CN106148560A - A kind of comparison authentication method for Herba Dendrobii with weight lip Herba Dendrobii - Google Patents
A kind of comparison authentication method for Herba Dendrobii with weight lip Herba Dendrobii Download PDFInfo
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Abstract
The invention discloses a kind of comparison authentication method for Herba Dendrobii with weight lip Herba Dendrobii.Provided by the present invention for identifying or the primer pair of auxiliary qualification Herba Dendrobii, it is specially the primer pair being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.It is demonstrated experimentally that use primer provided by the present invention, by rapid PCR methods achieve Herba Dendrobii and weight lip Herba Dendrobii quick, accurately differentiate, it is achieved the on-the-spot utilization of medical material molecular identificalion provides technical support.
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
Comparison authentication method in Herba Dendrobii with weight lip 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).Weight lip Herba Dendrobii (Dendrobium hercoglossum) be Herba Dendrobii belong to sibling species together, both morphological characteristics are more close, after making Herba Dendrobii extract both be difficult to lead to
Cross profile to differentiate, cause commercially weighing one of lip Herba Dendrobii adulterant becoming Herba Dendrobii.Due to Herba Dendrobii and weight
Lip Herba Dendrobii differentiates relatively difficult by profile, uses traditional authentication method to have certain limitation.Therefore, searching is needed badly a kind of fast
Speed, accurately authentication method, 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 have carried out identifying that (Li Dan, Li Zhenjian, Mao Ping, Yan Xuefeng, Chun Ze, Ma Xinrong, based on ITS sequence
The qualification of row Herba Dendrobii material and phylogenetic analysis, gardening journal, 2012,39(8): 1539), but the method needs to survey
Sequence, the longest, and use large-scale instrument, it is unfavorable for realizing quick, Site Detection.
Summary of the invention
First purpose of the present invention is to provide a kind of for identifying or the primer pair of auxiliary qualification Herba Dendrobii.
A kind of as follows with the operating procedure that the comparison of weight lip Herba Dendrobii is identified for Herba Dendrobii:
(1) DNA extracting testing sample to carrying out pcr amplification reaction, obtains pcr amplification product as template, employing primer；
Described primer is to for forward primer CP6s:5 '-GCCCATAAATGGGTTTC-3 '
Downstream primer CP6a:5 '-GCACATCCGAGCCTTA-3 '；
(2) agarose gel electrophoresis method detection pcr amplification product
If the DNA fragmentation containing 275 bp in pcr amplification product, containing Herba Dendrobii in the most described testing sample；If PCR expands
Not containing the DNA fragmentation of 275 bp in product, the most described testing sample is non-Herba Dendrobii.
The comparison identification technology scheme limited further is as follows:
In step (1) pcr amplification reaction, cumulative volume 25 L of PCR reaction system, the wherein DNA of 0.5 L testing sample, 1.0
L content is the forward primer CP6s of 10 pmol, and 1.0 L content are the downstream primer CP6a of 10 pmol, 2.5 L 10 ×
Buffer buffer, 1.5 L concentration are the magnesium chloride (MgCl of 25 mM2) aqueous solution, 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, 60 DEG C of 30 s, 72 DEG C of 30 s, 45 circulations；72
℃ 10 min。
The concrete operations of step (2) are as follows: take 5 LPCR amplified productions, with the agarose gel of 2%, at voltage 80-90V
Lower electrophoresis 30 minutes, observes under gel imaging system and takes pictures；If the DNA fragmentation containing 275 bp in pcr amplification product, then institute
State in testing sample containing Herba Dendrobii；If pcr amplification product not containing the DNA fragmentation of 275 bp, the most described testing sample
For non-Herba Dendrobii.
A kind of detection kit identified for the comparison of Herba Dendrobii with weight lip Herba Dendrobii, upper by PCR reaction system
Trip primer CP6s and downstream primer CP6a individually packs；By except DNA, forward primer CP6s and the downstream primer of testing sample
Other material mix homogeneously outside CP6a is individually packed；The material individually packed above is positioned over same test kit
In, form a detection kit.
The detection kit technical scheme limited further is as follows:
Identify the detection kit described in operational aspect according to the comparison limited further, when detecting every time, need from
Taking different materials in detection kit, the amount that various materials are taken must is fulfilled for the qualification operating technology side limited further
The requirement of each material amount in PCR reaction system in case.
It is demonstrated experimentally that use primer provided by the present invention, achieve Herba Dendrobii and weight lip stone by rapid PCR methods
Between dry measure used in former times quick, accurately differentiate, it is achieved the on-the-spot utilization of medical material molecular identificalion provides technical support.
