CN106282373A - A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii - Google Patents
A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii Download PDFInfo
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Abstract
The present invention relates to the contrast authentication method of a kind of Herba Dendrobii and Henan Herba Dendrobii.Contrast identifies that operating procedure is: (1) is extracted genomic DNA from two kinds of samples to be measured respectively and to carrying out pcr amplification reaction respectively, obtained two kinds of pcr amplification products as template, employing primer；(2) use agarose gel electrophoresis method two kinds of pcr amplification products of detection, if the DNA fragmentation containing 262 bp in one of which or another kind of pcr amplification product, then in one of which or another kind of testing sample containing or candidate contain Herba Dendrobii；If PCR primer does not contains the DNA fragmentation of 262 bp, the most one or another kind of testing sample does not contains or candidate does not contains Herba Dendrobii.The inventive method achieves Herba Dendrobii and Henan Herba Dendrobii quick, accurately differentiate.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to Chinese medicine and Materia Medica Identification field, use particularly to one
In the method identifying Herba Dendrobii and Henan Herba Dendrobii.
Background technology
Herba Dendrobii (Dendrobium huoshanense) it is that Dabie Mountains, Anhui Province district is distinctive, special-protection-by-the-State
Rare Chinese medicine, be under the jurisdiction of the orchid family (Orchidaceae) Dendrobium (Dendrobium).Henan Herba Dendrobii (Dendrobium henanense) be Herba Dendrobii belong to sibling species together, both morphological characteristics are more close, especially make after Herba Dendrobii extract both more
It is difficult to be differentiated by profile, causes commercially one of Henan Herba Dendrobii adulterant becoming Herba Dendrobii.Due to Huoshan stone
Dry measure used in former times differentiates relatively difficult with Henan Herba Dendrobii by profile, uses traditional authentication method to have certain limitation.Therefore, searching is needed badly
One authentication method fast and accurately, to guarantee the accuracy of Herba Dendrobii Med Mat Appreciation.Li Dan etc. utilize DNA bar code ITS
Sequence pair Herba Dendrobii and nearly edge species thereof carried out identifying (Li Dan, Li Zhenjian, Mao Ping, Yan Xuefeng, Chun Ze, Ma Xinrong, based on
The qualification of ITS sequence Herba Dendrobii material and phylogenetic analysis, gardening journal, 2012,39(8): 1539), but the method need into
Row order-checking, the longest, and use large-scale instrument, it is unfavorable for realizing quick, Site Detection.
Summary of the invention
It is an object of the invention to provide the contrast authentication method of a kind of Herba Dendrobii and Henan Herba Dendrobii.
A kind of Herba Dendrobii and the contrast of Henan Herba Dendrobii identify that operating procedure is as follows:
(1) from two kinds of samples to be measured, extract genomic DNA respectively as template, use primer to carrying out PCR amplification respectively
Reaction, obtains two kinds of pcr amplification products；
Described primer to for forward primer CP29s:5 '-TTCGTGGGATGGGGTCC-3 ',
Downstream primer CP29a:5 '-TGCACATCCGAGCCTAA-3 '；
(2) two kinds of pcr amplification products of agarose gel electrophoresis method detection
If the DNA fragmentation containing 262 bp bands in one of which pcr amplification product, then one of which or another kind of testing sample
In containing or candidate contain Herba Dendrobii；If PCR primer does not contains the DNA fragmentation of 262 bp bands, the most one or another kind of
Testing sample does not contains or candidate does not contains Herba Dendrobii.
The qualification operational aspect limited further is as follows:
In step (1) pcr amplification reaction, cumulative volume 25 L of PCR reaction system, wherein 0.5 L is one or another kind of to be measured
The DNA of sample, 1.0 L content are the forward primer CP29s of 10 pmol, and 1.0 L content are the downstream primer of 10 pmol
CP29a, 2.5 L 10 × buffer buffer, 1.5 L concentration are the magnesium chloride brine of 25 mM, and 0.5 L concentration is 10
The dNTPs of mM, 0.5 L concentration is the Taq archaeal dna polymerase of 5U/ L, and aseptic double-distilled water complements to 25 L.
