CN101849972A - Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio - Google Patents
Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio Download PDFInfo
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- CN101849972A CN101849972A CN 201010172605 CN201010172605A CN101849972A CN 101849972 A CN101849972 A CN 101849972A CN 201010172605 CN201010172605 CN 201010172605 CN 201010172605 A CN201010172605 A CN 201010172605A CN 101849972 A CN101849972 A CN 101849972A
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- glue
- cordyceps sinensis
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- banding
- high resolution
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- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 239000003292 glue Substances 0.000 claims abstract description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 12
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000011161 development Methods 0.000 claims abstract description 9
- 238000001502 gel electrophoresis Methods 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 30
- 238000004043 dyeing Methods 0.000 claims description 14
- 101710134784 Agnoprotein Proteins 0.000 claims description 6
- 238000010186 staining Methods 0.000 claims description 4
- 239000000834 fixative Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 5
- 238000011156 evaluation Methods 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 abstract 3
- 239000008098 formaldehyde solution Substances 0.000 abstract 1
- 241000190633 Cordyceps Species 0.000 description 14
- 230000000694 effects Effects 0.000 description 5
- 230000005855 radiation Effects 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000012850 discrimination method Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 238000006424 Flood reaction Methods 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- -1 polypropylene acrylamide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of the molecule biology, and particularly relates to a banding detection method for valuable endangered medical plant Cordyceps sinensis DAN fingerprint high resolution. The invention discloses a banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio, which comprises a PAGE gel electrophoresis step, a glue fixing step, a glue dying step and a glue color development step of Cordyceps sinensis DAN, and is characterized in that fixing liquid in the glue fixing step is of 0.5 percent acetic acid, dying liquid in the glue dying step is of 0.1 to 0.3 percent AgNO3, and developer solution in the glue color development step is of 1 to 4 percent strong base and 0.5 percent formaldehyde solution. The banding detection method can simplify the steps and can save the time, can save the experiment reagent and material in the experiment, can obtain high-efficient and accurate Cordyceps sinensis high resolution PAGE banding so as to set a molecule analysis foundation for establishing the valuable endangered traditional Chinese medicine - cordyceps sinensis resource sustainable development system and to provide reliable and high-efficient evaluation system for the Cordyceps sinensis character index - chemistry - molecule system.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of banding detection method that is used for the valuable endangered medical plant Cordyceps sinensis DNA fingerprint high resolution ratio.
Background technology
Cordyceps Cordyceps (Fr.) Link is a big class insect pathogenic fungus, and it is China's rare medicinal herbs that its specific bacterium infects the Cordyceps [Cordycepssinensis (Berk.) Sacc.] that has important medical value behind the specific insecticide.The Cordyceps nature and flavor are sweet, flat, go into lung, kidney channel, and energy lung benefiting kidney, cough-relieving, tonifying deficiency, beneficial vital essence, defying age, anticancer disease have the effect that strengthens immune function of human body and regulate reproduction.The natural cordyceps main product in Tibet, ground such as Yunnan, Sichuan and Qinghai-Tibet, germ plasm resource is abundant.But the puppet product that belong to together the kind that floods market at present is various, and the quality height is different.
At present, evaluation of medicinal fungi Cordyceps and discrimination method are in urgent need to be improved, traditional pharmacognosy discrimination method is many directly to be differentiated and identifies according to the apparent structure feature, and the common chemical analytical method is accurate inadequately, efficiently, thereby the sample size of required detection causes waste to preciousness Chinese medicine in imminent danger greatly.Dna fingerprint detects quality testing and the evaluation that now has been widely used in raw material of Chinese medicine, but the radiation of adopting in the existing dna fingerprint detection method shows the band method and the fluorescence banding method all has certain limitation, radiation shows the band method all has high pollution to environment and experimenter risk, and the used instrument costliness of fluorescence banding method, be difficult to popularize, and low pollution the and use general conventional polypropylene acrylamide gel electrophoresis (PAGE) silver and dye that to show the band method operating time long, step is many, employing reagent is various, easily cause modern background colour dense, resolution is low, band is fuzzy, and it is inaccurate to cause showing the band result.Simultaneously, the Cordyceps sinensis DNA fingerprint rich polymorphism, the operation that shows band is required very strictness, and prior art is difficult to obtain the apparent band of Cordyceps high-resolution PAGE of efficiently and accurately, thereby is difficult to provide reliably appraisement system efficiently for Cordyceps characteristic index-chemistry-molecular system.
Summary of the invention
Technical problem to be solved by this invention be for solve that existing Cordyceps is identified and discrimination method in consumption greatly, accurately efficient inadequately, radiation shows the band method and there are many defectives in the fluorescence banding method, and foundation can adapt to the banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio fully.
