CN105063034B - Molecular method quantification analyzes the specific primer and method of Chloranthus glaber and 3 kinds of adulterants - Google Patents
Molecular method quantification analyzes the specific primer and method of Chloranthus glaber and 3 kinds of adulterants Download PDFInfo
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Abstract
The present invention provides the specific primer and method of a kind of molecular method quantification analysis Chloranthus glaber and 3 kinds of adulterants, belongs to biological technical field, the upstream specific primer SgF of specific recognition Chloranthus glaber：5 ' TGATTCTGATAGATT TTTGAAGAATTGA 3 ' and anti-sense primer trnL Fb；The special sense primer ChF of specific recognition tree and oneself and Chloranthus henryi Hemsl simultaneously：5 ' ATCTGATCACCCTTACACTTACAAGC 3 ' and anti-sense primer trnL Ff.By this 2 pairs of primer structure structure quantitative fluorescent PCRs, the molecular method quantification analysis of the middle specific sample of biased sample is carried out.The present invention provides one kind can be accurate, quickly, simple quantitative analysis Chloranthus glaber and tree, Chloranthus henryi Hemsl and and oneself 3 kinds of adulterants technology, the melange quantitative analysis for related Chloranthus glaber medicine materical crude slice and this 3 kinds of adulterants provides technical support.
Description
Technical field
The invention belongs to biological technical field, and in particular to molecular method quantification analyzes the specificity of Chloranthus glaber and 3 kinds of adulterants
Primer and method, it is specially a kind of discriminating Chloranthus glaberSarcandra glabra (Thunb.) Nakai, tree
Chloranthus spicatus, and oneselfChloranthus serratus, with Chloranthus henryi HemslChloranthus henryi
Molecular method quantification analysis specificity labeled primers, and it is a kind of using the specific primer to Chloranthus glaber and tree, and oneself
With 3 kinds of melange quick quantitative analytic methods such as Chloranthus henryi Hemsl.
Background technology
Chloranthus glaberSarcandra glabra (Thunb.) Nakai, be commonly called as Glabrous Sarcandra Herb, be generally used for treat tinea versicolor,
Purpura, rheumatic arthralgia, wound.Modern study shows to have treating cancer, antitumor, anti-inflammatory, antiviral and non-specific exempt from
Epidemic disease strengthens first-class pharmacological action.However, wild plant germ plasm resource is reduced in recent years, due to tree, Chloranthus henryi Hemsl,
And own profile is extremely similar to Chloranthus glaber, it is easy to mutually obscures with Chloranthus glaber.But they have different attribute and medical value,
These easy mixed planting things also have a corresponding hepatotoxic effect, and oneself is in the toxicity research of serious hepatotoxicity wind agitation case report and animal
Notable hepatotoxicity wind agitation is had confirmed, which has limited its application clinically.
Although the fresh blade of Chloranthus glaber can be by micro- between the nonglandular hair vascular structure of vein, adjunct and leaf
Identification difference identifies from its adulterant, but the Chloranthus glaber and easy mixed planting thing of powder or crushing sheet form pass through morphology
It is very indiscernible with histological techniques.In the past few years, identified using chemical analysis technology such as TLC, HPLC, MS etc.
Chloranthus glaber and its related preparations have had described.Although these methods can supplement morphology or histology to a certain extent
The limitation of identification, but chemical fingerprint can only detection part compound, limited species composition information can only be provided, it is impossible to
The method for identifying Chloranthus glaber completely is provided to us, and is difficult to carry out quantitative analysis, particularly very much like with it
When species mutually mix.Therefore, Chloranthus glaber is established with quantitatively being divided when tree and the product mix such as oneself and Chloranthus henryi Hemsl
Analysis method, than just most important to monitoring quality to its Related product.