Pcr amplification reaction condition: 95 DEG C of 5 min；95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 30 s, 35 are followed
Ring；72 ℃ 10 min.
In step (2), take 5 one or another kind of pcr amplification products of L, with the agarose gel of 2%, at voltage 80-
Observe under electrophoresis 30 minutes, gel imaging system under 90V and take pictures；If containing 262 in one or another kind of pcr amplification product
The DNA fragmentation of bp, the most described testing sample is Herba Dendrobii；If pcr amplification product not containing the DNA fragmentation of 262 bp, then
Described testing sample is non-Herba Dendrobii.
A kind of detection kit identified with the contrast of Henan Herba Dendrobii for Herba Dendrobii, by the upstream in PCR reaction system
Primer CP29s and downstream primer CP29a individually packs, and will remove the DNA of testing sample, forward primer in PCR reaction system
Other material mix homogeneously outside CP29s and downstream primer CP29a is individually packed, and is placed by the material individually packed above
In same test kit, form one for the detection kit identifying Herba Dendrobii.
The technical scheme of the detection kit limited further is as follows:
According to the detection kit described in the qualification operational aspect limited further, when detecting every time, need from detection
Taking different materials in test kit, the amount that various materials are taken must is fulfilled in the qualification operational aspect limited further
The requirement of each material amount in PCR reaction system.
The Advantageous Effects of the present invention is embodied in following aspect:
1. The inventive method achieves Herba Dendrobii and Henan Herba Dendrobii quick, accurately differentiate.
2., compared with traditional form authentication method, the present invention need not morphological taxonomy basis and achieves Rapid identification
Herba Dendrobii and Henan Herba Dendrobii, solve the problem according to Morphological Identification more difficulty.
3. technology highly versatile, less demanding to instrument and equipment, easy popularization and application in real work, operating process letter
Single, shorter, the most highly sensitive and more stable.
Accompanying drawing explanation
Fig. 1 is Herba Dendrobii, Henan Herba Dendrobii and the phylogenetic tree of part sibling species.Wherein, above pedigree branch node
For posterior probability PP and bootstrapping support BS of MP tree of BI tree, being PP value on the left side of oblique line, the right of oblique line is BS value.
Fig. 2 is that universal primer expands gel electrophoresis figure.(DNA Marker, is followed successively by M:DNA molecular criteria from top to bottom
2000,1000,750,500,250 and 100 bp)；1: Herba Dendrobii；2: Henan Herba Dendrobii；3:: blank.
Fig. 3 is that Specific PCR primers is to CP29(forward primer CP29s and forward primer CP29a) amplification gel electrophoresis figure.
M:DNA molecular criteria (DNA Marker is followed successively by 2000,1000,750,500,250 and 100 bp from top to bottom)；1: with spy
The specific PCR primers result to CP29 1 Herba Dendrobii sample of amplification；2: Specific PCR primers expands 1 Henan stone to CP29
The result of dry measure used in former times；3: blank.
Fig. 4 is that Specific PCR primers is to CP29(forward primer CP29s and forward primer CP29a) amplification gel electrophoresis figure.
M:DNA molecular criteria (DNA Marker is followed successively by 2000,1000,750,500,250 and 100 bp from top to bottom)；1 to 7: suddenly
Mountain Herba Dendrobii DNA profiling concentration is followed successively by 156 ng/ μ l, 78 ng/ μ l, 39 ng/ μ l, 19 ng/ μ l, 10 ng/ μ l, 5 ng/ μ l
With 2 ng/ μ l；8 to 14: Henan Herba Dendrobii DNA profiling concentration is followed successively by 156 ng/ μ l, 78 ng/ μ l, 39 ng/ μ l, 19 ng/ μ
L, 10 ng/ μ l, 5 ng/ μ l and 2 ng/ μ l.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, for identifying the method for preparation and use of test kit of Herba Dendrobii
One, for identifying design and the synthesis of the primer pair of Herba Dendrobii
Search from GenBank or carry out checking order by this research and obtain the ITS sequence of Herba Dendrobii and adulterant, refer to table 1 below.
