CN104611424A - PCR-RFLP method for rapidly identifying radix tetrastigme and various counterfeits and adulterants of radix tetrastigme - Google Patents
PCR-RFLP method for rapidly identifying radix tetrastigme and various counterfeits and adulterants of radix tetrastigme Download PDFInfo
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Abstract
The invention belongs to the field of molecular markers and discloses a PCR-RFLP method for rapidly identifying radix tetrastigme and various counterfeits and adulterants of the radix tetrastigme. The method comprises the following steps: 1, extracting the NDA of the medicinal material (namely the radix tetrastigme); 2, carrying out PCR amplification by forward and reverse primers of internal transcribed spacer ITS2 sequences of a pair of amplified ribosomal DNAs; 3, digesting the PCR product by restriction enzyme NCO I; 4, carrying out agarose gel electrophoresis analysis. After the enzyme digestion of amplified products of the DNA of the radix tetrastigme, two DNA fragments with sizes about 335 bp and 200 bp are generated, and the various counterfeits and adulterants are not identified by the NCO I enzyme. By utilizing the differences between the DNA sequences of radix tetrastigme and the counterfeits and adulterants of the radix tetrastigme, a quick, convenient and reliable PCR-RFLP identification method is established and used for identifying whether counterfeits and adulterants are mixed in the radix tetrastigme or not, so that the technical problem that the truth and false cannot be identified by sensory or physical and chemical analysis methods is solved, and the medication safety of the radix tetrastigme is ensured.
Description
Technical field
The invention belongs to molecular marking technique field, particularly relate to a kind of PCR-RFLP method of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) and pseudo-mixed product thereof.
Background technology
Radix Apioris Fortunei (Radix Lespedezae Buergeri) (Tetrastigma hemsleyanum) has another name called gold thread hoist, it is Vitaceae Tetrastigma per nnial herb, its underground block root is main medicinal part, can be used for treating the various diseases such as high heat, hepatitis, rheumatic arthritis and viral meningitis, is also antitumor common drug.This plant wild resource is endangered at present, and it is very harsh to the requirement of growing environment, and artificial culture difficulty is large, and development and utilization is extremely restricted.Due to scarcity of resources, demand is large, Radix Apioris Fortunei (Radix Lespedezae Buergeri) price constantly rises, medicinal material market there is Root of Fortune Apios, the large multiple puppet such as sinomenium acutum, the sub-root of the rhizome of Chinese monkshood mixed product, quality of medicinal material extremely unstable, even there occurs patient and take the poisoning phenomenon of the fake and inferior Radix Apioris Fortunei (Radix Lespedezae Buergeri) medicinal material that is mixed with the sub-root of the rhizome of Chinese monkshood.For ensureing Chinese medicinal material quality and drug safety, extremely important to the qualification of the former plant of Radix Apioris Fortunei (Radix Lespedezae Buergeri) medicinal material base.
What the traditional identification of means of current Chinese medicinal materials adopted mostly is according to its formalness and microstructure, but because the most formalness of plant sub-root class medicinal material is similar, and after place of production roughing and process of preparing, be difficult to shape and the color of finding out itself, microscopic features also have destruction in various degree, cause qualification difficulty.Chinese medicine assortment matter Molecular Identification technology is not by growth and development stage, for the impact trying position, envrionment conditions, DNA sequence dna is directly utilized to carry out the qualification of species, there is unique accuracy and repeatability, and the primer is few, stability and versatility high, experimentation stdn, fast simple to operate.Chinese patent application 201310373809.4 discloses a kind of method identifying medicinal plant Radix Apioris Fortunei (Radix Lespedezae Buergeri), and this is the Radix Apioris Fortunei (Radix Lespedezae Buergeri) molecular assay method based on ISSR molecule marker.By a pair primer amplified, whether this technology successfully can differentiate that the common puppet of Radix Apioris Fortunei (Radix Lespedezae Buergeri) mixes product, but some nearly edge species also has positive findings to it, discrimination is not high, and do not verify the common puppets such as large sinomenium acutum, the sub-root of the rhizome of Chinese monkshood mix product qualification in suitability, need in addition to synthesize a pair Auele Specific Primer in advance.The Internal Transcribed Spacer (the internal transcribed spacer of plant core rDNA, ITS) comprise ITS1 and ITS2 two fragments of separating by 5.8SrDNA, pcr amplification has universal primer sequence, and it is simple to check order, ITS sequence variation is very fast, can provide more rich variant sites and informative site.The discriminating that PCR-RFLP is used for sibling species has some successful example, and have the advantages such as quick, simple, accurate, but this discriminating is to different species, the kind of primer used, pcr amplification condition, restriction enzyme and discrimination method are not identical.Still do not apply PCR-RFLP technology carries out molecular identificalion report to Radix Apioris Fortunei (Radix Lespedezae Buergeri) at present.
