CN109486984A - A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng - Google Patents
A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng Download PDFInfo
- Publication number
- CN109486984A CN109486984A CN201810882412.0A CN201810882412A CN109486984A CN 109486984 A CN109486984 A CN 109486984A CN 201810882412 A CN201810882412 A CN 201810882412A CN 109486984 A CN109486984 A CN 109486984A
- Authority
- CN
- China
- Prior art keywords
- ginseng
- sequence
- trnl
- sample
- trnf
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng, the combination bar code sequence ITS+trnL-trnF is spliced by ITS region sequence and trnL-trnF region sequence, with primer versatility is good and the variability of height, specific site abundant can be provided, and it is very low to the requirement of template when pcr amplification reaction, it is easily operated, it is had great advantages in the identification of DNA of medicine plants bar code, simultaneously have both by external environment selection pressure it is small, it is possible to provide hereditary information it is more；This method application combination bar code sequence ITS+trnL-trnF, it is analyzed by comparing, obtain distinguishing 6 informative sites of ginseng and American Ginseng, pass through aligned sequences and 6 informative sites, Molecular Identification simple and quick and effectively can be carried out to ginseng and American Ginseng in molecular biology level, have many advantages, such as to identify that accuracy is high, easy to operate, low in cost, is conducive to large-scale promotion use.
Description
[technical field]
The present invention relates to plant species identification technology field more particularly to a kind of combinations identified for ginseng and American Ginseng
Bar code sequence and its accurate identification method.
[background technique]
Ginseng (Panax ginseng) is the dry root and rhizome of Araliaceae ginseng, and the ginseng of cultivation is commonly called as " garden
Ginseng "；It sows the ginseng grown naturally under mountain forest wild state to claim " ginseng under forest ", practises and claim " seed sea ".Ginseng is as top grade
Tonic is most common, most important, most precious one of the traditional Chinese medicine of China using existing more than 2,000 years history.
Ginseng is that have very high medical value, i.e., in rare traditional Chinese medicine and one of most widely used, the most in-depth Chinese medicine of research
There is considerable value in terms of medical treatment and health, have effects that tonifying Qi, prevent prolapse, promote the production of body fluid, intelligence development, tranquilizing the mind, and qi-restoratives is imitated
Fruit is significant.
Modern clinic often treats the diseases such as shock caused by a variety of causes, heart failure, acute cerebrovascular damage with ginseng
Disease.Newest progress shows the bioactive ingredients such as ginseng triterpene saponin rich in and polysaccharide, these activity
Ingredient especially saponin component, with extensive pharmacological action, such as antitumor, anti-oxidant, anti-inflammatory, antiallergy, it is antifatigue,
Resisting stress, anti-radiation, anti-aging, anti-osteoporosis, immunological regulation, Adjust-blood lipid, hypoglycemic, liver protection, protection nervous centralis and the heart
Cerebrovascular system etc..
American Ginseng (Panax quinquefolium) also known as American ginseng, are the dry roots of Panax quinquefolium L. of araliaceae.West
American ginseng bitter, it is cool in nature, enter the heart, lung, kidney channel, function is based on help, boosting qi and nourishing yin, clearing heat and promoting fluid, for deficiency of vital energy yin deficiency, interior
Heat, cough and asthma phlegm blood, abnormal heat are tired of the diseases such as tired.American Ginseng is both a kind of rare traditional Chinese medicine with special pharmaceutical value and advanced
Excellent tonic product.Contain plurality of active ingredients in American Ginseng, there are various pharmacology such as antitumor, anticancer, lowering blood pressure and blood fat
Activity, at the same it is also tired and other effects with boosting qi and nourishing yin, reinforcing stomach reg fluid, heat-clearing solution.
