CN107739755A - Long-nosed pit viper capillary electrophoresis DNA finger-print and authentication method - Google Patents
Long-nosed pit viper capillary electrophoresis DNA finger-print and authentication method Download PDFInfo
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Abstract
The invention discloses one kind to be related to Chinese medicine long-nosed pit viper DNA identification technologies, and this method is specifically CTAB methods extraction long-nosed pit viper DNA, builds capillary electrophoresis fingerprint and authentication method.There is high resolution, easy to operate, quick, result is accurate, can be used in the identification of long-nosed pit viper sample.
Description
Technical field
The present invention relates to Materia Medica Identification technology, specifically a kind of long-nosed pit viper capillary electrophoresis DNA finger-print and mirror
Determine method.
Background technology
Long-nosed pit viper is traditional rare traditional Chinese medicine,《Chinese Pharmacopoeia》(version in 2015) provides that its certified products is that this product is that this product is Viperidae
Animal long-noded pit viper Agkistrodon acutus (Guenther) hirudo leech.Caught more than summer, season in autumn two, split snake abdomen,
Internal organ are removed, is cleaned, is strutted belly with bamboo chip, be coiled into the shape of a disc, bamboo chip is removed after drying.There is a wind-dispelling, dredging collateral, the only work(such as convulsion
Effect.For rheumatoid arthritis stubborn, numbness contracture, middle air port eye fish sticking its mouth out of the water oblique, hemiplegia, spasm of twitching, lockjaw, leprosy, mange.Due to
Its economic interests is driven, and in the market medicine variety is chaotic.
The discrimination method major sexual shape of long-nosed pit viper differentiates at present, microscopic features differentiate and physics and chemistry differentiates.Although in certain journey
These methods can be provided for the discriminating research and quality evaluation of long-nosed pit viper and investigated on degree, but need abundant identification of experience, subjectivity
It is stronger.Further, since the plasticity of such Chinese medicine character during drying and being made medicine materical crude slice is very big, physicochemical character is caused to reflect
Other effect is undesirable, it is difficult to meets accurate, the quick and objective requirement to long-nosed pit viper identification.Therefore, differentiate for the true and false of long-nosed pit viper
It is particularly important to provide a kind of method simple and practical, that accuracy is high.
DNA finger-prints refer to have germplasm specificity, can differentiate the electrophoresis pattern of difference between bion, tool
Have the advantages that high specificity, amount of samples are few, be highly suitable for Chinese medicine（Especially rare traditional Chinese medicine）The discriminating of easily mixed kind.Mesh
Before be widely used in the molecule labelling method of constructed dna finger-print mainly to include restriction fragment length polymorphic
Property (RFLP) analysis, random amplified polymorphic DNA analysis (RAPD) analysis, minisatellite DNA (VNTR) technology, microsatellite (SSR) technology
Analyzed with AFLP (AFLP).Due to RFLP, VNTR and SSR technical sophistication and it should be understood that experiment material
Genetic background, AFLP has been patented protection, therefore these technologies are being produced and are being somewhat limited using upper.
And RAPD technologies in the case of no any molecular biology research basis to a certain species due to can carry out
The research of the structure and genetic diversity of DNA finger-prints, and rich polymorphism, DNA dosages are few, economical and convenient, thus closely
It is widely applied over year in terms of cultivar resources identification.At present, RAPD technique construction species DNA is utilized both at home and abroad
The correlative study report of finger-print, but some deficiency below these research generally existings：
1. repeatability and less stable：Many factors can influence the amplification of RAPD analyses, cause analysis result to repeat
Property and less stable.
2. detection technique lags：RAPD amplified productions analysis at present mainly uses agarose gel electrophoresis and polyacrylamide
Two methods of gel electrophoresis.Agarose gel electrophoresis resolution ratio and sensitivity are relatively low, largely mask amplified production
Polymorphism, and DNA dosage is larger, and the dye toxicity used is also larger.Polyacrylamide gel electrophoresis resolution ratio and spirit
Sensitivity is higher, but cumbersome (including assembling, encapsulating, electrophoresis, unload the steps such as glue, dyeing), it is time-consuming that (whole process needs several
Hour arrives several days), make analysis efficiency relatively low.Therefore, currently used detection technique can not still meet that RAPD amplified productions are analyzed
The high-resolution that needs, high sensitivity, simplicity, quick etc. require, lack suitable detection means and seriously constrain RAPD skills
Application of the art in terms of species DNA fingerprinting is built.
