CN105039584A - American ginseng DNA detection reagent box and identification method - Google Patents
American ginseng DNA detection reagent box and identification method Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicine, in particular to an American ginseng DNA detection reagent box and an identification method. The American ginseng DNA detection reagent box comprises five parts, namely a sample pretreatment solution, a genome DNA extracting system, a PCR reaction system, a restriction enzyme reaction system and an observation result system. The identification method comprises six steps, namely detection sample pretreatment, genome DNA extraction, PCR primer design and synthesis, PCR reaction establishment, restriction enzyme reaction and result judgment. According to judgment standards, a 100 bp band and a 50 bp band occur simultaneously, the American ginseng is a salable product, and if only one band occurs or no band occurs, the American ginseng is a fake product. The PCR-AFLP identification method has the advantages of being simple, convenient, fast, reliable in detection result and the like, and the specificity of the American ginseng and the fake product can be identified accurately at the same time.
Description
Technical field
The present invention relates to Materia Medica Identification technology, specifically a kind of Radix Panacis Quinquefolii DNA detection test kit and authentication method.
Background technology
Radix Panacis Quinquefolii (PanaxquinquefoliusL.) is Araliaceae Panax (PanaxginsengC.A.Mey.) perennial root herbaceous plant, another name Radix Panacis Quinquefolii, American ginseng, Western ginseng, originate in southern Canada and the northern US of North America.Radix Panacis Quinquefolii is former is wild varieties, and rear kind domestication, countries in the world are all at introducing culture.After 1975, China introduces several seeds from the U.S. successively, and successful introduction, wherein three provinces in the northeast of China are for mainly to plant area.Radix Panacis Quinquefolii is famous and precious tonic herb, though genus equal with ginseng, drug effect and function exist very big-difference, though the two all has effect of tonifying QI to produce body fluid, Radix Panacis Quinquefolii taste cool in nature is sweet, and yin-nourishing is partial in tonifying Qi; Ginseng is warm in nature partially bitter, and tonifying Qi is relatively supporing yang.The morphological specificity of the two has many similarities, carefully do not distinguish and very easily obscure, the Radix Panacis Quinquefolii of especially domestic cultivation and cultivated ginseng are difficult to accurately differentiate according to morphological specificity, and in real work, we also run into and Radix Panacis Quinquefolii and ginseng are obscured and indistinguishable situation mutually.Because Radix Panacis Quinquefolii and ginseng price variance are comparatively large, proterties is similar with microscopic features, therefore market pretends to be the situation of Radix Panacis Quinquefolii more common with ginseng.Particularly in recent years because resource seriously reduces, price presents lasting ascendant trend always, and some adulterants and adulterant are occurred in a large number, and seriously confused normal market order, the two is obscured, and will have an immense impact on to curative effect.
Conventional Radix Panacis Quinquefolii discrimination method has Medicinal Materials Characters discriminating, microscopical identification and chemical identification method.Character identification mainly refers to from the macrofeature such as profile, size, color, epidermis characteristic, quality, cut surface character, smell of Radix Panacis Quinquefolii to judge kind.Although method is simple, experience impact is in this approach very large, subjective.Even if carried out very detailed character description, then judged by other people also more difficult.And normally incomplete as configuration of medicinal materials, therefore identify that difficulty is larger.Existing scholar differentiates American Ginseng and powder at present, microscopical identification for Radix Panacis Quinquefolii provides certain foundation, but the powder of Radix Panacis Quinquefolii does not have too large difference, just whether have between the shape of suberized cell, bast crack, the quantity of sparse, resin canal abscission ring of xylem vessel's arrangement, calcium oxalate cluster crystal number etc., only there is relative discriminating meaning, cannot the precise Identification true and false.Chemistry differentiates the qualitative reaction of mainly Radix Panacis Quinquefolii saponin Rb, Rg, Ro, but the kind of most of Radix Panacis Quinquefolii all contains Radix Panacis Quinquefolii saponin, characteristic is strong, thus only carries out qualitative reaction to Radix Panacis Quinquefolii saponin Rb, Rg, Ro, is difficult to the object reaching quality of medicinal material control.And in traditional Chinese medicine test method, generally adopt chemistry, infrared, the analytical procedure such as ultraviolet, liquid chromatography, to belong to the specificity that easily mixed medicinal material not of the same race differentiates together poor to equal for aforesaid method, and the method such as infrared, ultraviolet, liquid phase also exists again the drawbacks such as strong to the material specificity that chemical structure is similar, complex operation, expense are high, be difficult to meet Chinese medicinal materials, especially Chinese medicine easily mixed to the qualification of kind.Therefore applied morphology, histology and chemical composition identify to there is inconvenience, still can not differentiate fast and effectively Radix Panacis Quinquefolii.