With the development of molecular biology, DNA molecular marker such as RAPD, ISSR and SSR equimolecular marks, ITS
（Internal transcribed spacer）Etc. the method that DNA bar code technology provides reliable identification medicinal material.However,
These molecular marking techniques based on genomic DNA are easily influenceed by genomic DNA degraded, due to nuclear DNA ratio
Chloroplast DNA copy number is few, is easier to go to pot in finished medicinal material and leads to not enter by its molecular marking technique
Row differentiates.Chloroplast DNA trnL-F regions include trnL（UAA）Introne and interval, i.e. trnL（UAA）- trnF（GAA）
Area is as DNA bar code, and it can be readily available universal primer and be expanded, and this sequence has been demonstrated can be used for a variety of
Plant is widely used in the detection of plant product in kind of a horizontal identification.
This seminar is in early-stage Study, it was also found that for drying or the medicinal material of processed mistake, based on chloroplast DNA
The authentication technique in trnL-F regions is more stable more effective than the molecular marking technique based on nuclear DNA.This research is to careless coral
Coral, tree, Chloranthus henryi Hemsl and and oneself trnL-F region sequences be sequenced, analyze its sequential structure and feature, find single
Nucleotide polymorphisms (single nucleotide polymorphism SNP) molecular labeling, design identify Chloranthus glaber respectively,
Tree, Chloranthus henryi Hemsl and and oneself specific recognition primer, establish multiple PCR technique, identify the specificity of primer, in this base
On plinth, using these special primers pair, build real-time fluorescence quantitative PCR, can quickly and easily quantitative analysis Chloranthus glaber and tree,
Chloranthus henryi Hemsl and and oneself 3 kinds of adulterants method.The present invention provides for Chloranthus glaber and 3 molecular method quantification for obscuring kind analyses
Technical support, quantitative analysis is carried out to the related prepared slices of Chinese crude drugs and had very important significance, retrieval have no Chloranthus glaber and tree,
Chloranthus henryi Hemsl and and oneself etc. the molecular method quantification of 3 kinds of adulterants application for a patent for invention.
The content of the invention
The main object of the present invention is to provide one kind can be accurate, quickly, simple quantitative analysis Chloranthus glaber and tree, wide leaf
Tree and and oneself 3 kinds of adulterants technology, the melange quantitative analysis for related Chloranthus glaber medicine materical crude slice and this 3 kinds of adulterants provides
Technical support.
To achieve the above object, the present invention adopts the following technical scheme that：
Molecular method quantification analyzes the specific primer of Chloranthus glaber and 3 kinds of adulterants, and the upstream of specific recognition Chloranthus glaber is special
Property primer SgF：5 '-TGATTCTGATAGATT-TTTGAAGAATTGA -3 ' and anti-sense primer trnL-Fb：5’-
GGGACTTGAACCCTCACGATT- 3’；The special sense primer of specific recognition tree and oneself and Chloranthus henryi Hemsl simultaneously
ChF：5 '-ATCTGATCACCCTTACACTTACAAGC -3 ' and anti-sense primer trnL-Ff：5’- ATT TGA ACT GGT
GAC ACG AG - 3’。
The method that molecular method quantification analyzes Chloranthus glaber and 3 kinds of adulterants, structure quantitative fluorescent PCR carry out 40 circulations, reaction
System
It is as follows：Total amount is 20 μ L, and 10 ng DNA is extracted from plant as amplification masterplate, 10 2 × SYBR of μ L
Green Real-time PCR Master Mix (TaKaRa), 0.2 μM of upstream specific primer, 0.2 μM of downstream is general to draw
Thing, remaining is sterilized water；Response procedures are as follows：In real-time quantitative PCR instrument（ABI7900）Kind, 50 DEG C of pre-degenerations 2 minutes,
95 DEG C of pre-degenerations 10 minutes, are denatured 15 s at 95 DEG C, 58 DEG C of 15s of primer annealing and extend 30 s, 40 circulations at 72 DEG C.