Accurate species identification is the prerequisite of human cognitive and application bio-diversity and sustainable development.Panax
Plant is traditional Chinese medicine mostly, has the function of conditioning engine body, promotes longevity and antitumor, anti-aging etc., people are to Panax
The demand of plant is increasing, and panax species such as ginseng and American Ginseng all unusual phase either in shape or color
Seemingly, it has been difficult to be distinguished using the method that traditional morphological feature is identified, in addition panax species price difference
Not big, some criminals pretend to be American Ginseng using ginseng to play one's own game, and cheat consumption with the even false product of adulterated product
Person, not only compromises the equity of consumer, is also unfavorable for the maintenance of market order.Therefore, a kind of reliable and stable method is found
Ginseng and American Ginseng are distinguished, guarantee consumer's interests, Maintenance Market order etc. are all had a very important significance.
Currently, for the status that the Molecular Identification of ginseng and American Ginseng is all also only identified using single bar code,
And it is understood that each bar code of ribosomes and chloroplast DNA respectively has advantage and disadvantage in medicinal plant identification, therefore, based on core
The advantages of genome ITS region sequence and Chloroplast gene trnL-trnF region sequence, finds the item for being suitable for ginseng and American Ginseng
Shape code or combinations thereof constructs more perfect common sequence database, to the standard for realizing ginseng and American Ginseng medicinal plant species
Really identification has a very important significance.
[summary of the invention]
It is an object of the invention to for the existing method for only carrying out ginseng and American Ginseng identification using single bar code
Existing limitation improves, that is, provides a kind of combination bar code sequence identified for ginseng and American Ginseng, a combination thereof
The ITS region sequence of Matrix attachment region and the trnL-trnF region sequence of Chloroplast gene, while also providing a kind of using combobar
The method that shape code sequence ITS+trnL-trnF precisely identifies ginseng and American Ginseng.
A kind of combination bar code sequence identified for ginseng and American Ginseng of the present invention, the combination bar code sequence is by ITS
Region sequence and trnL-trnF region sequence are constituted, and are ITS+trnL-trnF, and nucleotide sequence is denoted as SEQ ID No:1.
ITS sequence is located at the internal gene transcribed spacers of rDNA, and the part is by ITS1,5.8S gene and the area ITS2 sequence
Column composition, primer versatility is good, and the sequence quantity included in ncbi database is more, and the variability with height can provide rich
Rich specific site, and requirement when pcr amplification reaction to template is very low, it is easily operated, it is identified in DNA of medicine plants bar code
In have great advantages, but ITS sequence have by the risk of fungal contamination.
The area trnL-trnF of Chloroplast gene belongs to noncoding region, and fragment length is moderate, is selected pressure by external environment
It is small, and the hereditary information provided is more, is applied to polant systematics research also widely at present.
For the above two o'clock situation, the present invention also provides a kind of methods for identifying ginseng and American Ginseng, use combobar
Shape code sequence ITS+trnL-trnF is precisely identified ginseng and American Ginseng on the one hand this method provides more variations
Site can more accurately identify ginseng and American Ginseng, and the ITS that on the other hand also can avoid to occur is by fungal contamination
Risk finally provides molecular biology evidence for the species identification of ginseng and American Ginseng Chinese medicine medicinal plant.
Above-mentioned purpose of the invention is achieved by following scheme:
The present invention provides a kind of combination bar code sequence identified for ginseng and American Ginseng, the combination bar code sequence by
ITS region sequence and trnL-trnF region sequence are spliced to form, and are ITS+trnL-trnF, and nucleotide sequence is denoted as SEQ ID No:
1。
A kind of method for identifying ginseng and American Ginseng of the present invention, is carried out using combination bar code sequence ITS+trnL-trnF
Identification, includes the following steps:
Step 1: extracting the DNA of sample to be tested, sample to be tested, which first passes through, just to carry out the DNA of sample to be tested after pretreatment and mention
It takes, the pretreatment is to take sample to be tested to be soaked in the ethanol solution that mass percent concentration is 75%, and put after 5min
It is placed in gnotobasis and air-dries, be then ground under conditions of liquid nitrogen is cooling powdered, which is to test sample
Product；The sample to be tested is carried out to the extraction of DNA using DNA extraction kit, or DNA is extracted using CTAB method, is obtained to be measured
Sample DNA.