Emerging molecular marking technique of nearly stage is that medicinal plant qualification and quality research provide a new approach.This technology develops rapidly and permeates with the field of Chinese medicines, merges, wherein AFLP(amplified fragment length polymorphism) in the qualification of Chinese medicinal materials (except mineral drug), have specificity strong, accuracy is high, the advantage such as reproducible, the imperfection from identification and assessment of Chinese medicines before molecular level solves on macroscopical identification level.Modern molecular biology progressively expands in the field of quality control of Chinese medicine, and method is perfect gradually, greatly accelerates the process of the modernization of Chinese medicine.
The method of Chinese Pharmacopoeia three kinds (Unibract Fritillary Bulb, long-nosed pit viper, Zaocys) applied molecular biology qualification.Our team relies on Jilin Technology Bureau innovation and development planning item and education department of Jilin Province to subsidize problem: " development & application of ginseng and preparation DNA detection technology thereof ", " gene biological information science mountain participates in Cultivated shen chloroplast genomic dna barcode authentication method ", develops DNA identification kit.Seminar utilizes mountain to join the advantage of cpDNA uniqueness, the genome portion sequence of application DNA cloning technology and sequencing technologies research mountain ginseng chloroplast DNA, the oligonucleotide fragment of design can participate in Cultivated shen in unique identification mountain, a kind of easy, quick, special qualification mountain ginseng patented technology (wild ginseng and Cultivated shen multiple PCR detection kit and authentication method, patent of invention: ZL201010118823.6 are successfully invented; 2014.6 authorize).Related ends is published in Chinese Pharmaceutical Journal " Ginseng under Forest and Cultivated shen amplified fragment length polymorphism finger print identification " [2013,48 (9): 677-679].
On this basis, the authenticate technology of the theory that seminar's applied molecular biology and identification and assessment of Chinese medicines merge mutually to Unibract Fritillary Bulb is studied, and high conservative property and the specificity of main appliable plant DNA are identified Unibract Fritillary Bulb.Cetyl trimethylammonium bromide method is adopted to extract genomic dna from Unibract Fritillary Bulb, utilize the relevant information in Genbank, according to the difference of the genomic dna sequence of Unibract Fritillary Bulb and other corresponding plants, choose specific gene sequences, application primerpremier5.0 primer-design software design Unibract Fritillary Bulb pair of primers, carry out PCR(polymerase chain reaction) amplification, and different according to specific restriction endonuclease restriction enzyme site in this amplified fragments, carry out endonuclease reaction.And foundation can be provided from molecular level to Unibract Fritillary Bulb qualification in this approach.Related ends is published in Chinese Pharmaceutical Journal " development of Unibract Fritillary Bulb DNA detection test kit and evaluation " [2014,49 (6): 501-504].
Domestic Cui Guang is red waits people by studying Radix Panacis Quinquefolii particular sequence site-tag, the method adopts single primer to carry out the APAPD(anchor primer amplification polymorphism DNA of pcr amplification according to known array) method, although stability and the specificity of amplification increase greatly, but PCR primer needs cloning and sequencing, analysis, cost is higher, be not suitable for rapid detection [Cui Guanghong, Huang Luqi, Tang Xiaojing, Deng: the novel method that acquisition ginseng, Radix Panacis Quinquefolii particular sequence site (STS) mark. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2007,32(11): 1012-1015.].The people such as Song Qinxin differentiate mononucleotide polymorphism site on ginseng and Radix Panacis Quinquefolii ITS gene by ALM-ASA, the method designs Auele Specific Primer for SNP site, the allele specific amplification utilizing adapter to connect mediation detects, high specificity, good stability, but need design dna adapter, process is loaded down with trivial details, be difficult to carry out applying [Song Qinxin as a kind of ordinary method, Zhang Xinyue, Bu Ying, mononucleotide polymorphism site on ginseng and Radix Panacis Quinquefolii ITS and 5.8s gene is differentiated Deng: ALM-ASA method. China Medicine University's journal, 2008, 39(3): 274-278.].Chen Zi easily waits people to utilize microsatellite marker to differentiate ginseng and Radix Panacis Quinquefolii, the method according to SSR (simple repeated sequence) molecule marker as microsatellite DNA, multipair primer need be designed for different SSR site to increase, detected by poly-propionic acid amide gel electrophoresis, although improve accuracy, but because primer is more, groping of PCR reaction conditions, the detection of poly-propionic acid amide gel electrophoresis is more time-consuming comparatively speaking, grasp for experiment condition also needs strict control, therefore in actual applications comparatively inconvenience [Chen Ziyi, Lv Xunan, Cheng Zhou, Deng: the application of microsatellite marker in ginseng and Radix Panacis Quinquefolii are differentiated. Fudan Journal (natural science edition), 2011, 50(2): 185-191.].Experiment is demonstrated some gene locuss of Radix Panacis Quinquefolii and can be differentiated by specific PCR and molecular marking technique.