Wherein, this research order-checking gained haplotype sequence (including Hap5, Hap6 and Hap7) refers to sequence 4 to sequence 6 institute in sequence table
Show.
Table 1 Herba Dendrobii, Henan Herba Dendrobii and the ITS sequence of part sibling species
| Numbering | Sample ID | Sample size | Haplotype | GenBank accession number | This institute obtains sequence numbering |
| 1 | Herba DendrobiiDendrobium catenatum | 1 | Hap1 | KP743543 | |
| 2 | Herba DendrobiiDendrobium catenatum | 1 | Hap2 | KF143439 | |
| 3 | Dendrobium nobileDendrobium nobile | 1 | Hap3 | EF618732 | |
| 4 | Dendrobium nobileDendrobium nobile | 1 | Hap4 | JN388579 | |
| 5 | Henan Herba DendrobiiDendrobium henanense | 1 | Hap5 | IHN1H | |
| 6 | Henan Herba DendrobiiDendrobium henanense | 6 | Hap6 | IHN1C; IHN1E; IHN1F; IHN1G; HNITS01; IHN1D | |
| 7 | Herba DendrobiiDendrobium huoshanense | 3 | Hap7 | KF143476 | IHS1A IHS2D |
| 8 | Herba DendrobiiDendrobium huoshanense | 1 | Hap8 | JN388567 |
Carry out with Bayesian Method (Bayesian inference, BI) and parsimony principle (maximum parsimony, MP) respectively
Phylogenetic Analysis, builds two kinds of phylogenetic tree of BI and MP.During constructing system tree, use rhombic lip Herba DendrobiiDendrobium leptocladum2 ITS sequence (GenBank accession number is respectively AF521612 and EU840697) as outer group.Wherein BI
Tree MrBayes version 3.1.2 software building, MP tree PAUP* version 4.0 beta 10 software building.Structure
When building BI tree, with MrModeltest version 2.3 software according to AIC(Akaike Information Criterion) inspection
Testing the optimality model of standard selection data, selected optimality model is (SYM+G).Markovian monte carlo method
(Markov Chains Monte Carlo, MCMC) is set to four chains and ran for 500000 generations.In order to determine that it restrains feelings
Condition, MCMC is separately operable twice.One sample of every 100 generations extraction, forms 10002 samples altogether.Learn by analysis, whole fortune
Row reaches steady after 20000 generations, and so, the most remaining sample number is 9602, with remaining sample reconstructing system tree and estimating
Its posterior probability values.When building MP tree, arranging bootstrapping number of repetition bootstrap nreps is 1000 times, uses heuristic searching
Rope analysis bootstrapping repeats data set, and heuristic search is set to be produced initial tree by random progressively additive process, is repeated 10 times, uses
TBR branch exchange.The phylogenetic tree built by BI method and MP method is shown, the phylogenetic tree trunk of 2 kinds of method structures is consistent
(Fig. 1).Posterior probability (posterior probability, the PP) value of BI tree is indicated at the node of Tu1Zhong Ge pedigree branch
Bootstrapping support (Bootstrap, BS) with MP tree.Result shows, the haplotype of Herba Dendrobii all sequences forms monosystem
(PP=1.00, BS=96).
On the premise of determining that Herba Dendrobii is monosystem group, with software Clustal X 1.81 to ITS sequence all in table 1
Carrying out para-position sequence and compare, finding differential fragment, design Herba Dendrobii detects Specific PCR primers, as follows:
Forward primer CP29s:5 '-TTCGTGGGATGGGGTcC-3 ' (sequence 1)；
Downstream primer CP29a:5 '-TGCACATCCGAGCCTaA-3 ' (sequence 2).