The upstream and downstream primer of step 2:ITS region sequence is respectively primer I TS4 and primer I TS5, trnL-trnF region sequence
Upstream and downstream primer be respectively primers F, primer R, use the primer I TS4 and primer I TS5 of ITS region sequence, and use
The primers F of trnL-trnF region sequence, primer R carry out pcr amplification reaction respectively, obtain sample to be tested ITS region sequence and to test sample
Product trnL-trnF region sequence, the nucleotides sequence of the primer I TS4 are classified as SEQ ID No:2, the nucleotide of the primer I TS5
Sequence is SEQ ID No:3；The nucleotides sequence of the primers F is classified as such as SEQ ID No:4；The nucleotide sequence of the primer R
For SEQ ID No:5.
The reaction system total volume of above-mentioned pcr amplification reaction is 20 μ L, which includes 2.5mmol/L MgCl2's
10 × PCR Buffer, 2 μ L, four kinds of 1.6 μ L of 2.5mmol/L mononucleotide, 10 μm of ol/L ITS region sequences upstream primer or
0.8 μ L of upstream primer of trnL-trnF region sequence, the upstream primer of 10 μm of ol/LITS region sequences or trnL-trnF region sequence
0.8 μ L, 5U/ μ L HiFi archaeal dna polymerase of downstream primer, 0.1 μ L and template DNA 50ng, surplus be sterile ultrapure water, described four
Kind 2.5mmol/L mononucleotide can be adenine, thymidine, guanine, cytimidine.
The process of above-mentioned pcr amplification reaction is successively are as follows: 95 DEG C of initial denaturations 3min, 94 DEG C of denaturation 1min, 56 DEG C of annealing 1min,
72 DEG C of extension 1min, 30 circulations, 72 DEG C of extension 10min.
Step 3: after sample to be tested ITS region sequence and sample to be tested trnL-trnF the region sequence splicing that step 2 is obtained,
Combination bar code sequence ITS+trnL-trnF is obtained, then imports software MEGA and is compared；Before splicing and importing software MEGA,
First sample to be tested ITS region sequence and sample to be tested trnL-trnF region sequence are compared respectively, after matching, excision is respectively drawn
Object end is spliced according to 5 ' -3 ' direction, is obtained combination bar code sequence ITS+trnL-trnF, is imported software MEGA later
It carries out analyzing the combination bar code sequence: if the 147th bit base is A, the 266th from 5 ' ends in SEQ ID No:1 sequence
Bit base is A, the 445th bit base is C, the 456th bit base is T, the 892nd bit base is C, the 978th bit base is T, then identifies
For ginseng；If the 147th bit base is C from 5 ' ends in SEQ ID No:1 sequence, the 266th bit base is G, the 445th alkali
Base is T, the 456th bit base is C, the 892nd bit base is G, the 978th bit base is C, then is accredited as American Ginseng.
In above-mentioned steps 3, the combination bar code sequence ITS+trnL-trnF obtained from sample to be tested must satisfy the 147th
Bit base is A, the 266th bit base is A, the 445th bit base is C, the 456th bit base is T, the 892nd bit base is C, the 978th
When base is this 6 conditions of T, which can just be determined as ginseng, be unsatisfactory for above-mentioned 6 conditions when whole, or meet small
When above-mentioned 6 conditions, determine that the sample to be tested is not ginseng.
In above-mentioned steps 3, the combination bar code sequence ITS+trnL-trnF obtained from sample to be tested must satisfy the 147th
Bit base is C, the 266th bit base is G, the 445th bit base is T, the 456th bit base is C, the 892nd bit base is G, the 978th
When base is this 6 conditions of C, which can just be determined as American Ginseng, when whole is unsatisfactory for above-mentioned 6 conditions, or satisfaction
When less than above-mentioned 6 conditions, determine that the sample to be tested is not American Ginseng.