3. authentication method is unreasonable：When being identified currently with RAPD technologies species, typically in electrophoresis pattern
The representative amplified band of selected section, according to the having of these bands, without being identified.Although this method makes result point
Analysis becomes simple, but reduces the polymorphism of amplified production, adds the probability of false positive results appearance.And artificial choosing
Select part amplified band and give up other bands, also reduce the objectivity of analysis result.In addition, this authentication method can only be anti-
Reflect the presence or absence of amplified band, it is impossible to reflect the strong and weak difference of amplified band.
For these reasons, currently with the correlation of RAPD technique construction species DNA fingerprintings and progress species identification
Report only has theoretical significance, can not still carry out practical application.
The content of the invention
It is existing to solve it is an object of the invention to provide a kind of long-nosed pit viper capillary electrophoresis DNA finger-print and authentication method
The defects of technology.This method high resolution, high sensitivity, easy to operate, quick, safety, result are accurate, can be used in long-nosed pit viper sample
The identification of product.
The purpose of the present invention is realized by following technical scheme：
A kind of method for building up of long-nosed pit viper capillary electrophoresis DNA finger-print, it is characterized in that, it includes following step：
1) DNA is extracted：Long-nosed pit viper piece is taken, its mitochondrial DNA is extracted by CTAB methods；
2) PCR is expanded：10-mer primers are screened, using 300 pairs of primers, above-mentioned DNA expanded；
3) PCR primer is identified：Amplified production carries out capillary electrophoresis analysis, records electrophoresis pattern；
4) foundation of long-nosed pit viper DNA fingerprinting：According to the amplification situation of each primer in electrophoresis pattern, select representational polymorphic
Property RAPD electrophoresis patterns structure long-nosed pit viper DNA fingerprinting.
A kind of long-nosed pit viper DNA fingerprint authentication method, it is characterized in that, it includes following step：
1) long-nosed pit viper DNA standard finger-prints are first established, method is as follows：
(a) DNA is extracted：Long-nosed pit viper control medicinal material is taken, its mitochondrial DNA is extracted by CTAB methods；
(b) PCR is expanded：10-mer primers are screened, using 300 pairs of primers, above-mentioned DNA expanded；
(c) PCR primer is identified：Amplified production carries out capillary electrophoresis analysis, records electrophoresis pattern；
(d) foundation of long-nosed pit viper DNA standard finger-prints：According to the amplification situation of each primer in electrophoresis pattern, select representative
Polymorphism RAPD electrophoresis patterns structure long-nosed pit viper DNA standard finger-prints；
2) determination sample DNA fingerprinting：Long-nosed pit viper sample to be measured is determined in above-mentioned identical method, obtains sample DNA finger-print；
3) result is identified：Utilize the phase of similarity analysis comparison sample DNA finger-print and long-nosed pit viper DNA standard finger-prints
Like degree, authenticity is carried out to long-nosed pit viper sample according to similarity.
The present invention compared with prior art, has advantages below：
1. the present invention by RAPD technologies it is simple and easy to do, contain much information the characteristics of and HPCE is quick, high resolution, spirit
The characteristics of sensitivity is high combines, and uses it for the DNA fingerprinting of Chinese medicine long-nosed pit viper and the foundation of fingerprint identification method, this
Belong to pioneering in the application aspect of long-nosed pit viper identification.
2. the present invention is using High Performance Capillary Electrophoresis analysis RAPD amplified productions, with currently used Ago-Gel
Electrophoresis is compared with polyacrylamide gel electrophoresis, HPCE have high sensitivity, high resolution, analyze speed it is fast,
Repeatability it is good with stability, amount of samples is small, cost is low, without using radio isotope and poisonous reagent the advantages that.This method
Overcome the shortcomings that agarose gel electrophoresis resolution ratio and sensitivity are low, DNA dosages are big and polyacrylamide gel electrophoresis operation
Cumbersome, the shortcomings that time-consuming, it is a kind of detection technique more suitable for the analysis of RAPD amplified productions.
3. the present invention builds long-nosed pit viper DNA standard finger-prints with the original electrophoresis pattern of long-nosed pit viper control medicinal material, without people
For delete or change, ensure that the integrality of finger-print, make qualification result more accurate and objective.
