip DNA fingerprint authentication method, and it comprises following step:
1) first set up deer flagellum cons electrophoresis DNA standard finger-print, method is as follows:
(a) deer whip DNA extraction: get deer whip control medicinal material, by its Mitochondrial DNA of alkaline lysis method of extracting;
(b) pcr amplification of deer whip DNA: carry out the RAPD amplification take the high purity deer whip DNA that obtains as template, the primer of employing is primer 1 (GAGAGCCAAC), primer 2 (CTACGGAGGA), primer 3 (ACGGATCCTG).
(c) analysis of pcr amplification product: adopt capillary electrophoresis technique to carry out polymorphism analysis to amplified production, record electrophoretogram;
(d) foundation of deer flagellum cons electrophoresis DNA standard finger-print: in spotted deer whip and red deer whip electrophoretogram, its special DNA fingerprint is arranged all, can distinguish for the examination material with other, this electrophoretogram is deer flagellum cons electrophoresis DNA standard finger-print.
2) working sample DNA fingerprinting: measure deer whip sample to be measured with above-mentioned identical method, get sample capillary electrophoresis DNA finger printing;
3) result is identified: utilize the similarity of similarity analysis comparison sample DNA finger printing and deer whip DNA standard finger-print, according to similarity, deer whip sample is carried out authenticity.
The present invention compared with prior art has the following advantages:
1. the present invention's RAPD technology is simple and easy to do, characteristics that contain much information and the characteristics that HPCE is quick, resolving power is high, highly sensitive combine, and use it for the DNA fingerprinting of Chinese medicinal materials deer whip and the foundation of fingerprint identification method, this application facet of identifying at the deer whip belongs to pioneering.
2. the present invention uses efficient capillary technical Analysis RAPD amplified production, has overcome that classic flat-plate gel electrophoresis resolving power is low, process is numerous and diverse and tediously long, and the sample demand is large.HPCE has highly sensitive, and analysis speed is fast, favorable reproducibility and good stability, does not use the advantages such as nocuity reagent.A kind of being more suitable in the detection technique of RAPD amplified production analysis.
The present invention analyzes and builds deer whip DNA standard finger-print with the amplified production of deer whip and adulterant thereof, use capillary electrophoresis that the RAPD result is analyzed, avoid artificially classic flat-plate gel electrophoresis result being deleted and being chosen, guaranteed the integrity of finger printing, applications similar degree analysis software is analyzed sample, according to similarity, deer whip sample is carried out authenticity, result is objective, more easily realizes automated analysis.
Description of drawings
Fig. 1 deer whip (spotted deer) capillary electrophoresis DNA standard finger-print
Fig. 2 deer whip (red deer) capillary electrophoresis DNA standard finger-print
Embodiment
The invention will be further described below in conjunction with embodiment, below embodiment only be used for explanation the present invention and be not limitation of the present invention.
Embodiment 1
The foundation of deer flagellum cons electrophoresis DNA fingerprinting
1.DNA extract
(1) get deer whip control medicinal material, with drying after 70% alcohol and distilled water clean surface, uv irradiating 30min pulverizes, and takes 1.0g, changes in the 25ml centrifuge tube.
(2) add lysate 5ml, incubated overnight in 56 ℃ of water-baths is to organizing the complete digestion dissolving.
(3) Digestive system is used the saturated phenol extracting of equal-volume once, equal-volume phenol-chloroform extracting twice, and chloroform-primary isoamyl alcohol (24: 1) extracting is once.
(4) add the dehydrated alcohol of-20 ℃ of precoolings of 2 times of volumes, mixing is placed in cryogenic refrigerator and spends the night.4 ℃ of centrifugal 20min of 15000rpm, vacuum-drying is dissolved in the TE damping fluid.
2.PCR amplification
Carry out the RAPD amplification take spotted deer whip, red deer whip DNA as template respectively, primer is primer 1 (GAGAGCCAAC), primer 2 (CTACGGAGGA) and primer 3 (ACGGATCCTG).
The PCR reaction system is: 10 * PCR buffer (contains Mg
2+) 3 μ l, dNTPs 2.4 μ l, random primer 1 μ l, 0.2U Tap archaeal dna polymerase, 0.5ug DNA profiling.
Loop parameter is set to: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 80sec, 36 ℃ of annealing 1min, 72 ℃ are extended 2min, 45 rear 72 ℃ of extension 10min of circulation.95 ℃ of sex change 3min of first circulation, 4 ℃ of preservations.
3.PCR Product Identification
Pcr amplification product in step 2 is carried out capillary electrophoresis analysis, and concrete steps are as follows:
3.1 instrument, reagent
Agilent capillary electrophoresis system (Agilent HP 3D/CE); Agilent DAD detector (Agilent diode-arraydetector); Agilent chem workstation (Agilent ChemStation software package).