Compared with prior art, the invention has the following beneficial effects:
A kind of combination bar code sequence identified for ginseng and American Ginseng of the present invention, has primer versatility good and height
Variability, specific site abundant can be provided, and requirement when pcr amplification reaction to template is very low, it is easily operated, in medicine
With DNA of plants bar code identification in have great advantages, while have both by external environment selection pressure it is small, it is possible to provide heredity
Information is more.
A kind of method for identifying ginseng and American Ginseng of the present invention is used using combination bar code sequence ITS+trnL-trnF
After ITS region sequence and the splicing of trnL-trnF region sequence, 6 informative sites for distinguishing ginseng and American Ginseng are obtained, comparison group is passed through
Bar code sequence ITS+trnL-trnF and its 6 informative sites are closed, it can be simple and quick and effectively in molecular biology level
On Molecular Identification is carried out to ginseng and American Ginseng, have many advantages, such as that identification accuracy is high, easy to operate, low in cost, be conducive to
Large-scale promotion uses.
[specific embodiment]
It is illustrated below by way of specific embodiment.
The particular technique being not specified in example or condition person, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Production firm person is not specified in reagent or instrument used in this example, can pass through purchase
It obtains, and reagent is that analysis is pure.
Embodiment 1: the acquisition of Panax sample ITS region sequence and trnL-trnF region sequence
1, the DNA of sample to be tested is extracted
(1) more parts of ginsengs and American Ginseng sample are collected from different sources, as shown in table 1.
Table 1: ginseng and American Ginseng sample acquire information
| Serial number | Sample | Sampling point | Locality |
| 1 | Ginseng | Blade | Dandong |
| 2 | Ginseng | Root | Dandong |
| 3 | Ginseng | Root | Liaoyang |
| 4 | Ginseng | Root | Liaoyang |
| 5 | Ginseng | Root | Fushun |
| 6 | Ginseng | Root | Fushun |
| 7 | American Ginseng | Blade | Jilin Jingyu |
| 8 | American Ginseng | Root | Jilin Ji'an |
| 9 | American Ginseng | Root | Beijing Huairou |
| 10 | American Ginseng | Root | Beijing Huairou |
| 11 | American Ginseng | Root | Shandong Wendeng City |
| 12 | American Ginseng | Root | Jilin Fusong |
(2) DNA extraction is carried out using sample to be tested of the CTAB method to table 1, concrete operations are as follows:
1) sample to be tested is immersed in 75% ethyl alcohol 5 minutes, is taken out later, and be placed on natural wind in gnotobasis
It is dry.
2) 2g sample to be tested is taken, liquid feeding nitrogen is ground to powdered, is placed in 10mL centrifuge tube, 65 DEG C of preheatings are then added
In 65 DEG C of water-bath 1h after 3 × CTAB extracting solution 4.5mL, SDS 0.5mL mixing, and gently shakes and shake up every 15-20min；Institute
3 × CTAB the extract recipe stated are as follows: 3%CTAB, 0.1mol/L Tris-HCl, 1.4mol/LNaCl, 2%PVPP,
25mmol/L EDTA, autoclave sterilization；Wherein, 2% beta -mercaptoethanol is to sterilize, be added after cooling, hundred in above-mentioned system
Dividing ratio is volume accounting.
3) it after the water bath is over, is centrifuged 5min under conditions of 12000rpm, middle layer clear liquid is taken to dispense to 1.5mL centrifuge tube,
Add isometric Tris saturated phenol-chloroform-isoamyl alcohol (25:24:1), is centrifuged 10min under the conditions of 12000rpm after mixing, takes
Layer clear liquid adds isometric chloroform-isoamyl alcohol (24:1) to new 1.5mL centrifuge tube, after mixing again under conditions of 12000rpm from
Heart 10min, takes supernatant liquor, adds 0.6 times of volume isopropanol, then plus 3mol/L sodium acetate, until sodium acetate ultimate density is
0.3mol/L precipitates 1h under the conditions of -20 DEG C, and is centrifuged 10min under conditions of 12000rpm, gives up supernatant liquor, obtains
Precipitating is precipitated with 70% ethanol washing of 1mL pre-cooling, is centrifuged 5min under the conditions of 12000rpm later, then give up supernatant liquor,
It is precipitated, is washed repeatedly 2-3 times.Precipitating is air-dried after washing, is added 50 μ L sterile waters or 1 × TE solution to be dissolved, is set
It is saved in -20 DEG C, obtains sample to be tested DNA.