On this basis, declared education department of Jilin Province and subsidized problem " Radix Panacis Quinquefolii DNA qualitative and quantitative detection technique ", set up the DNA detection technology of Chinese medicinal materials based on plant ribosome DNA, successfully develop Radix Panacis Quinquefolii Chinese medicinal materials DNA identification kit.
Rrna is present in intracellular a kind of organoid, rDNA (nuclearribosomeDNA, nrDNA) ITS sequence analysis has become the highly effective means of relation in the section of discussion on a molecular scale, subfamily, race, in botanical system growth with the research of classification, play vital role.In higher plant cell core, nrDNA is the rRNA gene (rDNA) in higher plant, is the tandem sequence unit highly repeated.18S, 5.8S and 26SrDNA are bound up, as a transcription unit.18S-26SrDNA basic structure is made up of 18SrDNA, 26SrD-NA, 5.8SrDNA and the gene the Internal Transcribed Spacer (ITS) between three, ITS1 and ITS2 two fragments that wherein ITS district is separated by 5.8SrDNA form.18S-26S core rDNAITS district repeats at Matrix attachment region camber, and there occurs synchronous evolution in site and between site by not waiting between exchange and these repeating units of transcription frequency, namely the sequence between different I TS copy is tending towards close or completely the same, and suffered selective pressure is less, substitution rate is very fast, rate of evolution is fast, adds that fragment length variation is very little, is applicable to very much carrying out various molecule manipulation.ITS region due to nrDNA has more nucleotide variation information, and length has good conservative property, differentiates between the kind being thus widely used in Chinese medicine, and qualification result is more accurate, reliable.
Patent PCR-based-AFLP method of the present invention is used for Radix Panacis Quinquefolii and differentiates.The ultimate principle of PCR-AFLP uses pcr amplification target DNA, and amplified production cuts into different size fragment with specificity endonuclease digestion again, directly differentiates in gel electrophoresis.Not homoallelic restriction enzyme site distribution is different, produces the DNA fragmentation band of different lengths.Technique substantially increases content and the relative specificity of target DNA, and method is easy, and the somatotype time is short.This patent uses this method the specificity of Radix Panacis Quinquefolii and Ginseng DNA sequence to be shown by special DNA band collection of illustrative plates, and then complete the discriminating of Radix Panacis Quinquefolii and ginseng, establish Radix Panacis Quinquefolii and Ginseng DNA PCR-AFLP authentication method, have the advantages that to save time, reduce costs and raise the efficiency, and provide scientific basis for the qualification of Chinese medicinal materials and analysis.
Summary of the invention
Patent of the present invention adopts bioinformatics technique to screen Radix Panacis Quinquefolii rDNA genome, utilize the relevant information in Genbank, according to the difference of the ribosomal dna sequence of Radix Panacis Quinquefolii and other corresponding plants, choose ITS gene order, application primerpremier5.0 design software, design can distinguish the specific cleavage site of Radix Panacis Quinquefolii and adulterant effectively, PCR-AFLP technology is utilized to add primer in a reaction system, amplified fragments is cut through enzyme, produces and has specific two object fragments.There is provided one accurately can differentiate Radix Panacis Quinquefolii and adulterant specific molecular markers, Radix Panacis Quinquefolii easy to use and adulterant PCR-AFLP detection kit, have the advantages that to save time, reduce costs and raise the efficiency, and provide that it is easy, quick, the reliable authentication method of detected result.