Specific method is as follows：
（1）Specific recognition primer is designed, and verifies its specificity
According to Chloranthus glaber, the tree that experiment gained is sequenced early stage, and the trnL-F gene orders of oneself and Chloranthus henryi Hemsl,
Its special SNP site is analyzed, separately designs the upstream specific primer SgF of Chloranthus glaber（5’-
TGATTCTGATAGATTTTTGAAGAATTGA - 3’）, design can specific recognition tree, and oneself and wide leaf gold grain simultaneously
Blue special sense primer ChF（5’- ATCTGATCACCCTTACACTTACAAGC - 3’）With downstream universal primer trnL-Ff
（5’- ATT TGA ACT GGT GAC ACG AG - 3’）, build multiplex PCR system：Total amount is 20 μ L, takes 10 ng plants
The DNA of middle extraction adds 1 μ L 1 × TransStart TopTaq DNA polymerase reaction liquid as masterplate, and 2 μ L 10 ×
Two of TransStart TopTaq Buffer, dNTP 2.5mM, 0.2 μM of anti-sense primer trnL-Ff and upstream specifically draw
Each 0.1 μM of thing SgF, ChF, remaining is sterilized water.Multiplex PCR system is built, response procedures are as follows:Divide in 95 DEG C of pre-degenerations 2
Clock, 30 s are denatured at 95 DEG C, primer annealing 30s and extends 2 minutes at 72 DEG C at 50 DEG C, carried out 35 and circulate, finally
One circulates in 72 DEG C and extends 7 minutes, to ensure complete extension product.Only Chloranthus glaber achieves 575 bp, tree,
And oneself and Chloranthus henryi Hemsl generate specific 216bp, biased sample then produces the band of 575 bp and 216bp etc. 2 simultaneously.Confirm
The specificity of primer.
（2）Quantitative analysis method：
The upstream specific primer SgF of Chloranthus glaber（5’- TGATTCTGATAGATT-TTTGAAGAATTGA - 3’）With under
Swim primer trnL-Fb（5’- GGGACTTGAACCCTCACGATT- 3’）Form the primer pair, simultaneously of specific recognition Chloranthus glaber
Specific recognition tree and the special sense primer ChF of oneself and Chloranthus henryi Hemsl（5’-
ATCTGATCACCCTTACACTTACAAGC - 3’）With anti-sense primer trnL-Ff（5’- ATT TGA ACT GGT GAC ACG
AG - 3’）The primer pair of 3 kinds of samples of specific recognition tree and oneself and Chloranthus henryi Hemsl etc. is formed, builds real-time quantitative respectively
PCR system.Build quantitative fluorescent PCR and carry out 40 circulations, reaction system is as follows：Total amount is 20 μ L, and 10 are extracted from plant
Ng DNA, which is used as, expands masterplate, 10 μ L 2 × SYBR Green Real-time PCR Master Mix (TaKaRa), and 0.2
μM upstream specific primer and 0.2 μM of downstream universal primer, remaining is sterilized water；Response procedures are as follows：In real-time PCR
Device（ABI7900）In, 50 DEG C of pre-degenerations 2 minutes, 95 DEG C of pre-degenerations 10 minutes；15 s, 58 DEG C of primer annealing are denatured at 95 DEG C
15s and 72 DEG C extend 30 s, 40 circulation.Primer SgF and trnL-Fb are used for the quantitative analysis of Chloranthus glaber in product mix；
The quantitative analysis for the Chloranthus (tree and oneself and Chloranthus henryi Hemsl) that primer ChF and trnL-Ff are used in product mix.
The concentration of all primers is 0.2 μM.Real time quantitative PCR method can be by relative quantitative analysis method R for quantitative analysis method
=2-△△CtMethod is derived, due to being expanded in same DNA sample using its STb gene as template, the total masterplate DNA phases of biased sample
The Ct answered（total）Value is constant, i.e., the Ct in a specific biased sample（total）Value is constant.Then dopant number
The ratio of amount can be calculated in same sample by this formula:
Ct（total）) be total mixing sample Ct values, Ct (Sg) be primer SgF, trnL-Fb Ct values, that is, represent grass
The Ct values of coral content, Ct (Ch) are primer trnL-Ff, ChF Ct values, that is, represent the Ct values of three kinds of adulterant contents.