2, PCR amplification
(1) ITS region sequence is carried out to the resulting sample to be tested DNA of sample in table 1 respectively and trnL-trnF region sequence expands
Increase reaction, the upstream and downstream primer of ITS region sequence is respectively the upstream and downstream of primer I TS4 and primer I TS5, trnL-trnF region sequence
Primer is respectively primers F and primer R.
1) ITS sequence is carried out to sample to be tested DNA using primer I TS4 and primer I TS5 to carry out amplification reaction, obtain to be measured
Sample ITS sequence.
The nucleotides sequence of primer I TS4 is classified as SEQ ID No:2, specifically:
5’TCCTCCGCTTATTGATATGC 3’。
The nucleotides sequence of primer I TS5 is classified as SEQ ID No:3, specifically:
5’GGAAGTAAAAGTCGTAACAAGG 3’。
2) trnL-trnF sequence is carried out respectively to DNA sample to be measured using primers F and primer R to carry out amplification reaction, obtain
Sample to be tested trnL-trnF sequence.
The nucleotides sequence of primers F is classified as SEQ ID No:4, specifically:
F:5 ' GGTTCAAGTCCCTCTATCCC 3 '.
The nucleotides sequence of primer R is classified as SEQ ID No:4, specifically:
R:5 ' ATTTGAACTGGTGACACGAG 3 '.
Above-mentioned primer I TS4, primer I TS5, primers F and primer R are closed by Sangon Biotech (Shanghai) Co., Ltd.
At.
(2) PCR reaction system: the reaction system total volume of PCR amplification is 20 μ L, which includes 2.5mmol/L
The upstream of 10 × PCR Buffer, 2 μ L of MgCl2, four kinds of 1.6 μ L of 2.5mmol/L mononucleotide, 10 μm of ol/L ITS region sequences
The upstream primer or trnL-trnF of 0.8 μ L of upstream primer of primer or trnL-trnF region sequence, 10 μm of ol/L ITS region sequences
0.8 μ L, 5U/ μ L HiFi archaeal dna polymerase of downstream primer, the 0.1 μ L and template DNA 50ng of region sequence, surplus are sterile ultrapure
Water.Four kinds of 2.5mmol/L mononucleotides can be adenine, thymidine, guanine, cytimidine.
(3) pcr amplification reaction process is successively are as follows: 95 DEG C of initial denaturation 3min, 94 DEG C of denaturation 1min, 56 DEG C of annealing 1min, and 72
DEG C extend 1min, 30 circulation, 72 DEG C of extension 10min.
PCR amplification uses Beijing Quanshijin Biotechnology Co., Ltd HiFi kit in the present embodiment.
3, PCR product purifying, connection and conversion
Pcr amplification product uses DNA gel QIAquick Gel Extraction Kit (TaKaRaMiniBEST AgaroseGel DNA
Extraction Kit) it is tapped and recovered.The PCR amplification of ginseng and American Ginseng rDNA-ITS sequence and trnL-trnF sequence
Product carries out ampicillin selection from 1% agar gel competent cell later.
4, it is sequenced
Picking monoclonal colonies are sent to Bioisystech Co., Ltd, farsighted Boxing section and are sequenced, the primer of sequencing with it is above-mentioned
PCR primer is consistent.For the reliability for ensuring DNA bar code sequence, needs to carry out positive and negative sequencing or repeat to be sequenced, it then will be positive and negative
Sequencing result carries out splicing and obtains sample to be tested DNA combination bar code sequence ITS+trnL-trnF.
5, sequence assembly
The present embodiment using application software SeqMan to sample to be tested ITS sequence and sample to be tested trnL-trnF sequence into
Row sequence assembly and check and correction.