DNA bar code technology is by analyzing the DNA short-movie section of a standard goal gene, thus carries out the technology of species identification quickly and accurately.DNA bar code concept is first proposed by Canadian zoologist Paul Hebert for 2003, utilize the relatively short stranded DNA fragment having enough variations and easily amplification, a kind of new biological status identification system set up in specificity in kind and the diversity between planting, thus achieve and species are identified fast and accurately and identifies.The ideal sequence of DNA bar code has 3 basic judging criterions: (1) sequence variations level is suitable for, and different plant species can be distinguished from each other, and intraspecific variablity is less simultaneously; (2) the sequence high conservative at two ends, variable region, can design the universal primer of stable amplification in numerous species; (3) extension increasing sequence is as far as possible short, and what a reaction can complete order-checking.
DNA bar code is not by growth and development stage, for the impact trying position, envrionment conditions, DNA sequence dna is directly utilized to carry out the qualification of species, there is unique accuracy and repeatability, and the primer is few, stability and versatility high, experimentation stdn, and share by database realizing and DNA sequence dna can be converted to two-dimensional bar image applications in practice.Focus candidate sequence has rbcL, matK, psbA-trnH, ITS2 etc. so far, but does not also obtain extensively general DNA of plants bar code standards fragment.The research such as Fu Yuansen finds that ITS, bcL+matK+ITS are qualification Radix Apioris Fortunei (Radix Lespedezae Buergeri) and the best barcode of other Tetrastigma plant, Radix Apioris Fortunei (Radix Lespedezae Buergeri) can by ITS, ITs+rbcL, ITS+matK, ITs+trnH-psbA, matK+rbcL; BcL+trnH-psbA and 2+x combines barcode precise Identification (" Idioplasm identification of medicinal plant Radix Apioris Fortunei (Radix Lespedezae Buergeri) and tissue culture fast-propagation research ", Zhejiang University's master thesis, 2011).First the method needs to extract DNA of plants, then carries out pcr amplification, by could judge the kind of this plant to amplified production order-checking and comparison, is not suitable for Rapid identification.Only have other Tetrastigma vegetable wool branch precipice to climb the ITS2 gene order of rattan, shoulder pole rattan etc. in current Genbank, also do not have the related gene sequence of Radix Apioris Fortunei (Radix Lespedezae Buergeri) ITS2, therefore this area needs the rapid identification method setting up Chinese medicine Radix Apioris Fortunei (Radix Lespedezae Buergeri) especially.
PCR-RFLP method provided by the invention can realize the Rapid identification of Radix Apioris Fortunei (Radix Lespedezae Buergeri), improves the accuracy of qualification result.
Summary of the invention
In order to set up the rapid identification method of Chinese medicine Radix Apioris Fortunei (Radix Lespedezae Buergeri) and pseudo-mixed product thereof, the DNA bar code realizing Chinese medicine Radix Apioris Fortunei (Radix Lespedezae Buergeri) detects, and the present invention realizes object of the present invention by the following method.
Establish a kind of PCR-RFLP method that quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) and multiple puppet thereof mix product, comprise and extract medicinal material DNA and agarose gel electrophoresis analysis, carry out pcr amplification with forward and reverse primer of the Internal Transcribed Spacer (ITS2) sequence of pair for amplification rDNA and use digestion with restriction enzyme PCR primer, judge the true and false of medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) according to enzymic digestion product agarose gel electrophoresis analytical results and whether be mixed with pseudo-mixed product.
Pcr amplification gene of the present invention is the Internal Transcribed Spacer (ITS2) of rDNA, has the gene order described in SEQID NO.1.
Pcr amplification primer of the present invention has SEQID NO.2 and the nucleotide sequence described in SEQID NO.3.
The restriction enzyme used is Nco I, and its recognition sequence is CCATGG.
The gene segment size of pcr amplification is 400 ~ 600bp, wherein preferably 490 ~ 550bp, and particularly preferred is 533bp.
After digestion with restriction enzyme there is 335bp and 200bp band in the analysis of medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample electrophoresis, and DNA cloning product being not the limited property endonuclease digestion of its pseudo-mixed product.
PCR reaction conditions of the present invention is: 95 DEG C of denaturation 5min; Then enter circulation, 95 DEG C of sex change 30 seconds, 45 ~ 55 DEG C of annealing 30 seconds, 72 DEG C extend 40 seconds, 25 ~ 35 circulations; Last 72 DEG C extend 10 minutes.Wherein preferred annealing temperature is 55 DEG C, and preferred PCR circulation is 35.