The theoretical a length of 262 bp(sequences 3 of PCR primer)
Two, for identifying the assembling of the test kit of Herba Dendrobii
Step one is designed after the forward primer CP29s and downstream primer CP29a of synthesis individually pack, by PCR reactant
In system, other material mix homogeneously in addition to DNA, forward primer CP29s and the downstream primer CP29a of testing sample is individually packed,
The material individually packed above is positioned in same test kit, forms one for the detection examination identifying Herba Dendrobii
Agent box.
Three, the method whether containing Herba Dendrobii in testing sample is identified
The test kit using step 2 identifies whether contain Herba Dendrobii in testing sample according to the method comprised the steps:
1, PCR amplification
Genomic DNA is extracted as template, the forward primer CP29s of employing step one design synthesis and downstream from testing sample
Primer CP29a(sequence 1 and sequence 2) proceed as follows PCR amplification:
PCR reacts cumulative volume 25 L, including following reagent: 0.5 L template DNA, and 1.0 L forward primer CP29s(10 pmol),
1.0 L downstream primer CP29a(10 pmol), 2.5 L 10 × buffer buffer, 1.5 L concentration are the MgCl of 25 mM2Water
Solution, 0.5 L concentration is the dNTPs of 10 mM, and 0.5 L concentration is the Taq archaeal dna polymerase of 5U/ L, and aseptic double-distilled water is supplied
To 25 L.
After PCR reactant liquor has been prepared, shake mixing gently, PCR pipe is put in PCR instrument, carry out PCR amplification, the most instead
Answer condition as follows: 95 DEG C of 5 min；95 DEG C of 30 s, 60 DEG C of 30 s, 72 DEG C of 30 s, 45 circulations；72 ℃ 10
min。
2, PCR primer detection
Use agarose gel electrophoresis method detection PCR primer, specific as follows:
Taking 5 L amplified productions, use the agarose gel of 2%, electrophoresis 30 minutes under voltage 80-90V, under gel imaging system
Observe and take pictures.According to the size of purpose band, determine as follows and whether testing sample contains Herba Dendrobii: if obtaining
Obtain the purpose band (sequence 3) that size is about 262 bp, containing Herba Dendrobii in the most described testing sample；If there is no size
It is the purpose band (sequence 3) of 262 bp, the most described testing sample does not contains Herba Dendrobii.
Embodiment 2, the test kit using embodiment 1 to prepare identify the specificity analyses of Herba Dendrobii
Testing sample: Herba Dendrobii (picking up from Huoshan) and Henan Herba Dendrobii (picking up from Huoshan).All meet China to plant
The diagnostic characteristics of thing will and pertinent literature describes.By identifying, the material object of each sample is consistent with title, quality conformance with standard.
One, from testing sample, genomic DNA is extracted
Take about 50 mg drying samples (certainly may be used without 100 mg fresh sample and carry out extracting genome DNA) respectively, use CTAB
Method extracts STb gene.Specific as follows:
Dry medical material without going mouldy is placed in pulverizer and grinds, cross 40 mesh sieves.Powder is transferred to the trace of 2.0 mL
In centrifuge tube, add 900 L sterilized extract with CTAB liquid (formula: 2% (2g/100ml) CTAB, 100 mmol/L Tris-
HCl pH=8.0,20 mmol/L EDTA, 1.4 mol/L NaCl), 0.02 g PVP 40000,10 L beta-mercaptoethanol fills
Divide vibration mixing, 65 DEG C of water-bath 1.5 h-2 h, period jog 2-3 time.Take out after end and be cooled to room temperature, add 900 L chlorine
Imitative-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, 12000 g are centrifuged 10 min.Take supernatant, addition equal-volume chloroform-different
Amylalcohol (volume ratio 24:1), mixing of fully vibrating, 12000 g are centrifuged 10 min.Take supernatant, add the isopropyl of 2/3 volume pre-cooling
Alcoholic solution, places 0.5 more than h for-20 DEG C.Taking out, 12000 g are centrifuged 10 min, abandon supernatant, precipitate by 70 %(volume fractions)
Washing with alcohol twice, 37 DEG C volatilize ethanol, dissolve with appropriate aquesterilisa ,-20 DEG C of preservations.