The advantage of the invention is that：
Due to carrying out quantitative analysis according only to some activity characteristic component contents, easily by external conditions such as plant growth environment
Influence, it is difficult to carry out accurate quantitative analysis.Content of this method based on hereditary material DNA carries out molecular method quantification, is not produced by medicinal material
The influence of the external factors such as ground, it is more accurately, also more simple and convenient relative to stoichiometric method.
It is more stable in the product after drying and processing because chloroplast DNA is more more than the copy number of genomic DNA,
Detection object of this method by the use of chloroplast DNA as quantitative analysis, Billy is by the use of genomic DNA as detection object, to processing
Related product carry out molecular method quantification it is more reliable and stable.
This method can carry out molecular method quantification analysis, 3 kinds of accurate quantitative analysis simultaneously to Chloranthus glaber and 3 kinds of common adulterants
The presence ratio of adulterant, it is more efficient, it is more convenient.
Brief description of the drawings
Fig. 1 utilizes the product gel electrophoresis of primer trnL-Ff, SgF, ChF multiplex PCR（M :2 log DNA ladder；
1-8：Chloranthus glaber；9-11：And oneself；12-13：Chloranthus henryi Hemsl；14-15：Chloranthus glaber and own mixture；16：Chloranthus glaber, wide leaf
Tree mixture）.
Fig. 2 pair of primers SgF, trnL-Fb Ct/x curve maps（R2=0.9989)。
Fig. 3 pair of primers ChF, trnL-Ff Ct/x curve maps（R2=0.9982)。
Embodiment
1 instrument
PCR instrument (Eppendorf, model 5332), electrophoresis system (Beijing Liuyi Instrument Factory, model DYY-12),
Cryogenic freezing centrifuge (Eppendorf, model 5810R), gel image analyser (BIO-RAD ChemiDoc XRS) are micro-
Measure pipettor (Eppendorf).
2 reagents
2 × CTAB extract solutions, 1 × TAE buffer solutions, agarose (Promega companies), ethidium bromide (Fluka
Company), TransStart TopTaq DNA Polymerase (Beijing Quan Shi King Companies), three chlorine first alkane ﹑ are without water second alcohol ﹑
Isopropanol is that domestic analysis is pure.
3 materials
This research experiment sample be identified and collected by Fujian University of Traditional Chinese Medicine Wei Yicong lecturer pick up from different sources
Sample：13 kinds of Chloranthus glabers, four kinds of trees, four kinds of Chloranthus henryi Hemsls and three kinds and own sample are received from Chinese different sources
Collection.Five kinds of Chloranthus glaber sample powders and three kinds of tree sample powders are collected from local market to be got.
Extraction：
(1) take the fresh Chloranthus glabers of 5 g and Chloranthus plant leaf blade to shred, be placed in the mortar by sterilization, add 0.5
GPVP powder, add liquid nitrogen and grind rapidly, powder is collected in sack, be placed in -20 DEG C of preservations, preserve then put for a long time
In -80 DEG C；
(2) take 10 mLCTAB solution to be preheated in 65 DEG C of water, then add 0.2 mL (200 μ L) mercaptoethanol in preheating
In CTAB（Mercaptoethanol：CTAB solution=1:50）；
(3) step is taken（1）In the g of powder 0.2 managed in 2 mLEP（Centrifuge tube）, be rapidly added 1 ml preheatings contains mercapto
The CTAB solution of base ethanol（Added during sample low temperature）, vibrate 2 minutes and put it into 65 DEG C of water-baths, water-bath about 1 hour；（Often
Vibration in 20 minutes once, suitably can be vibrated several times more）
(4) toward adding 600 μ L chloroform-isoamyl alcohol mixed liquors in pipe（Chloroform：Isoamyl alcohol volume ratio=24:1, it is necessary to divulging information
Cupboard operates）, shake up 5 minutes, then be placed on 15-20 DEG C and centrifuged 10 minutes with 12000rpm；
(5) 600 μ L of supernatant liquid are taken, add 1.2mL（2 times）The absolute ethyl alcohol of precooling or 360 μ L（0.6 times）Isopropanol,
- 20 DEG C are placed in precipitate about 3 hours or stay overnight；
(6) take out EP pipes to centrifuge 10 minutes with 12000 rmp under the conditions of 4 DEG C, remove supernatant, be inverted in blotting paper
Several minutes；
(7) 700 μ L70% ethanol are added, are turned upside down for several times, washing precipitation, with 12000 under the conditions of 4 DEG C
Rmp is centrifuged 10 minutes, is removed supernatant, is repeated, is finally placed in super-clean bench and dries up；（Pay attention to：It can not dry up excessively）
(8) 30-50 μ L deionized waters are added in EP pipes（ddH2O）Or 1 × TE solution, place 3 hours or mistake in 4 DEG C
At night, it is fully dissolved, if hand be difficult to dissolve, can be mixed with mixing machine, promote its dissolving；
(9) can detect whether it contains genomic DNA with Ago-Gel, after purified.