The object of the invention is to be realized by following technical scheme:
A kind of discriminating Radix Panacis Quinquefolii and adulterant PCR-AFLP detection kit, it is characterized in that, it comprises:
1. sample pretreatment solution
0.03g got by each sample, scrubs clean rear pretreatment liquid (mainly containing deionized water) and rinses well, drying at room temperature, with more than ultra violet lamp 30min, use scissors to shred to about 1 ~ 2mm by sample, be placed in centrifuge tube, weigh stand-by;
2. extracting genome DNA system
(a) lysate: 2%CTAB, 100mmol/LTris-HC1,30mmol/LEDTA, 2.5mmol/LNaCI,
3%PVP-40；
(b) precipitated liquid: chloroform-isoamyl alcohol (24:1);
(c) washings: 70% ethanol;
3.PCR reaction system
(a) identification reaction system: be totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, testing sample DNA2 ~ 4 μ L, remaining is bi-distilled water;
(b) positive control reaction system: be totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, Radix Panacis Quinquefolii DNA2 ~ 4 μ L, remaining is bi-distilled water;
(c) negative control reaction system: be totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, adulterant DNA2 ~ 4 μ L, remaining is bi-distilled water;
D () design response procedures will differentiate that each 100 μ L of total system of (a) identification reaction system of Radix Panacis Quinquefolii and adulterant PCR detection kit, (b) positive control reaction system and (c) negative control reaction system add in reaction tubes respectively, be placed in PCR reaction instrument (commercially available, disposable type) in undertaken by follow procedure: 94 DEG C of denaturation 5min, 94 DEG C of 30s, annealing temperature 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C extend 7min, 4 DEG C;
4. restriction endonuclease reaction system
A () enzyme cuts identification reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, treat that enzyme cuts sample P CR product D NA1 ~ 2 μ L, remaining is bi-distilled water;
B () enzyme cuts positive control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, Radix Panacis Quinquefolii PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
C () enzyme cuts negative control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, adulterant PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
D each 50 μ L of total system differentiating that (a) enzyme of Radix Panacis Quinquefolii and adulterant PCR detection kit cuts identification reaction system, (b) enzyme cuts positive control reaction system and (c) enzyme cuts negative control reaction system add in reaction tubes by () design response procedures respectively, be placed in thermostat water bath (commercially available, disposable type) and undertaken by follow procedure: 37 DEG C of enzymes cut 10-15min;
5. result observe system
(a) sepharose: 2% agarose;
(b) sample-loading buffer: 6 × loadingbuffer;
C the DNAMarker(of () standard molecular weight: 50bp ~ 500bp can select the standard molecular weight of other kind, require maximum more than 500bp, can not to contain 50bp and 100bp).
Radix Panacis Quinquefolii DNA identification method, is characterized in that, it comprises following steps:
1. detect Sample pretreatment
0.03g got by each sample, scrubs clean rear pretreatment liquid (mainly containing deionized water) and rinses well, drying at room temperature, with more than ultra violet lamp 30min, use scissors to shred to about 1 ~ 2mm by sample, be placed in centrifuge tube, weigh stand-by;
2. extracting genome DNA
A the cracking of () sample adds 1ml lysate in the sample through pre-treatment, be placed in water-bath, 65 DEG C of water-bath 30min, rotating speed 100r/min;
Sample 13000r/min4 DEG C of centrifugal 10min after the cracking of (b) precipitation, Aspirate supernatant, adds equal-volume precipitated liquid, fully mixes, and 13000r/min4 DEG C of centrifugal 2min, abandons supernatant, stay precipitation;
C () washing uses washings washing precipitation two to three times, drying precipitated, is dissolved in sterilizing distilled water ,-80 DEG C of preservations, as detection DNA profiling;
3.PCR design of primers and synthesis
(a) design Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, containing Radix Panacis Quinquefolii DNA sequence dna, following Primer:
Radix Panacis Quinquefolii Primer
5'-TTGCGCCCGAAGCCATTA-3'
5'-AGACACGGGAGGCATTATCC-3'
B () synthetic Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, adopts ABI3900 high-throughput synthesizer (entrusting the raw work in Shanghai to complete);
4. set up PCR reaction
A () identification reaction is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, testing sample DNA2 ~ 4 μ L, remaining is bi-distilled water;
B the reaction of () positive control is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, Radix Panacis Quinquefolii DNA2 ~ 4 μ L, remaining is bi-distilled water;
C the reaction of () negative control is totally 100 μ L, containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, adulterant DNA2 ~ 4 μ L, remaining is bi-distilled water;
D () design response procedures will differentiate that each 100 μ L of total system of (a) identification reaction system of Radix Panacis Quinquefolii and adulterant PCR detection kit, (b) positive control reaction system and (c) negative control reaction system add in reaction tubes, be placed in PCR reaction instrument (commercially available, disposable type) in undertaken by follow procedure: 94 DEG C of denaturation 5min, 94 DEG C of 30s, annealing temperature 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C extend 7min, 4 DEG C;
5. set up restriction endonuclease reaction
A () enzyme cuts identification reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, treat that enzyme cuts sample P CR product D NA1 ~ 2 μ L, remaining is bi-distilled water;
B () enzyme cuts positive control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, Radix Panacis Quinquefolii PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
C () enzyme cuts negative control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, adulterant PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
D each 50 μ L of total system differentiating that (a) enzyme of Radix Panacis Quinquefolii and adulterant PCR detection kit cuts identification reaction system, (b) enzyme cuts positive control reaction system and (c) enzyme cuts negative control reaction system add in reaction tubes by () design response procedures respectively, be placed in thermostat water bath (commercially available, disposable type) and undertaken by follow procedure: 37 DEG C of enzymes cut 10-15min;
6. result judges
Product 2.0% agarose gel electrophoresis, the DNAMarker(standard molecular weight of molecular weight 50bp ~ 500bp, commercially available) do reference, Labworks image acquisition and analysis software is (commercially available, can buy) observation and analysis result, occur that 100bp and 50bp band is certified products Radix Panacis Quinquefolii simultaneously, only occur one or do not occur being adulterant Radix Panacis Quinquefolii (Fig. 1).