For this reason, the invention discloses a kind of banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio, the PAGE gel electrophoresis step, glue fixing step, glue staining procedure, the glue development step that comprise Cordyceps sinensis DNA, it is characterized in that fixative is 0.5% acetic acid in the described glue fixing step, dyeing liquor is 0.1%-0.3%AgNO in the described glue staining procedure
3, developer solution is 1%-4% highly basic and 0.5% formalin in the described glue development step.
In some embodiments, PAGE glue is the PAGE glue of 3%-6% in the PAGE gel electrophoresis step of described Cordyceps sinensis DNA, is beneficial to the representing of polymorphism of Cordyceps sinensis DNA.
In a preferred embodiment, described dyeing liquor is 0.3%AgNO
3, dna fingerprint polymorphism effect is the 60-80 band, and apparent band is more clear, and background is moderate, rich polymorphism.
In some embodiments, described highly basic can be N
aOH or KOH, wherein preferred 1%-2%N
aOH.
A kind of banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio has been set up in this invention.Because having adopted dna molecular fingerprint method and PAGE banding detection method differentiates, the Cordyceps amount of samples only needs 50ng, saved endangered plants medical material consumption greatly, avoided radiation to show limitation such as the high pollution of band method and fluorescence banding method and costliness simultaneously, and (fixing, dyeing, colour developing, stop) argentation of traditional 4 steps saved the process of cessation reaction, shortened the time improving under the specific prerequisite.Banding detection method of the present invention reduces medicine pollution, the strategy of its highly basic low concentration can better reduce the background colour that Cordyceps sinensis DNA fingerprint collection of illustrative plates PAGE shows band, improved resolution, weak base characteristics such as expend consuming time have been overcome, simultaneously, employing in addition, the present invention has saved sodium thiosulfate and acetum, feasible operation need not to carry out in fume hood, makes detection method safer and economical.The present invention provides a kind of high-resolution banding detection method of modern molecular biology technique for the researchs such as evaluation resolution of Cordyceps.The present invention has shortened search time, research efficient and accuracy have been improved, more suit the actual requirement that Cordyceps is identified and differentiated, the Cordyceps high-resolution PAGE that obtains efficiently and accurately shows band, thereby establish the analysis of molecules basis for setting up precious Chinese crude drug in imminent danger-Cordyceps resources sustainable development system, for Cordyceps characteristic index-chemistry-molecular system provides reliably appraisement system efficiently.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The present invention disposes the ratio of liquid and the concrete steps of operation are:
Embodiment 1
(1) the PAGE gel electrophoresis step of Cordyceps sinensis DNA: the PAGE glue with 3%-6% detects the Cordyceps sinensis DNA fingerprint collection of illustrative plates.
Table 1 polyacrylamide concentration table
According to the polyacrylamide gel of last table preparation variable concentrations, with reference to conventional method extracting Cordyceps sinensis DNA, carry out the PAGE gel electrophoresis.
(2) glue fixation procedure: its objective is make nucleic acid can better and Ag
+The specificity combination, set time 15-30min, concentration of fixation fluid be without ethanol 10%, and directly use 0.5% acetic acid, obviously improves specificity.
(3) dyeing liquor adopts 0.1%-0.4%AgNO
3Solution formula.The PAGE offset plate is taken off and separate, being sidelong into ddH of PAGE glue of Cordyceps sinensis DNA fingerprint will be arranged
2The middle rinsing 3-6s of 0 (2000ml) puts into the PAGE plate container that fills dyeing liquor again and carries out dna fingerprint dyeing, and dyeing course is for shaking 10-15min, result such as following table 2 on shaking table:
Table 2 coloured differently concentration dna fingerprint polymorphism effect table
| AgNO concentration (%) | Dna fingerprint polymorphism effect |
| ??0.1 | The 50-60 bar, clear, band is slightly shallow |
| ??0.2 | The 60-70 bar, more clear, small-molecular weight DNA fails to show fully |
| ??0.3 | The 60-80 bar, more clear, background is moderate, rich polymorphism |
| ??0.4 | Smear, the more recognition not too easily of band |
(4) developer solution adopts 1%-4%NaOH solution formula and 0.5% formalin.PAGE plate after the dyeing is immersed ddH
2Among the O, rinsing 2 times, each 3-6s puts into colour developing liquid again and carries out the dna fingerprint development.The dna fingerprint band is used ddH20 clear 2 times after all occurring at once, each 3-6s.
(5) cleaning PAGE plate and dried glue.