Accompanying drawing explanation
Fig. 1 is Herba Dendrobii, weight lip 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: weight lip Herba Dendrobii；3:: blank.
Fig. 3 is that Specific PCR primers is respectively SH-CP6s and SH-CP6a to SH-CP6(upstream and downstream primer) amplification gel
Electrophoretogram.M:DNA molecular criteria (DNA Marker is followed successively by 2000,1000,750,500,250 and 100 bp from top to bottom)；
1: with Specific PCR primers, SH-CP6 is expanded the result of 1 Herba Dendrobii sample；2: with Specific PCR primers to SH-CP6
Expand the result of 1 weight lip Herba Dendrobii；3: blank.
Fig. 4 is that Specific PCR primers is respectively SH-CP6s and SH-CP6a to SH-CP6(upstream and downstream primer) amplification gel
Electrophoretogram.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: Herba Dendrobii DNA profiling concentration be followed successively by 143 ng/ μ l, 71 ng/ μ l, 36 ng/ μ l, 18 ng/ μ l, 9 ng/ μ l, 4
Ng/ μ l and 2 ng/ μ l；8 to 14: weight lip Herba Dendrobii DNA profiling concentration be followed successively by 143 ng/ μ l, 71 ng/ μ l, 36 ng/ μ l, 18
Ng/ μ l, 9 ng/ μ l, 4 ng/ μ l and 2 ng/ μ l.
Detailed description of the invention
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, weight lip 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 | |
| 9 | Weight lip Herba DendrobiiDendrobium hercoglossum | 1 | Hap9 | JN388576 | |
| 10 | Weight lip Herba DendrobiiDendrobium hercoglossum | 1 | Hap10 | EU840693 | |
| 11 | Dendrobium loddigesiiDendrobium loddigesii | 1 | Hap11 | KF143481 | |
| 12 | Dendrobium loddigesiiDendrobium loddigesii | 1 | Hap12 | HQ114220 | |
| 13 | Rhombic lip Herba DendrobiiDendrobium leptocladum | 1 | Hap13 | AF521612 | |
| 14 | Rhombic lip Herba DendrobiiDendrobium leptocladum | 1 | Hap14 | EU840697 |
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, employment face Herba DendrobiiDendrobium macrophyllum1 ITS sequence (GenBank accession number is respectively AY239979) as outer group.Wherein BI tree is used
MrBayes version 3.1.2 software building, MP tree PAUP* version 4.0 beta 10 software building.Build BI
During tree, with MrModeltest version 2.3 software according to AIC(Akaike Information Criterion) inspection mark
The accurate optimality model selecting data, selected optimality model is (GTR+G).Markovian monte carlo method (Markov
Chains Monte Carlo, MCMC) it is set to four chains and ran for 1000000 generations.In order to determine its convergence situation, MCMC
It is separately operable twice.One sample of every 100 generations extraction, forms 20002 samples altogether.Learning by analysis, whole service exists
Reaching steady after 100000 generations, so, the most remaining sample number is 18002, with remaining sample reconstructing system tree and estimating it
Posterior probability values.When building MP tree, arranging bootstrapping number of repetition bootstrap nreps is 1000 times, uses heuristic search
Analyzing bootstrapping and repeat data set, 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:
SH-CP6s:5 '-GCCCATAAATGGGTTTC-3 ' (sequence 1)；
SH-CP6a:5 '-GCACATCCGAGCCTTA-3 ' (sequence 2).
The theoretical a length of 275 bp(sequences 3 of PCR primer)
Two, for identifying the assembling of the test kit of Herba Dendrobii
By in PCR reaction system, forward primer CP6s and downstream primer CP6a that step one designs synthesis individually pack；
Other material mix homogeneously in addition to DNA, forward primer CP6s and the downstream primer CP6a of testing sample is individually packed；Will be with
On the material individually packed be positioned in same test kit, i.e. obtain the present invention for Herba Dendrobii and weight lip Herba Dendrobii
The detection kit that comparison is identified.