The present invention differentiates that Radix Panacis Quinquefolii and adulterant identification kit utilize to differentiate Radix Panacis Quinquefolii and adulterant rDNA restriction endonuclease specific position, accurately can distinguish the specificity differentiating Radix Panacis Quinquefolii and adulterant; The PCR-AFLP authentication method set up has the advantages such as easy, quick, detected result is reliable.
Accompanying drawing explanation
Fig. 1 is Radix Panacis Quinquefolii test kit detected result schematic diagram:
Wherein: be 1. standard molecular weight; 2-10. is certified products Radix Panacis Quinquefolii result, amplifies 100bp and 50bp simultaneously; 11. is positive control, amplifies 100bp and 50bp simultaneously; 12-16. is negative findings, does not occur or only occurs 1 amplified production; 17. is negative control, does not occur amplified production;
Fig. 2 embodiment one Radix Panacis Quinquefolii test kit detected result schematic diagram:
Wherein: be 1. standard molecular weight; 2-7. is certified products Radix Panacis Quinquefolii result, amplifies 100bp and 50bp simultaneously; 8. be positive control, amplify 100bp and 50bp simultaneously; 9-13. is negative findings, does not occur or only occurs 1 amplified production; 14. is negative control, does not occur amplified production;
Fig. 3 embodiment two carries out qualification result schematic diagram to commercially available American ginseng medicine:
Wherein: be 1. standard molecular weight; 12. is positive control, amplifies 100bp and 50bp; 6, be 9. certified products Radix Panacis Quinquefolii result, amplify 100bp and 50bp; 2-5: ginseng; 7-8: ginseng; 10-11: ginseng; 13-14: ginseng; 15-16. is negative findings, does not occur amplified production; 17. is negative control, does not occur amplified production.
Embodiment
Below in conjunction with embodiment, the invention will be further described, below the embodiment only not limitation of the present invention for illustration of the present invention.
embodiment 1
1. detect Sample pretreatment
Detect (two kinds, sample, 1 is Radix Panacis Quinquefolii certified products, and 2 is ginseng, Radix Panacis Quinquefolii adulterant, there is provided by Nat'l Pharmaceutical & Biological Products Control Institute and identify), get 0.03g for every part, scrub clean rear pretreatment liquid and rinse well, drying at room temperature, with more than ultra violet lamp 30min, use scissors to shred to about 1 ~ 2mm by sample, be placed in centrifuge tube, weigh stand-by;
2. extracting genome DNA
A the cracking of () sample adds 1ml lysate in the sample through pre-treatment, be placed in water-bath, 65 DEG C of water-bath 30min, rotating speed 100r/min;
Sample 13000r/min4 DEG C of centrifugal 10min after the cracking of (b) precipitation, Aspirate supernatant, adds equal-volume precipitated liquid, fully mixes, and 13000r/min4 DEG C of centrifugal 2min, abandons supernatant, stay precipitation;
C () washing uses washings washing precipitation two to three times, drying precipitated, is dissolved in sterilizing distilled water ,-80 DEG C of preservations, as detection DNA profiling;
3.PCR design of primers and synthesis
(a) design Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, containing Radix Panacis Quinquefolii DNA sequence dna, following Primer:
Radix Panacis Quinquefolii Primer
5'-TTGCGCCCGAAGCCATTA-3'
5'-AGACACGGGAGGCATTATCC-3'
B () synthetic Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, adopts ABI3900 high-throughput synthesizer (entrusting the raw work in Shanghai to complete);
4. set up PCR reaction
A () identification reaction is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, testing sample DNA2 ~ 4 μ L, remaining is bi-distilled water;
B the reaction of () positive control is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, Radix Panacis Quinquefolii DNA2 ~ 4 μ L, remaining is bi-distilled water;
C the reaction of () negative control is totally 100 μ L, containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, adulterant DNA2 ~ 4 μ L, remaining is bi-distilled water;
D () design response procedures will differentiate that each 100 μ L of total system of (a) identification reaction system of Radix Panacis Quinquefolii and adulterant PCR detection kit, (b) positive control reaction system and (c) negative control reaction system add in reaction tubes, be placed in PCR reaction instrument (commercially available, disposable type) in undertaken by follow procedure: 94 DEG C of denaturation 5min, 94 DEG C of 30s, annealing temperature 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C extend 7min, 4 DEG C;
5. set up restriction endonuclease reaction
A () enzyme cuts identification reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, treat that enzyme cuts sample P CR product D NA1 ~ 2 μ L, remaining is bi-distilled water;