Vltra tears (HPMC), tetrabutyl ammonium phosphate (TBAP) are that chromatographically pure, ethylenediamine tetraacetic acid (EDTA) (EDTA), Tutofusin tris (Tris), SODIUM PHOSPHATE, MONOBASIC, sodium hydroxide are analytical pure, deionized water.Spotted deer deer whip control medicinal material (Jilin Lu Chang provides and identifies through Jilin medicine inspecting institute), red deer deer whip control medicinal material (Jilin medicine inspecting institute provides and identifies), commercially available deer whip (Jilin ginseng whip market purchase at random), common adulterant deer whip (Jilin medicine inspecting institute and the Chinese Academy of Agricultural Sciences left special product institute provide and identify through Jilin medicine inspecting institute).
3.2 sample preparation
Get above-mentioned pcr amplification product 10 μ l, with 10 times of electrophoretic buffer dilutions, through 0.45 μ m filtering with microporous membrane, filtrate is as need testing solution.
3.3 deposition condition
Chromatographic column: 50cm * 75 μ m, the not coating quartz capillary column of useful length 40cm (Hebei sharp Feng chromatogram Yongnian device company limited);
Electrophoretic buffer: 20mM phosphate buffered saline buffer (pH 7.3)-15mM TBAP-2mM EDTA-0.6% (w/v) HPMC;
Separation voltage :-8KV; Temperature: 25 ℃; Detect wavelength; 260nm;
Sampling condition: electrokinetic injection ,-10kV, 15s.
4. the foundation of deer whip DNA fingerprinting
With capillary electrophoresis technique, amplified production is carried out polymorphism analysis according to step 3, record electrophoretogram, select the electrophoretogram of representative (stability and repeatability is good, sample peak number amount is moderate, resolution good, analysis time is short and each sample is distinguished obviously each other), build deer whip DNA fingerprinting.Spotted deer whip, red deer whip and common adulterant deer whip all have its special DNA fingerprinting, can be distinguished from each other out them.Record respectively the electrophoretogram of spotted deer whip and red deer whip, as deer flagellum cons electrophoresis DNA standard finger-print, this standard finger-print can be used for the evaluation (seeing Fig. 1, Fig. 2) of deer whip sample.
Embodiment 2
Deer whip DNA fingerprint authentication method of the present invention is used for the evaluation of certain deer whip sample
Suppose existing a collection of deer whip sample can not determine whether be real spotted deer whip, therefore, as follows it is identified.
Deer whip DNA standard finger-print in the time of will identifying certain sample, is at first used its Mitochondrial DNA of alkaline lysis method of extracting after setting up; As template, with primer 1 (GAGAGCCAAC), primer 2 (CTACGGAGGA) and primer 3 (ACGGATCCTG) carry out pcr amplification to it with the DNA that extracts; Amplified production carries out capillary electrophoresis analysis and records electrophoretogram, namely gets the DNA fingerprinting of this batch testing sample; Utilize the similarity of similarity analysis comparison testing sample DNA fingerprinting and spotted deer whip DNA standard finger-print, according to similarity, testing sample is identified.
Qualification result: if both similarity is greater than 95% (containing 95%), this batch sample is exactly real spotted deer whip; If both similarity is less than 95%, this batch sample is not just real spotted deer whip so.
Embodiment 3
Deer whip DNA fingerprint authentication method of the present invention is used for the evaluation of certain deer whip sample
Suppose existing a collection of deer whip sample can not determine whether be real red deer whip, therefore, build the DNA fingerprinting of this batch sample according to method of the present invention, then utilize the similarity of similarity analysis comparison testing sample DNA fingerprinting and red deer whip DNA standard finger-print, according to similarity, testing sample is identified.
Qualification result: if both similarity is greater than 95% (containing 95%), this batch sample is exactly real red deer whip; If both similarity is less than 95%, this batch sample is not just real red deer whip so.
Claims (6)
1. the construction process of a deer flagellum cons electrophoresis DNA fingerprinting, is characterized in that operating as follows: the extraction of deer whip DNA; The pcr amplification of deer whip DNA; The analysis of pcr amplification product; The foundation of deer flagellum cons electrophoresis DNA fingerprinting.
2. a deer whip DNA fingerprint authentication method, is characterized in that operating as follows: the foundation of deer whip DNA standard finger-print; The foundation of sample DNA finger printing; Utilizing similarity software to carry out result identifies.
3. as claim 1 and deer flagellum cons electrophoresis DNA fingerprinting claimed in claim 2 and authentication method, it is characterized in that: described deer whip DNA standard finger-print is spotted deer deer whip DNA fingerprinting or red deer deer whip DNA fingerprinting.