The digestion with restriction enzyme reaction conditions of pcr amplification product is: 10 μ L PCR primer, 2 μ L 10 × enzyme cutting buffering liquids, 8 μ L sterilizing ultrapure waters, 5 ~ 25U restriction enzyme, 20 ~ 37 DEG C of constant temperature 2 ~ 8 hours, wherein preferred restriction enzyme add-on is 20U, and temperature of reaction 37 DEG C, the time is 4 hours.
The PCR-RFLP method of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) provided by the invention and pseudo-mixed product thereof can realize the Rapid identification of Radix Apioris Fortunei (Radix Lespedezae Buergeri), and improve the accuracy of qualification result, compared with prior art tool has the following advantages and positively effect:
1, method provided by the present invention can realize the authenticity of Radix Apioris Fortunei (Radix Lespedezae Buergeri) fast and effectively.Compared with traditional Morphological Identification method and other molecule marking method, effectively can shorten the qualification time of Radix Apioris Fortunei (Radix Lespedezae Buergeri), adopt the inventive method qualification needs 6 ~ 8h time, reach the object of Rapid identification, because utilize DNA sequence dna difference to carry out the qualification of species, there is unique accuracy and repeatability, and the primer is few, stability and versatility high, add the resolving power to nearly edge species, considerably increase the accuracy of qualification.
2, the invention provides the reaction conditions of a pair PCR universal primer needed for qualification, a kind of restriction enzyme and endonuclease reaction condition, compared with existing Radix Apioris Fortunei (Radix Lespedezae Buergeri) molecular identification method, the present invention belongs to nearly edge species together to the mixed product of all Radix Apioris Fortunei (Radix Lespedezae Buergeri) puppets that market finds at present and its and all can realize effectively differentiating.
Accompanying drawing explanation
Fig. 1 is that agarose gel electrophoresis analyzes PCR primer result;
Swimming lane 1:DNA Marker, 400bp, 500bp, 700bp, 900bp, 1500bp from top to bottom;
Swimming lane 2: from Lishui of Zhejiang Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 3: from plant resources in Wenling Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 4: from Xiangshan of Zhejiang Province Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 5: from Guangxi Tianlin County Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 6: to work in peace and contentment Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample from Guangxi; Swimming lane 7: from Shaoyang, Hunan Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 8: from yiyang, hunan Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 9: from Yuanling, Hunan Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 10: from Wan An, Jiangxi Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 11: from Shangrao, Jiangxi Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 12: Root of Fortune Apios sample; Swimming lane 13: rhizome of Chinese monkshood sample;
Swimming lane 14: large sinomenium acutum sample; Swimming lane 15: Radix Apioris Fortunei (Radix Lespedezae Buergeri) kindred plant shoulder pole rattan sample;
Swimming lane 16: Radix Apioris Fortunei (Radix Lespedezae Buergeri) kindred plant Mao Zhiya climbs rattan sample.
Fig. 2 is Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample P CR product order-checking partial sequence;
Fig. 3 is that agarose gel electrophoresis analyzes digestion with restriction enzyme PCR primer result;
Swimming lane 1:DNA Marker, 400bp, 400bp, 500bp, 700bp, 900bp, 1500bp from top to bottom;
Swimming lane 2: from Lishui of Zhejiang Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 3: from plant resources in Wenling Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 4: from Xiangshan of Zhejiang Province Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 5: from Guangxi Tianlin County Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 6: to work in peace and contentment Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample from Guangxi; Swimming lane 7: from Shaoyang, Hunan Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 8: from yiyang, hunan Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 9: from Yuanling, Hunan Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 10: from Wan An, Jiangxi Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample; Swimming lane 11: from Shangrao, Jiangxi Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample;
Swimming lane 12: Root of Fortune Apios sample; Swimming lane 13: rhizome of Chinese monkshood sample;
Swimming lane 14: large sinomenium acutum sample; Swimming lane 15: Radix Apioris Fortunei (Radix Lespedezae Buergeri) kindred plant shoulder pole rattan sample;
Swimming lane 16: Radix Apioris Fortunei (Radix Lespedezae Buergeri) kindred plant Mao Zhiya climbs rattan sample.
Fig. 4 is the PCR-RFLP identity process figure of medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) and pseudo-mixed product thereof.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but and non-limiting the present invention.
The extraction of embodiment one medicinal material DNA
Collect Radix Apioris Fortunei (Radix Lespedezae Buergeri) and various medicinal material sample, adopt and extract general CTAB (cetyl trimethylammonium bromide) method of DNA of plants or use plant genome DNA extraction agent box to extract sample DNA.