B () enzyme cuts positive control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, Radix Panacis Quinquefolii PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
C () enzyme cuts negative control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, adulterant PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
D each 50 μ L of total system differentiating that (a) enzyme of Radix Panacis Quinquefolii and adulterant PCR detection kit cuts identification reaction system, (b) enzyme cuts positive control reaction system and (c) enzyme cuts negative control reaction system add in reaction tubes by () design response procedures respectively, be placed in thermostat water bath (commercially available, disposable type) and undertaken by follow procedure: 37 DEG C of enzymes cut 10-15min;
6. result judges
PCR primer 2.0% agarose gel electrophoresis, the DNAMarker of standard molecular weight 50bp ~ 500bp does reference, Labworks image acquisition and analysis software observation and analysis result (Fig. 2).
embodiment 2 is identified commercially available American ginseng medicine
1. material
5 kinds, commercially available Radix Panacis Quinquefolii sample, Radix Panacis Quinquefolii certified products a kind (provided by drug inspection office, Jilin and identify)
2. method
2.1 detect Sample pretreatment every part detection sample gets 0.03g, scrubs clean rear pretreatment liquid and rinses well, drying at room temperature, with more than ultra violet lamp 30min, use scissors to shred to about 1 ~ 2mm by sample, be placed in centrifuge tube, weigh stand-by;
2.2 extracting genome DNA
A the cracking of () sample adds 1ml lysate in the sample through pre-treatment, be placed in water-bath, 65 DEG C of water-bath 30min, rotating speed 100r/min;
Sample 13000r/min4 DEG C of centrifugal 10min after the cracking of (b) precipitation, Aspirate supernatant, adds equal-volume precipitated liquid, fully mixes, and 13000r/min4 DEG C of centrifugal 2min, abandons supernatant, stay precipitation;
C () washing uses washings washing precipitation two to three times, drying precipitated, is dissolved in sterilizing distilled water ,-80 DEG C of preservations, as detection DNA profiling;
2.3PCR design of primers and synthesis
(a) design Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, containing Radix Panacis Quinquefolii DNA sequence dna, following Primer:
Radix Panacis Quinquefolii Primer
5'-TTGCGCCCGAAGCCATTA-3'
5'-AGACACGGGAGGCATTATCC-3'
B () synthetic Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, adopts ABI3900 high-throughput synthesizer (entrusting the raw work in Shanghai to complete);
2.4 set up PCR reaction
A () identification reaction is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, testing sample DNA2 ~ 4 μ L, remaining is bi-distilled water;
B the reaction of () positive control is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, Radix Panacis Quinquefolii DNA2 ~ 4 μ L, remaining is bi-distilled water;
C the reaction of () negative control is totally 100 μ L, containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, adulterant DNA2 ~ 4 μ L, remaining is bi-distilled water;
D () design response procedures will differentiate that each 100 μ L of total system of (a) identification reaction system of Radix Panacis Quinquefolii and adulterant PCR detection kit, (b) positive control reaction system and (c) negative control reaction system add in reaction tubes, be placed in PCR reaction instrument (commercially available, disposable type) in undertaken by follow procedure: 94 DEG C of denaturation 5min, 94 DEG C of 30s, annealing temperature 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C extend 7min, 4 DEG C;
2.5 set up restriction endonuclease reaction
A () enzyme cuts identification reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, treat that enzyme cuts sample P CR product D NA1 ~ 2 μ L, remaining is bi-distilled water;
B () enzyme cuts positive control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, Radix Panacis Quinquefolii PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
C () enzyme cuts negative control reaction system: be totally 50 μ L, and wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, adulterant PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
D each 50 μ L of total system differentiating that (a) enzyme of Radix Panacis Quinquefolii and adulterant PCR detection kit cuts identification reaction system, (b) enzyme cuts positive control reaction system and (c) enzyme cuts negative control reaction system add in reaction tubes by () design response procedures respectively, be placed in thermostat water bath (commercially available, disposable type) and undertaken by follow procedure: 37 DEG C of enzymes cut 10-15min;
3. result judges
PCR primer 2.0% agarose gel electrophoresis, the DNAMarker of standard molecular weight 50bp ~ 500bp does reference, Labworks image acquisition and analysis software observation and analysis result (Fig. 3).