Primer specificity discriminance analysis
According to Chloranthus glaber, the tree that experiment gained is sequenced early stage, and the trnL-F gene orders of oneself and Chloranthus henryi Hemsl,
Its special SNP site is analyzed, separately designs Chloranthus glaber and the upstream specific primer SgF of oneself and Chloranthus henryi Hemsl（5’-
TGATTCTGATAGATTTTTGAAGAATTGA - 3’）、ChF（5’- ATCTGATCACCCTTACACTTACAAGC - 3’）Point
Not with downstream universal primer trnL-Ff（5’- ATT TGA ACT GGT GAC ACG AG - 3’）Build multiplex PCR system：
Total amount is 20 μ L, takes the DNA extracted in 10 ng plants to add 1 μ L1 × TransStart TopTaq DNA as template and gather
Synthase reaction solution (TransGen biotechnologys), 2 μ L 10 × TransStart TopTaq Buffer, dNTP 2.5mM, 0.2
μM anti-sense primer trnL-Ff and two specific primer SgF, ChF of 0.1 μM of upstream, remaining is sterilized water.Build multiplex PCR
System, response procedures are as follows：In 95 DEG C of pre-degenerations 2 minutes, be denatured in 95 DEG C of 30 s, 50 DEG C of primer annealing 30s and
72 DEG C extend 2 minutes, carry out 35 circulations.Last circulates in 72 DEG C and extended 7 minutes, to ensure complete extension production
Product.Only Chloranthus glaber achieves 575 bp, and oneself and Chloranthus henryi Hemsl generate specific 216bp, and biased sample then produces simultaneously
The band of 575 bp and 216bp etc. 2, is shown in Fig. 1.
Molecular method quantification analysis is carried out using specific primer
To determine that the specific primer is appropriate for the molecular method quantification analysis of biased sample, we use the grass of gradient dilution
The mixture D NA samples of coral and its easy mixed planting thing have carried out real-time PCR detections.Tested sample DNA carries out continuous ten times of dilutions
（1/10/100/1000/10000 times of dilution）, 2 μ L samples DNA are incorporated as masterplate in total amount is 20 μ L reaction systems.Phase
A curve is built for specific DNA Ct values, the results showed that, the Ct values of 2 pairs of special primers exist with the logarithm of starting template
Linear relationship, and R2 >0.99, illustrate that designing this 2 pairs of primers can quantitative determine to unknown sample, therefore, designed reality
When quantitative fluorescent PCR be effectively to the biased sample carry out molecular method quantification method.Such as（Fig. 2）With（Fig. 3）It is shown, pair of primers
The Ct values of SgF, trnL-Fb amplification form regression curve with the logarithm value of sample relative quantity：
Y = -3.0885 log(X) + 39.015, R2= 0.9989。
Equally, the Ct values of pair of primers ChF and trnL-Ff amplifications form regression curve with the logarithm value of sample relative quantity：
Y= -3.5698 log(X) + 37.004, R2= 0.9982。
During this investigation it turned out, the Ct values and 4 diluted concentrations (1/10/ of DNA profiling of 2 pairs of primers of the mixed sample
100/1000 times dilution) linear relationship be present, therefore, can be by accurate quantitative analysis in the dilution intervals.Chloranthus glaber with
Content ratio of the chloroplast DNA of adulterant in the easy mixed planting thing of same sample can be calculated by this method, i.e., 3 kinds are obscured sample
Condition can be calculated the relative amount of Chloranthus glaber sample by below equation:。
In our current research, easy mixed planting thing chloroplast DNA content is above 12.94 times of Chloranthus glaber.It is these results indicate that real
When PCR be that a sensitive enough and accurate method is assessed the chloroplast DNA of Chloranthus glaber and its 3 kinds of adulterants in biased sample and contained
The ratio of amount, and according to this content assess the ratio of Chloranthus glaber and its easy mixed planting thing biased sample.Because specific
It relative to Chloranthus glaber and its easy mixed planting thing is identical that mixing sample Chloroplast DNA degradation rate, which is, therefore this method can have
Effect carries out molecular method quantification analysis to the biased sample of Chloranthus glaber and its easy mixed planting thing.