6, sequence alignment
With MEGA7.0 software to after sequencing each sample to be tested ITS sequence and sample to be tested trnL-trnF sequence carry out
It compares, then cuts off respective primer end, spliced according to 5 ' -3 ' direction, obtain the composite sequence of ITS+trnL-trnF,
If 5 ' ends of the sequence shown in the SEQ ID No:1, the 147th of ginseng be A, the 266th be A, the 445th be C, the
456 be T, the 892nd be C, the 978th be T；And the 147th of American Ginseng be C, the 266th be G, the 445th be T,
456 be C, the 892nd be G, the 978th be C.Therefore, accurate area can be carried out according to the specificity of above-mentioned six base positions
Divide ginseng and American Ginseng.
Embodiment 2
Compared with Example 1, except that by embodiment 1 ginseng and American Ginseng be prepared into medicine materical crude slice, with the medicine materical crude slice
For sample, DNA is extracted, is identified, as a result also can specifically detect six above-mentioned base positions.
Embodiment 3
Compared with Example 1, except that by embodiment 1 ginseng and American Ginseng be prepared into powder, with the powder
For sample, DNA is extracted, is identified, as a result also can specifically detect six above-mentioned base positions.
Claims (6)
1. a kind of combination bar code sequence identified for ginseng and American Ginseng, it is characterised in that the combination bar code sequence by
ITS region sequence and trnL-trnF region sequence are spliced to form, and are ITS+trnL-trnF, and nucleotide sequence is denoted as SEQ ID No:
1。
2. a kind of method for identifying ginseng and American Ginseng, it is characterised in that right using combination bar code sequence ITS+trnL-trnF
Sample to be tested is identified, is included the following steps:
Step 1: sample to be tested pretreatment extracts the DNA of sample to be tested, obtains sample to be tested DNA；
The upstream and downstream primer of step 2:ITS region sequence be respectively primer I TS4 and primer I TS5, trnL-trnF region sequence it is upper,
Downstream primer is respectively primers F, primer R, using the primer I TS4 and primer I TS5 of ITS region sequence, and uses trnL-trnF
The primers F and primer R of region sequence carry out pcr amplification reaction respectively, obtain sample to be tested ITS region sequence and sample to be tested trnL-
TrnF region sequence, the nucleotide sequence of the primer I TS4 are denoted as SEQ ID No:2, the nucleotide sequence note of the primer I TS5
For SEQ ID No:3, the nucleotide sequence of the primers F is denoted as SEQ ID No:4, and the nucleotide sequence of the primer R is denoted as
SEQ ID No:5；
Step 3: sample to be tested ITS region sequence and sample to be tested trnL-trnF region sequence that step 2 obtains are compared respectively
It is right, after matching, after cutting off respective primer end, spliced according to 5 ' -3 ' direction, obtains combination bar code sequence ITS+
TrnL-trnF, if the 147th bit base is A from the 5 ' ends of sequence SEQ ID No:1, the 266th bit base is A, the 445th
Base is C, the 456th bit base is T, the 892nd bit base is C, the 978th bit base is T, then is accredited as ginseng；If from sequence
It is C that the 147th bit base has been held in the 5 ' of SEQ ID No:3, the 266th bit base is G, the 445th bit base is T, the 456th bit base is
C, the 892nd bit base is G, the 978th bit base is C, then is accredited as American Ginseng.
3. a kind of method for identifying ginseng and American Ginseng according to claim 2, it is characterised in that sample to be tested first passes through pre-
The DNA that sample to be tested is just carried out after processing is extracted, and obtains sample to be tested DNA, the pretreatment is that sample to be tested is soaked in matter
5min in the ethanol solution that percent concentration is 75% is measured, is placed in gnotobasis and air-dries later, then in liquid nitrogen cooling
Under the conditions of be ground to fine powdered, which is sample to be tested.