Claims (5)
1. a Radix Panacis Quinquefolii DNA detection test kit, is characterized in that, it comprises sample pretreatment solution, extracting genome DNA system, PCR reaction system, restriction endonuclease reaction system, result observe system:
(1) sample pretreatment solution
0.03g got by each sample, scrubs clean rear pretreatment liquid (mainly containing deionized water) and rinses well, drying at room temperature, with more than ultra violet lamp 30min, use scissors to shred to about 1 ~ 2mm by sample, be placed in centrifuge tube, weigh stand-by;
(2) extracting genome DNA system
(a) lysate: 2%CTAB, 100mmol/LTris-HC1,30mmol/LEDTA, 2.5mmol/LNaCI,
3%PVP-40；
(b) precipitated liquid: chloroform-isoamyl alcohol (24:1);
(c) washings: 70% ethanol;
(3) PCR reaction system
(a) identification reaction system: be totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, testing sample DNA2 ~ 4 μ L, remaining is bi-distilled water;
(b) positive control reaction system: be totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, Radix Panacis Quinquefolii DNA2 ~ 4 μ L, remaining is bi-distilled water;
(c) negative control reaction system: be totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, adulterant DNA2 ~ 4 μ L, remaining is bi-distilled water;
(4) restriction endonuclease reaction system
(a) identification reaction system: be totally 50 μ L, wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, treat that enzyme cuts sample P CR product D NA1 ~ 2 μ L, remaining is bi-distilled water;
(b) positive control reaction system: be totally 50 μ L, wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, Radix Panacis Quinquefolii PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
(c) negative control reaction system: be totally 50 μ L, wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, adulterant PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
(5) result observe system
(a) sepharose: 2% agarose;
(b) sample-loading buffer: 6 × loadingbuffer;
C the DNAMarker(of () standard molecular weight: 50bp ~ 500bp can select the standard molecular weight of other kind, require maximum more than 500bp, can not to contain 50bp and 100bp).
2. Radix Panacis Quinquefolii DNA identification method, is characterized in that, it comprise detect Sample pretreatment, extracting genome DNA, PCR primer Design and synthesis, set up PCR reaction, restriction endonuclease reaction, result judges six steps:
(1) Sample pretreatment is detected
0.03g got by each sample, scrubs clean rear pretreatment liquid and rinses well, drying at room temperature, with more than ultra violet lamp 30min, uses scissors to shred to about 1 ~ 2mm by sample, is placed in centrifuge tube, weighs stand-by;
(2) extracting genome DNA
A the cracking of () sample adds 1ml lysate in the sample through pre-treatment, be placed in water-bath, 65 DEG C of water-bath 30min, rotating speed 100r/min;
Sample 13000r/min4 DEG C of centrifugal 10min after the cracking of (b) precipitation, Aspirate supernatant, adds equal-volume precipitated liquid, fully mixes, and 13000r/min4 DEG C of centrifugal 2min, abandons supernatant, stay precipitation;
C () washing uses washings washing precipitation two to three times, drying precipitated, is dissolved in sterilizing distilled water ,-80 DEG C of preservations, as detection DNA profiling;
(3) PCR primer Design and synthesis
(a) design Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, containing Radix Panacis Quinquefolii DNA sequence dna, following Primer:
Radix Panacis Quinquefolii Primer
5'-TTGCGCCCGAAGCCATTA-3'
5'-AGACACGGGAGGCATTATCC-3'
B () synthetic Radix Panacis Quinquefolii rDNA a pair specific oligonucleotide primers, adopts ABI3900 high-throughput synthesizer (entrusting the raw work in Shanghai to complete);
(4) PCR reaction is set up
A () identification reaction is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, 0.1mM primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, testing sample DNA2 ~ 4 μ L, remaining is bi-distilled water;
B the reaction of () positive control is totally 100 μ L, wherein containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, Radix Panacis Quinquefolii DNA2 ~ 4 μ L, remaining is bi-distilled water;
C the reaction of () negative control is totally 100 μ L, containing damping fluid 8 μ L, 12.5mMdNTP4 ~ 8 μ L, and 0.1mM Radix Panacis Quinquefolii primer 2 ~ 4 μ L, Taq DNA polymerase 4 ~ 8 μ L, adulterant DNA2 ~ 4 μ L, remaining is bi-distilled water;