4. deer whip DNA fingerprint authentication method as claimed in claim 2 is characterized in that: the primer that adopts in described RAPD amplification is primer 1 (GAGAGCCAAC), primer 2 (CTACGGAGGA), primer 3 (ACGGATCCTG).
5. deer whip DNA fingerprint authentication method as claimed in claim 2, it is characterized in that: it is the similarity of utilizing similarity analysis comparison sample DNA finger printing and deer whip DNA standard finger-print that result is identified, according to similarity, deer whip sample is carried out authenticity.
6. the application of a deer flagellum cons electrophoresis DNA fingerprinting, is characterized in that: the authenticity that can be applicable to deer whip sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210593298 CN103122380A (en) | 2012-12-22 | 2012-12-22 | Penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as establishment method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210593298 CN103122380A (en) | 2012-12-22 | 2012-12-22 | Penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as establishment method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103122380A true CN103122380A (en) | 2013-05-29 |
Family
ID=48453521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210593298 Pending CN103122380A (en) | 2012-12-22 | 2012-12-22 | Penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as establishment method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103122380A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525935A (en) * | 2013-10-22 | 2014-01-22 | 张敏 | Identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as PCR (Polymerase Chain Reaction) identification method |
| CN107699614A (en) * | 2017-11-27 | 2018-02-16 | 北华大学 | Zaocys dhumnade capillary electrophoresis DNA finger-print and authentication method |
-
2012
- 2012-12-22 CN CN 201210593298 patent/CN103122380A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525935A (en) * | 2013-10-22 | 2014-01-22 | 张敏 | Identification primers for penis cervi of New Zealand wapiti and penis cervi of sika deer as well as PCR (Polymerase Chain Reaction) identification method |
| CN107699614A (en) * | 2017-11-27 | 2018-02-16 | 北华大学 | Zaocys dhumnade capillary electrophoresis DNA finger-print and authentication method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lenz et al. | Bacterial profiling of soil using genus‐specific markers and multidimensional scaling | |
| CN104911256B (en) | A kind of Radix Angelicae Sinensis SCAR molecular labelings and its authentication method and specific primer pair | |
| CN102586448B (en) | Deer antler capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and identification method | |
| WO2000060124A3 (en) | Fixed address analysis of sequence tags | |
| CN104017859A (en) | Method for identifying sugarcane germplasm resources based on SSR (Simple Sequence Repeats) and CE (capillary electrophoresis) technique | |
| CN102732973A (en) | Construction method for DNA fingerprint database of high flux cotton variety | |
| CN105063028B (en) | SSR primer sets and the method using primer sets structure malt finger-print | |
| CN108486275B (en) | Method for identifying begonia varieties by SSR (simple sequence repeat) fingerprint spectrum and application of method | |
| CN104034820A (en) | Method for rapidly distinguishing brand of kirschwasser | |
| CN106555000A (en) | A kind of method of plant derived component in plant identification protein beverage | |
| CN103122381A (en) | Radix bupleuri capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum and identification method | |
| CN103074428B (en) | A kind of mycobacterium tuberculosis TB detection kits | |
| CN103540678A (en) | Method for constructing sugarcane capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and authenticating sugarcane variety resource | |
| CN103122380A (en) | Penis cervi capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum as well as establishment method and application | |
| CN109735647A (en) | Identify specific primer, kit and the method for aweto | |
| CN103525908A (en) | Method for rapidly detecting chicken, duck and pig blood components in blood jelly | |
| CN109486983A (en) | Buta-buta, Yunnan agalloch eaglewood, Aguilaria malaccensis Lamk SNP marker and application | |
| CN106701746B (en) | High-throughput malt Purity technology based on Capillary Electrophoresis and SSR marker | |
| CN102827933B (en) | Kit for qualitative detection of pinewood nematode and detection method thereof | |
| CN108754007B (en) | Identification method of poppy by using SSR molecular marker | |
| CN107937567B (en) | Specific primers for identifying bombyx batryticatus and identification method thereof | |
| CN104073551A (en) | Method for detecting authenticity of sugarcane seeds based on capillary electrophoresis and SSR (Simple Sequence Repeat) marker | |
| CN101974621B (en) | LAMP detection method for babesia bovis | |
| Siles et al. | Genetic fingerprinting of grape plant (Vitis vinifera) using random amplified polymorphic DNA (RAPD) analysis and dynamic size-sieving capillary electrophoresis | |
| CN101139605B (en) | Nucleotides sequence, molecule probe and method for identifying zhejiang fritillary variant-dong fritillary |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130529 |