By the above Herba Dendrobii extracted of universal primer TY1s and TY1a detection and the genomic DNA quality of Henan Herba Dendrobii.
TY1s:5 '-GATGGATGAACCCTCAAATC-3 '；
TY1a:5 '-GGCAGCCAACGAGAAGA-3 '.
Reacting total system is 25 L, including DNA profiling 0.5 L(5-50ng), forward primer 1 L(10 pmol), downstream
Primer 1 L(10 pmol), 10 × PCR Buffer 2.5 L, MgCl2(25 mM) 1.5 L, dNTPs(10 mM) 0.5 L, Taq
Archaeal dna polymerase (5U/ L) 0.5 L, ddH2O complements to 25 L.Universal primer PCR reaction condition is: 95 DEG C of 5 min, 35 follow
Ring (each circulation includes 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C 30 s), 72 DEG C of 10 min.
Found that all samples all can amplify DNA band (Fig. 2).
Two, PCR amplification
The genomic DNA extracted from testing sample with step one, as template, uses the primer pair of embodiment 1 step one design
CP29(forward primer CP29s and downstream primer CP29a) carry out PCR amplification.Concrete reaction system and reaction condition are with implementing
Example 1 step 31.Experiment is arranged using distilled water for template as negative control simultaneously.Experiment is in triplicate.
After reaction terminates, according to the method for step 32 in embodiment 1, testing sample is identified.
Result is as it is shown on figure 3, as can be seen from the figure:
Utilize the primer of the present invention to CP29(forward primer CP29s and downstream primer CP29a) to Herba Dendrobii and Henan Herba Dendrobii
Detecting, Herba Dendrobii can amplify the purpose band that clip size is about 262 bp.And Henan Herba Dendrobii does not amplifies mesh
Band.This shows that Herba Dendrobii can be identified accurately by this reaction system with Henan Herba Dendrobii.Size is about 262 bp
Purpose band reclaim order-checking, its sequence is being just sequence 3 in sequence table.
Embodiment 3, the test kit using embodiment 1 to prepare identify the sensitive analysis of Herba Dendrobii
Testing sample: Herba Dendrobii (picks up from Huoshan).By identifying, sample object is consistent with title, and quality meets mark
Accurate.
One, from testing sample, genomic DNA is extracted
Take about 50 mg drying samples (certainly may be used without 100mg fresh sample and carry out extracting genome DNA) respectively, use CTAB
Method extracts STb gene.Concrete operations see embodiment 2 step one.
Two, PCR amplification
Genomic DNA step one extracted from Herba Dendrobii carries out doubling dilution, obtains genomic DNA concentration and is respectively
156 ng/ μ l, 78 ng/ μ l, 39 ng/ μ l, 19 ng/ μ l, 10 ng/ μ l, 5 ng/ μ l and the serial dilutions of 2 ng/ μ l, with
Serial dilutions is respectively template, uses the primer of embodiment 1 step one design to enter (primer SH-CP29s and SH-CP29a)
Performing PCR expands.Concrete reaction system and reaction condition are with embodiment 1 step 31.Experiment is arranged with distilled water as template simultaneously
As negative control.Experiment is in triplicate.
After reaction terminates, according to the method for step 32 in embodiment 1, testing sample is identified.
Result as shown in Figure 4, as can be seen from the figure:
Utilize the primer of the present invention to CP29(forward primer CP29s and downstream primer CP29a) Herba Dendrobii is detected, when
Template concentrations is still to be able to during 39 ng/ μ l detect that size is about 262 bp purpose band clearly.Size is about 262 bp
Purpose band reclaim order-checking, its sequence is being just sequence 3 in sequence table.