D () design response procedures will differentiate that each 100 μ L of total system of (a) identification reaction system of Radix Panacis Quinquefolii and adulterant PCR detection kit, (b) positive control reaction system and (c) negative control reaction system add in reaction tubes, be placed in PCR reaction instrument (commercially available, disposable type) in undertaken by follow procedure: 94 DEG C of denaturation 5min, 94 DEG C of 30s, annealing temperature 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C extend 7min, 4 DEG C;
(5) restriction endonuclease reaction system is set up
(a) identification reaction system: be totally 50 μ L, wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, treat that enzyme cuts sample P CR product D NA1 ~ 2 μ L, remaining is bi-distilled water;
(b) positive control reaction system: be totally 50 μ L, wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, Radix Panacis Quinquefolii PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
(c) negative control reaction system: be totally 50 μ L, wherein containing damping fluid 5 μ L, restriction endonuclease Hinf I 1 ~ 2 μ L, adulterant PCR primer DNA1 ~ 2 μ L, remaining is bi-distilled water;
D each 50 μ L of total system differentiating that (a) enzyme of Radix Panacis Quinquefolii and adulterant PCR detection kit cuts identification reaction system, (b) enzyme cuts positive control reaction system and (c) enzyme cuts negative control reaction system add in reaction tubes by () design response procedures respectively, be placed in thermostat water bath (commercially available, disposable type) and undertaken by follow procedure: 37 DEG C of enzymes cut 10-15min;
(6) result judges
Product 2.0% agarose gel electrophoresis, the DNAMarker(standard molecular weight of molecular weight 50bp ~ 500bp, commercially available) do reference, Labworks image acquisition and analysis software is (commercially available, can buy) observation and analysis result, occur that 100bp and 50bp band is certified products Radix Panacis Quinquefolii simultaneously, only occur one or do not occur being adulterant Radix Panacis Quinquefolii.
3., as claim 1 and a kind of Radix Panacis Quinquefolii DNA detection test kit according to claim 2 and authentication method, it is characterized in that, described Radix Panacis Quinquefolii DNA detection test kit and authentication method are Canadian Radix Panacis Quinquefolii.
4. the authentication method of Radix Panacis Quinquefolii DNA detection test kit as claimed in claim 2, it is characterized in that, result qualification utilizes comparative sample amplified fragments DNA after enzyme is cut, occur 50 ~ 100bp two bar segment, carries out authenticity according to occurring that criterion is completely the same to Radix Panacis Quinquefolii sample.
5. claim 1 and a kind of Radix Panacis Quinquefolii DNA detection test kit according to claim 2 and an authentication method, can be applicable to the authenticity of Radix Panacis Quinquefolii sample.
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| CN105505928A (en) * | 2016-02-04 | 2016-04-20 | 杭州科兴生物化工有限公司 | Nucleotide sequence, specific primer and method for identifying physalis angulata |
| CN105624291A (en) * | 2016-01-19 | 2016-06-01 | 中国食品药品检定研究院 | Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate |
| CN109385487A (en) * | 2018-09-11 | 2019-02-26 | 浙江省食品药品检验研究院 | The recombinase-mediated amplification Constant Temperature Detection method and kit of your detail herbal medicine American Ginseng |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624291A (en) * | 2016-01-19 | 2016-06-01 | 中国食品药品检定研究院 | Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate |
| CN105624291B (en) * | 2016-01-19 | 2019-01-11 | 中国食品药品检定研究院 | It whether there is Araliaceae ingredient in test sample and whether mix pseudo- method |
| CN105505928A (en) * | 2016-02-04 | 2016-04-20 | 杭州科兴生物化工有限公司 | Nucleotide sequence, specific primer and method for identifying physalis angulata |
| CN105505928B (en) * | 2016-02-04 | 2018-07-31 | 杭州科兴生物化工有限公司 | A kind of nucleotide sequence, specific primer and method differentiating Ku Zhi |
| CN109385487A (en) * | 2018-09-11 | 2019-02-26 | 浙江省食品药品检验研究院 | The recombinase-mediated amplification Constant Temperature Detection method and kit of your detail herbal medicine American Ginseng |
| CN109385487B (en) * | 2018-09-11 | 2022-04-01 | 浙江省食品药品检验研究院 | Recombinase-mediated amplification isothermal detection method and kit for American ginseng as Chinese medicinal herb |
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