4. because DNA fingerprinting of the present invention represents in graph form, it appears that more directly perceived, understandable；And due to sample
Similarity analysis software is utilized when product are identified, authenticity is carried out to long-nosed pit viper sample according to similarity, it is easier to realize certainly
Dynamicization is analyzed.
Brief description of the drawings
Fig. 1 long-nosed pit viper DNA standard finger-prints.
Embodiment
With reference to embodiment, the invention will be further described, example below be merely to illustrate the present invention and not pair
The limitation of the present invention.
Embodiment 1
The foundation of long-nosed pit viper capillary electrophoresis DNA finger-print
1.DNA is extracted
Take this product（About 300mg）It is placed in mortar, adds appropriate liquid nitrogen grinding to powdery；It is pre- in 65 DEG C of water-baths to add 700 μ l
2%CTAB extraction buffers after heat, are gently agitated for mixing；Liquid will be ground to be placed in 1.5 ml sterile centrifugation tube, 65 DEG C of water
45min is incubated in bath or insulating box, during which gently overturns and mixes every 10min；After cooling down 2min, isometric chlorine is added
It is imitative：Isoamyl alcohol (24:1), concussion mixes, centrifugation（10000 turns/min）10min, supernatant is taken to be transferred to 600 μ l isopropanol
In centrifuge tube, the 30s that fluctuates is to there are DNA floccules；Centrifugation（10000 turns/min）1min；Filtered fluid is discarded, is not poured out white
Color DNA is precipitated, and being stood upside down on filter paper stands 60s, adds 720 μ l 75% ethanol and 80 μ l 5M sodium acetate, and room temperature places 30
Min, centrifuge (10000 turns/min）1min；Filtered fluid is discarded, adds the ethanol of 800 μ l 75%, after standing 30min, centrifugation
(10000 turns/min）30s；Filtered fluid is discarded, is uprightly stood several minutes, adds 50 0.5 × TE of ul (containing RNase) bufferings
Liquid, it is placed in 37 DEG C of h of insulating box about 15；- 20 DEG C are put to save backup.
2.PCR is expanded
Template is done with long-nosed pit viper to screen the 10-mer primers of 300 OPERON companies, and it is stable, more therefrom to select 15 amplifications
The abundant primer of state property, then long-nosed pit viper sample is expanded respectively.
PCR reaction systems are：10 × PCR buffer solutions 2.5 μ l, dNTP (2.5mmol/L) 2 μ l, diagnostic primerses（10 μm of ol/L) it is each
0.5 μ l, high-fidelity Taq DNA polymerase 5U/m1) 0.2 μ l, the μ l of template 2.
Loop parameter is arranged to：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 80sec, 36 DEG C of annealing 1min, 72 DEG C of extension 2min, 45
72 DEG C of extension 10min after individual circulation.
3.PCR products are identified
Pcr amplification product in step 2 is subjected to capillary electrophoresis analysis, comprised the following steps that：
3.1 instruments, reagent
Agilent capillary electrophoresis system (Agilent HP 3D/CE)；Agilent DAD detectors (Agilent diode-
array detector)；Agilent chem workstation (Agilent ChemStation software package).
Sodium tetraborate, sodium carbonate, hydroxypropyl-β-cyclodextrin, sodium dihydrogen phosphate, sodium hydroxide are the pure, deionized water of analysis.
Long-nosed pit viper control medicinal material (Nat'l Pharmaceutical ＆ Biological Products Control Institute provides and identified through Jilin medicine inspecting institute), commercially available long-nosed pit viper (city
The random buying in field), common adulterant long-nosed pit viper (Nat'l Pharmaceutical ＆ Biological Products Control Institute provides simultaneously to be identified through Jilin medicine inspecting institute).
3.2 sample treatment
The above-mentioned μ l of PCR amplified productions 10 are taken, dilute 10 times with electrophoretic buffer, through 0.45 μm of filtering with microporous membrane, filter
Liquid is as need testing solution.
3.3 deposition condition
Chromatographic column：The quartz capillary column (the sharp Feng chromatograms device Co., Ltd of Hebei Yongnian) of cm × 75 μm of 51 cm × 67.4；
Electrophoretic buffer：Mmol/L sodium carbonate -50mmol/L the hydroxypropyl-β-cyclodextrins of 60 mmol/L sodium tetraborates -50（pH
9.2）
Separation voltage：20 kV；
Temperature；25℃；
Detection wavelength：245nm；
Sampling condition：Electrokinetic injection, 15kV, 5S.