Three, the method whether containing Herba Dendrobii in testing sample is identified
The detection kit using step 2 identifies in testing sample whether contain Huoshan stone according to the method comprised the steps
Dry measure used in former times:
1, PCR amplification
Genomic DNA is extracted as template, the forward primer CP6s of employing step one design synthesis and downstream from testing sample
Primer CP6a proceeds as follows PCR and expands:
PCR reacts cumulative volume 25 L, including following reagent: 0.5 L template DNA, the forward primer of 1.0 L10 concentration pmol
CP6s(10 pmol), the downstream primer CP6a(10 pmol of 1.0 L concentration pmol), 2.5 L 10 × buffer buffer,
1.5 L concentration are the MgCl of 25 mM2Aqueous solution, 0.5 L concentration is the dNTPs of 10 mM, 0.5 L Taq archaeal dna polymerase
(5U/ L), aseptic double-distilled water complements 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 275 bp, containing Herba Dendrobii in the most described testing sample；If there is no size
It is the purpose band (sequence 3) of 275 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 Anhui) and weight lip Herba Dendrobii (picking up from Guangxi).All meet Chinese Plants will and phase
The diagnostic characteristics closing document 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 weight lip 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 TY1s1 L(10 pmol),
Downstream primer TY1a1 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 5
Min, 35 circulations (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
(forward primer CP6s and downstream primer CP6a) carries out PCR amplification.Concrete reaction system and reaction condition are with embodiment 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:
Herba Dendrobii and weight lip Herba Dendrobii are examined by the primer utilizing the present invention by (forward primer CP6s and downstream primer CP6a)
Surveying, Herba Dendrobii can amplify the purpose band that clip size is about 275 bp.And weight lip Herba Dendrobii does not amplifies purpose band.
This shows that Herba Dendrobii can be identified accurately by this reaction system with weight lip Herba Dendrobii.Size is about the purpose bar of 275 bp
Band reclaims order-checking, and 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 Anhui).By identifying, sample object 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 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
143 ng/ μ l, 71 ng/ μ l, 36 ng/ μ l, 18 ng/ μ l, 9 ng/ μ l, 4 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 (forward primer CP6s and downstream primer
CP6a) PCR amplification is carried out.Concrete reaction system and reaction condition are with embodiment 1 step 31.Experiment is arranged with double steamings simultaneously
Water is that template is 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:
Herba Dendrobii is detected, when template is dense by the primer utilizing the present invention by (forward primer CP6s and downstream primer CP6a)
Degree is still to be able to during 36 ng/ μ l detect that size is about 275 bp purpose band clearly.Size is about the purpose of 275 bp
Band reclaims order-checking, and its sequence is being just sequence 3 in sequence table.
Claims (4)
1. the comparison authentication method for Herba Dendrobii with weight lip Herba Dendrobii, it is characterised in that operating procedure is as follows:
(1) DNA extracting testing sample to carrying out pcr amplification reaction, obtains pcr amplification product as template, employing primer；
Described primer is to for forward primer CP6s:5 '-GCCCATAAATGGGTTTC-3 '
Downstream primer CP6a:5 '-GCACATCCGAGCCTTA-3 '；
(2) agarose gel electrophoresis method detection pcr amplification product
If the DNA fragmentation containing 275 bp in pcr amplification product, containing Herba Dendrobii in the most described testing sample；If PCR expands
Not containing the DNA fragmentation of 275 bp in product, the most described testing sample is non-Herba Dendrobii.
The most according to claim 1 for the comparison authentication method of Herba Dendrobii with weight lip Herba Dendrobii, it is characterised in that: step
(1), in pcr amplification reaction, cumulative volume 25 L of PCR reaction system, the wherein DNA of 0.5 L testing sample, 1.0 L content are
The forward primer CP6s of 10 pmol, 1.0 L content are the downstream primer CP6a of 10 pmol, 2.5 L 10 × buffer bufferings
Liquid, 1.5 L concentration are the magnesium chloride (MgCl of 25 mM2) aqueous 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, aseptic double-distilled water complements to 25 L；
Pcr amplification reaction condition: 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。
The most according to claim 1 for the comparison authentication method of Herba Dendrobii with weight lip Herba Dendrobii, it is characterised in that: step
(2) concrete operations are as follows: take 5 LPCR amplified productions, with the agarose gel of 2%, and electrophoresis 30 minutes under voltage 80-90V,
Observe under gel imaging system and take pictures；If the DNA fragmentation containing 275 bp in pcr amplification product, in the most described testing sample
Containing Herba Dendrobii；If not containing the DNA fragmentation of 275 bp in pcr amplification product, the most described testing sample is non-Herba Dendrobii.
4. the detection kit identified for the comparison of Herba Dendrobii described in claim 1 with weight lip Herba Dendrobii, its feature exists
In: the forward primer CP6s in PCR reaction system and downstream primer CP6a is individually packed；By except testing sample DNA,
Other material mix homogeneously outside forward primer CP6s and downstream primer CP6a is individually packed；The thing that will individually pack above
Matter is positioned in same test kit, forms a detection kit.
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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 |
| 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 |
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