The common several method of table 3 shows the band result relatively
| Method | Key step | (min) consuming time | Background | Operation sequence | Fluid preservation | Show the band effect |
| Method one | Dyeing+colour developing | ??15-30 | Shallow | Simply | Now join first usefulness | The small-molecular weight band is difficult for differentiating |
| Method two | Fixing+dyeing+colour developing+termination | ??55-70 | Deeply | Loaded down with trivial details | Now join first usefulness | Macromolecule band muddiness |
| The inventive method | Fixing+dyeing+colour developing | ??25-35 | Moderate | Simpler | Recycle | Polymorphism is good |
The present invention is a kind of PAGE banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio of optimization.Its principle is to have utilized silver ion and nucleotide combination, and silver ion is met the formaldehyde reduction under alkaline environment, thereby manifests the dna fingerprint fragment in the gel.Show the band method with isotope and compare, this method has reduced isotopic harm.The fluorescent staining method of expense costliness is compared, and this method has been saved time and cost, and easily popularizes.Simultaneously, this invention is compared with traditional four step rule, has reduced the time, improved efficient, with two step method (is the method for a step compared with merging fixing and dyeing), this invention can detect the polymorphism of Cordyceps small-molecular weight, has obtained high-resolution Cordyceps sinensis DNA fingerprint.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating various aspects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of list of references mentioned above is listed this paper in as a reference all in full.
Claims (5)
1. the banding detection method of a Cordyceps sinensis DNA fingerprint high resolution ratio, the PAGE gel electrophoresis step, glue fixing step, glue staining procedure, the glue development step that comprise Cordyceps sinensis DNA, it is characterized in that fixative is 0.5% acetic acid in the described glue fixing step, dyeing liquor is 0.1%-0.3%AgNO in the described glue staining procedure
3, developer solution is 1%-4% highly basic and 0.5% formalin in the described glue development step.
2. banding detection method as claimed in claim 1 is characterized in that PAGE glue is the PAGE glue of 3%-6% in the PAGE gel electrophoresis step of described Cordyceps sinensis DNA.
3. banding detection method as claimed in claim 1 is characterized in that described dyeing liquor is 0.3%AgNO
3
4. banding detection method as claimed in claim 1 is characterized in that described highly basic can be N
aOH or KOH.
5. banding detection method as claimed in claim 1 is characterized in that described N
aOH is 1%-2%N
aOH.
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| CN2010101726050A CN101849972B (en) | 2010-05-07 | 2010-05-07 | Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio |
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| CN2010101726050A CN101849972B (en) | 2010-05-07 | 2010-05-07 | Banding detection method of Cordyceps sinensis DNA fingerprint high resolution ratio |
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| CN101849972A true CN101849972A (en) | 2010-10-06 |
| CN101849972B CN101849972B (en) | 2012-05-30 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101354348A (en) * | 2008-08-28 | 2009-01-28 | 云南省农业科学院甘蔗研究所 | Ultra-rapid silver dye detecting method of sugarcane molecular marker PAGE gel |
| CN101477001A (en) * | 2009-01-12 | 2009-07-08 | 河南科技大学 | DNA silver staining method |
| CN101560556A (en) * | 2009-01-16 | 2009-10-21 | 中国农业科学院棉花研究所 | Method for detecting DNA fingerprint purity in cotton crossbreeds |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101354348A (en) * | 2008-08-28 | 2009-01-28 | 云南省农业科学院甘蔗研究所 | Ultra-rapid silver dye detecting method of sugarcane molecular marker PAGE gel |
| CN101477001A (en) * | 2009-01-12 | 2009-07-08 | 河南科技大学 | DNA silver staining method |
| CN101560556A (en) * | 2009-01-16 | 2009-10-21 | 中国农业科学院棉花研究所 | Method for detecting DNA fingerprint purity in cotton crossbreeds |
Non-Patent Citations (4)
| Title |
|---|
| 《江西农业学报》 20081231 张妤艳 等 两种聚丙烯酰胺凝胶电泳染色方法对SRAP标记的影响 40-41 1-5 第20卷, 第12期 2 * |
| 《沈阳农业大学学报》 20060228 关海涛 等 两种聚丙烯酰胺凝胶银染方法的比较 86-87 1-5 第37卷, 第1期 2 * |
| 《细胞生物学杂志》 19991231 李宏业 等 改良聚丙烯酰胺凝胶电泳检测DNA 201-203 1-5 第21卷, 第4期 2 * |
| 《解放军药学学报》 20090630 张阵阵 等 冬虫夏草cDNA-AFLP银染法显带研究 189-192 1-5 第25卷, 第3期 2 * |
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