4. a kind of method for identifying ginseng and American Ginseng according to claim 2, it is characterised in that the reaction of the PCR amplification
System total volume is 20 μ L, which includes 10 × PCR Buffer, 2 μ L of 2.5mmol/L MgCl2, four kinds
Draw the upstream of 1.6 μ L of 2.5mmol/L mononucleotide, the upstream primer of 10 μm of ol/L ITS region sequences or trnL-trnF region sequence
0.8 μ L, the 5U/ μ L of downstream primer of 0.8 μ L of object, the upstream primer of 10 μm of ol/L ITS region sequences or trnL-trnF region sequence
HiFi archaeal dna polymerase 0.1 μ L and template DNA 50ng, surplus are sterile ultrapure water；Four kinds of 2.5mmol/L mononucleotides
It can be adenine, thymidine, guanine, cytimidine.
5. a kind of method for identifying ginseng and American Ginseng according to claim 2, it is characterised in that the pcr amplification reaction mistake
Cheng Yici are as follows: 95 DEG C of initial denaturation 3min, 94 DEG C of denaturation 1min, 56 DEG C of annealing 1min, 72 DEG C of extension 1min, 30 recycle, and 72 DEG C
Extend 10min.
6. a kind of method for identifying ginseng and American Ginseng according to claim 2, it is characterised in that by sample to be tested in step 2
Combination bar code sequence ITS+trnL-trnF is obtained after ITS region sequence and the splicing of sample to be tested trnL-trnF region sequence, then is led
Enter software MEGA, and is compared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810882412.0A CN109486984A (en) | 2018-08-03 | 2018-08-03 | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810882412.0A CN109486984A (en) | 2018-08-03 | 2018-08-03 | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109486984A true CN109486984A (en) | 2019-03-19 |
Family
ID=65689156
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810882412.0A Pending CN109486984A (en) | 2018-08-03 | 2018-08-03 | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109486984A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021218206A1 (en) * | 2020-04-28 | 2021-11-04 | 宁夏农林科学院 | Dna-barcoding-based method for rapid identification of chinese wolfberry |
| CN114292940A (en) * | 2021-12-15 | 2022-04-08 | 西北大学 | Combined barcode sequences and methods for identification of rehmannia and rehmannia plants |
| CN114317797A (en) * | 2021-12-15 | 2022-04-12 | 西北大学 | DNA barcode sequence combination for identifying rehmannia and identification method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505045A (en) * | 2011-11-10 | 2012-06-20 | 中国医学科学院药用植物研究所 | Method for fast identifying ginseng and American ginseng |
| CN106119394A (en) * | 2016-08-26 | 2016-11-16 | 刘明涛 | The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue and the application of nucleotide sequence |
| CN107287300A (en) * | 2017-06-27 | 2017-10-24 | 中国林业科学研究院木材工业研究所 | A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application |
-
2018
- 2018-08-03 CN CN201810882412.0A patent/CN109486984A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505045A (en) * | 2011-11-10 | 2012-06-20 | 中国医学科学院药用植物研究所 | Method for fast identifying ginseng and American ginseng |
| CN106119394A (en) * | 2016-08-26 | 2016-11-16 | 刘明涛 | The nucleotide sequence authentication method of a kind of generation latitude lettuce tongue and the application of nucleotide sequence |
| CN107287300A (en) * | 2017-06-27 | 2017-10-24 | 中国林业科学研究院木材工业研究所 | A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application |
Non-Patent Citations (5)
| Title |
|---|
| TABERLET P等: "Universal primers for amplification of three non-coding regions of chloroplast DNA", 《PLANT MOL BIOL》 * |
| 周林 等: "一株市售人参的分子鉴定", 《人参研究》 * |
| 宋晓娜 等: "基于trnL-trnF序列的人参和西洋参DNA条形码鉴定研究", 《中国中药杂志》 * |
| 朱玲 等: "基于ITS和trnl-trnF序列的新疆扁桃种质资源亲缘关系研究", 《西北农业学报》 * |
| 黄璐琦 著: "《道地药材品质保障技术研究》", 31 January 2018, 上海科学技术出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021218206A1 (en) * | 2020-04-28 | 2021-11-04 | 宁夏农林科学院 | Dna-barcoding-based method for rapid identification of chinese wolfberry |
| CN114292940A (en) * | 2021-12-15 | 2022-04-08 | 西北大学 | Combined barcode sequences and methods for identification of rehmannia and rehmannia plants |