The Ct values of table 1 draw the relative populations of sample
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Fujian University of Traditional Chinese Medicine
<120>Molecular method quantification analyzes the specific primer and method of Chloranthus glaber and 3 kinds of adulterants
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
tgattctgat agatttttga agaattga
28
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
gggacttgaa ccctcacgat t
21
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence
<400> 3
atctgatcac ccttacactt acaagc
26
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
atttgaactg gtgacacgag
20
Claims (2)
1. molecular method quantification analyzes the specific primer of Chloranthus glaber and 3 kinds of adulterants, it is characterised in that：Specific recognition gold grain simultaneously
Blue and oneself and 3 kinds of adulterants of Chloranthus henryi Hemsl upstream specific primer ChF：5’- ATCTGATCACCCTTACACTTACAAG
C - 3’。
2. a kind of method that molecular method quantification using primer described in claim 1 analyzes Chloranthus glaber and 3 kinds of adulterants, its feature exist
In：Build quantitative fluorescent PCR and carry out 40 circulations, reaction system is as follows：Total amount is 20 μ L, extracts 10 ng's from plant
DNA is as amplification masterplate, and 10 μ L 2 × SYBR Green Real-time PCR Master Mix, 0.2 μM of upstream is specifically drawn
Thing, 0.2 μM of downstream universal primer, remaining is sterilized water；Response procedures are as follows：In real-time quantitative PCR instrument, 50 DEG C of pre-degenerations
2 minutes, 95 DEG C of pre-degenerations 10 minutes；95 DEG C denaturation 15 s, 58 DEG C of 15s of primer annealing and 72 DEG C extend 30 s, 40 are followed
Ring；Described downstream universal primer is 5 '-ATT TGA ACT GGT GAC ACG AG -3 '.
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Non-Patent Citations (5)
| Title |
|---|
| Molecular authentication and quantitative analysis of Sarcandra glabra and adulterated chloranthus products using SNP markers.;Wei Y et al.;《Mitochondrial DNA A DNA Mapp Seq Anal.》;20150910;第27卷(第5期);3618-3625 * |
| Phylogeny of Chloranthus (Chloranthaceae) Based on Nuclear Ribosomal ITS and Plastid trnL-F sequence data;Hong-Zhi Kong et al.;《American Journal of Botany》;20020630;第89卷(第6期);940-946 * |
| 不同居群草珊瑚的ITS序列分析及模式识别研究;魏艺聪 等;《中草药》;20141028;第45卷(第20期);2982-2986 * |
| 基于双向位点特异性PCR 的金银花真伪鉴别方法研究;蒋超 等;《中国中药杂志》;20121215;第37卷(第2期);3752-3757 * |
| 草珊瑚与金粟兰叶片双位点特异性PCR鉴别方法研究;魏艺聪 等;《中国中药杂志》;20140901;第39卷(第17期);3259-3262 * |
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