Claims (4)
1. a Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii, it is characterised in that contrast identifies that operating procedure is as follows:
(1) from two kinds of samples to be measured, extract genomic DNA respectively as template, use primer to carrying out PCR amplification respectively
Reaction, obtains two kinds of pcr amplification products；
Described primer to for forward primer CP29s:5 '-TTCGTGGGATGGGGTCC-3 ',
Downstream primer CP29a:5 '-TGCACATCCGAGCCTAA-3 '；
(2) two kinds of pcr amplification products of agarose gel electrophoresis method detection
If the DNA fragmentation containing 262 bp bands in one of which or another kind of pcr amplification product, then one of which or another kind
Testing sample contains or candidate contains Herba Dendrobii；If PCR primer does not contains the DNA fragmentation of 262 bp bands, then a kind of
Or another kind of testing sample does not contains or candidate does not contains Herba Dendrobii.
A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii, it is characterised in that: step
(1) in pcr amplification reaction, cumulative volume 25 L of PCR reaction system, wherein 0.5 one or another kind of testing sample of L
DNA, 1.0 L content are the forward primer CP29s of 10 pmol, and 1.0 L content are the downstream primer CP29a of 10 pmol, 2.5
L 10 × buffer buffer, 1.5 L concentration are the magnesium chloride brine of 25 mM, and 0.5 L concentration is the dNTPs of 10 mM
, 0.5 L concentration is the Taq archaeal dna polymerase of 5U/ L, and aseptic double-distilled water complements to 25 L；
Pcr amplification reaction condition: 95 DEG C of 5 min；95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 30 s, 35 circulations；72
℃ 10 min。
A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii, it is characterised in that: step
(1) in, 5 one or another kind of pcr amplification products of L are taken, with the agarose gel of 2%, electrophoresis 30 points under voltage 80-90V
Clock, observes under gel imaging system and takes pictures；If the DNA fragmentation containing 262 bp in one or another kind of pcr amplification product, then
Described testing sample is Herba Dendrobii；If not containing the DNA fragmentation of 262 bp in pcr amplification product, the most described testing sample is
Non-Herba Dendrobii.
4. the detection kit identified with the contrast of Henan Herba Dendrobii for Herba Dendrobii described in claim 1, it is characterised in that:
Forward primer CP29s in PCR reaction system and downstream primer CP29a is individually packed, treats PCR reaction system is removed
Other material mix homogeneously outside the DNA of test sample product, forward primer CP29s and downstream primer CP29a is individually packed, and will divide above
The most individually the material of packaging is positioned in same test kit, forms one for the detection kit identifying Herba Dendrobii.
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| CN107400718A (en) * | 2017-08-29 | 2017-11-28 | 南京师范大学 | The primer sets and method of Dendrobidium huoshanness and maple bucket product are identified based on Real timeARMS qPCR |
| CN107400718B (en) * | 2017-08-29 | 2020-04-03 | 南京师范大学 | Primer group and method for identifying dendrobium huoshanense and dendrobium candidum products based on Real-timeARMS-qPCR |
| CN107619879A (en) * | 2017-11-13 | 2018-01-23 | 皖西学院 | A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum |
| CN107630104A (en) * | 2017-11-13 | 2018-01-26 | 皖西学院 | A kind of phylogenetic tree and authentication method for being used to identify Dendrobidium huoshanness or dendrobium candidum |
| CN110951912A (en) * | 2019-12-31 | 2020-04-03 | 安徽师范大学 | Method for identifying dendrobium huoshanense based on DNA bar code |
| CN113176227A (en) * | 2021-04-27 | 2021-07-27 | 皖西学院 | Method for rapidly predicting adulteration of dendrobium huoshanense in dendrobium hunan |
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