4. the foundation of long-nosed pit viper DNA fingerprinting is according to the amplification situation of each primer of each sample in step 3 gained electrophoresis pattern,
Select representative（Stability and repeatability are good, sample peak moderate number, separating degree are good, analysis time is short and each sample
It is distinguishable from one another obvious) electrophoresis pattern, build long-nosed pit viper DNA fingerprinting.Long-nosed pit viper and common adulterant have its special DNA fingerprint
Collection of illustrative plates, they can be distinguished from each other open.The electrophoresis pattern of long-nosed pit viper is recorded, as long-nosed pit viper DNA standard finger-prints, this standard
Finger-print can be used for the identification of long-nosed pit viper sample (see Fig. 1).
Embodiment 2
The long-nosed pit viper DNA fingerprint authentication method of the present invention is used for the identification of some long-nosed pit viper sample
Assuming that existing a collection of long-nosed pit viper sample cannot determine whether it is real long-nosed pit viper, therefore, it is reflected as follows
It is fixed.After long-nosed pit viper DNA standard finger-prints are established, when identifying some sample, its line grain is extracted with CTAB methods first
Body DNA；By the use of the DNA of extraction as template, with the primer pair filtered out, it enters performing PCR amplification；Amplified production carries out capillary electricity
Swimming is analyzed and records electrophoresis pattern, produces the DNA fingerprinting of this batch of testing sample；It is to be measured using similarity analysis comparison
The similarity of sample DNA finger-print and long-nosed pit viper DNA standard finger-prints, is identified testing sample according to similarity.Mirror
Determine result：If the two similarity was more than for 95% (containing 95%), this batch of sample is exactly real long-nosed pit viper；If the two similarity is less than
95%, then this batch of sample is not just real long-nosed pit viper.
Claims (2)
- A kind of 1. Chinese medicine long-nosed pit viper DNA identification method, it is characterised in that：Methods described using CTAB methods extraction long-nosed pit viper DNA with RAPD technologies are combined, and can fast and accurately differentiate long-nosed pit viper and its true and false.
- A kind of 2. Chinese medicine long-nosed pit viper DNA identification method as described in the appended claim 1, it is characterised in that：Comprise the following steps：1) DNA is extracted：Long-nosed pit viper piece is taken, its DNA is extracted by CTAB methods；2) PCR is expanded：PCR cumulative volumes are 25 μ l, comprising 10 × PCR buffer solutions 2.5 μ l, dNTP (2.5mmol/L) 2 μ l, are differentiated Primer（10 μm of ol/L) each 0.5 μ l, high-fidelity Taq DNA polymerase 5U/m1) 0.2 μ l, the μ l of template 2, aseptic double-distilled water 17.3 μl；Above-mentioned DNA is expanded；3) PCR primer is identified：Amplified production carries out capillary electrophoresis analysis, records electrophoresis pattern；4) long-nosed pit viper DNA fingerprinting is built.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613758A (en) * | 2009-06-30 | 2009-12-30 | 中国中医科学院中药研究所 | Identify PCR method and the special primer thereof of long-nosed pit viper |
| CN102586448A (en) * | 2012-03-06 | 2012-07-18 | 北华大学 | Deer antler capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and identification method |
-
2017
- 2017-11-27 CN CN201711202918.4A patent/CN107739755A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613758A (en) * | 2009-06-30 | 2009-12-30 | 中国中医科学院中药研究所 | Identify PCR method and the special primer thereof of long-nosed pit viper |
| CN102586448A (en) * | 2012-03-06 | 2012-07-18 | 北华大学 | Deer antler capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and identification method |
Non-Patent Citations (4)
| Title |
|---|
| YING JIA,ET AL: "cDNA cloning, expression and fibrin(ogen)olytic activity of two low-molecular weight snake venom metalloproteinases", 《TOXICON》 * |
| 张乐等: "常用蛇类药材鉴别研究进展", 《中国实验方剂学杂志》 * |
| 王义权等: "蛇类药材分子遗传标记鉴别的初步研究", 《药学学报》 * |
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Application publication date: 20180227 |