| CN114317797A (en) * | 2021-12-15 | 2022-04-12 | 西北大学 | DNA barcode sequence combination for identifying rehmannia and identification method |
| CN114292940B (en) * | 2021-12-15 | 2022-11-08 | 西北大学 | Combined barcode sequences and methods for identification of rehmannia and rehmannia plants |
| CN114317797B (en) * | 2021-12-15 | 2023-01-10 | 西北大学 | DNA barcode sequence combination for identifying rehmannia and identification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103060318B (en) | SSR (Simple Sequence Repeat) core primer group developed based on whole genome sequence of foxtail millet and application of SSR core primer group | |
| Pardo et al. | Molecular phylogeny and systematics of Genista (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trn L-trn F intergenic spacer) | |
| CN109486984A (en) | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng | |
| CN113637794B (en) | SSR molecular marker of new variety of mulberry, namely Guangdong mulberry 201, and core primer group, kit and application thereof | |
| CN103966210B (en) | The mark combination of peach SSAP molecular labeling primer sets splitting or integrating and the application on Peach cultivars analysis of genetic diversity thereof | |
| CN113430300B (en) | SSR molecular marker of mulberry variety Yuehen 123, core primer group and kit thereof, and application of SSR molecular marker | |
| CN103468709B (en) | Chloroplast genome of dendrobium huoshanense and germplasm identification method | |
| CN112980999B (en) | SSR molecular marker of mulberry variety Yuehei 74 and core primer group, kit and application thereof | |
| Zhao et al. | Molecular genetic diversity and population structure in Lycium accessions using SSR markers | |
| Lu et al. | Molecular phylogeny of the “true citrus fruit trees” group (Aurantioideae, Rutaceae) as inferred from chloroplast DNA sequence | |
| CN106011228A (en) | EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof | |
| US20210198756A1 (en) | Dna barcode for variety identification of wolfberry and identification method therefor | |
| CN103484558A (en) | Molecular identification method of Yunnan manyleaf Paris rhizome | |
| CN108754018B (en) | Screening method and application of acanthopanax senticosus target gene SSR molecular marker | |
| CN112080557A (en) | DNA barcode-based method for identifying producing area of cordyceps sinensis | |
| CN106434645A (en) | ITS (internal transcribed spacer) sequence of dalbergia odorifera and method for identifying dalbergia odorifera by ITS sequence | |
| CN108411002B (en) | PCR primer and method for identifying hybrid of echinus purpureus and echinus intermedius | |
| CN107254544B (en) | Chinese medicinal material rhizoma atractylodis germchit identification method based on DNA bar code technology | |
| Yang et al. | Development of a molecular marker to discriminate Korean Rubus species medicinal plants based on the nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL-F intergenic region sequences | |
| TAVAKOLI et al. | The phylogeny of Calligonum and Pteropyrum (Polygonaceae) based on nuclear ribosomal DNA ITS and chloroplast trnL-F sequences | |
| CN104450951B (en) | The primer of plant soil-borne diseases Fusarium oxysporum Rapid identification and method | |
| CN107794309A (en) | Rare wood identification method | |
| CN104928395B (en) | A kind of DNA fingerprinting and its acquisition methods of potato and its primer special | |
| CN115386649A (en) | A pair of Panax specific primers and molecular biological identification method of Panax Chinese medicinal material in Chinese patent medicine | |
| CN104830971A (en) | American ginseng molecule ID and identification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200617 Address after: No.10-3, denang East Road, Zhongshan port community, Torch Development Zone, Zhongshan City, Guangdong Province Applicant after: Guangdong Shengong Life Technology Co., Ltd Address before: 528401 No. 10, East Road, Torch Development Zone, Zhongshan, Guangdong. Applicant before: GUANGDONG TINBO GINSENG AND PILOSE ANTLER Co.,Ltd. |
|
| TA01 | Transfer of patent application right |