1, sample thief is about 0.5g in mortar, adds liquid nitrogen and is ground into fine powder, carefully proceeds in 2ml centrifuge tube;
2,2%CTAB extract (the CTAB 4g of 700 μ L, 65 DEG C of preheatings is added, NaCl 16.364g, 1M Tris-HCl 20ml (pH8.0), 0.5M EDTA 8ml, first dissolves with 70ml ddH2O, then is settled to 200ml sterilizing, 0.2-1% beta-mercaptoethanol is added) after cooling, after abundant mixing, be incubated 40min in 65 DEG C of water-baths, frequently jolt gently therebetween;
3, take out centrifuge tube, the centrifugal 10min of 12000r/min, with micropipet sucking-off supernatant liquor, puts into clean centrifuge tube, adds isopyknic chloroform-isoamyl alcohol (24:1), fully mixes, the centrifugal 10min of 12000r/min;
4, supernatant liquor is transferred in another centrifuge tube, add chloroform-isoamyl alcohol (24:1), repeat to wash 2 ~ 3 times with method;
5, get supernatant, add the Virahol of 0.6 times of volume-20 DEG C of precoolings, place-20 DEG C of 2h;
6, the centrifugal 10min of 12000r/min, gets precipitation, respectively washs once respectively with 75% ethanol and dehydrated alcohol, air-dry rear TE (10mM Tris-HCl, 1mM EDTA, PH8.0) buffer solution;
7, DNA sample quality inspection: get 5 μ L DNA sample, 1.0% agarose gel electrophoresis, voltage 120V, electrophoresis time is about 30min, dyes gel 0.5 μ g/mL EB after electrophoresis terminates, detects DNA band under ultraviolet gel imaging system.
The pcr amplification of embodiment two medicinal material DNA
1, according to the Internal Transcribed Spacer design primer sequence of rDNA, forward primer has the nucleotide sequence described in SEQID NO.1: 5 ' ATGCGATACTTGGTGTGAAT 3 '; Reverse primer has the nucleotide sequence described in SEQID NO.2: 5 ' GACGCTTCTCCAGACTACAAT 3 '.
2, pcr amplification is 25 μ L reaction systems, consists of the following composition:
3, amplification program: 95 DEG C of denaturation 5min; Then enter circulation, 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 40sec, 35 circulations; Finally extend 72 DEG C of 10min.
4, pcr amplification product quality inspection: get 5 μ L DNA sample, 2% agarose gel electrophoresis, voltage 120V, electrophoresis time is about 30min, dyeed by gel 0.5 μ g/mL EB after electrophoresis terminates, under ultraviolet gel imaging system, detect DNA band, result is as Fig. 1.
The sequential analysis of embodiment three PCR primer
Get PCR primer and carry out DNA sequencing, adopt two-way order-checking, part order-checking peak figure is shown in Fig. 2, and sequencing results has the nucleotide sequence described in SEQID NO.3, this sequence does not comprise 5 ' and the primer sequence that 3 ' holds.Determine from Lishui of Zhejiang, Wenling, Xiangshan, Guangxi Tianlin County, to work in peace and contentment, Shaoyang, Hunan, Yiyang, Yuanling, the Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample in Wan An, Jiangxi, Shangrao and other Closely related variety Root of Fortune Apios, the rhizome of Chinese monkshood, large sinomenium acutum, Radix Apioris Fortunei (Radix Lespedezae Buergeri) kindred plant shoulder pole rattan, Mao Zhiya climb the pcr amplification product sequence of rattan, through sequential analysis, Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample sequence has restriction enzyme Nco I recognition sequence CCATGG, and other nearly edge samples not this enzyme recognition site near 315bp.
Embodiment four restriction enzyme digestion digestion reaction
Following composition is added in 0.5ml sterile centrifugation tube:
37 DEG C of constant temperature enzymes cut 4 hours, wherein the consisting of of 10X damping fluid: 10mM Tris-HCL, 100mM KCL, 1mM EDTA, 1mM DTT, 0.2mg/mL BSA, 50% glycerine.
The analysis of embodiment five agarose gel electrophoresis
The endonuclease reaction product getting appropriate step 4 is separated by 2% agarose electrophoresis, voltage 120V, electrophoresis time is about 40min, dyeed by gel 0.5 μ g/mL EB after electrophoresis terminates, if sample has two bands, size is about 335bp and 200bp respectively, the results are shown in Figure 3, then test sample is Radix Apioris Fortunei (Radix Lespedezae Buergeri), if still for the band of about 500bp or band molecular weight are different from above-mentioned size, then and trial-product non-Radix Apioris Fortunei (Radix Lespedezae Buergeri) source or mix and has other pseudo-mixed product.
The PCR-RFLP qualification of embodiment six medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) and pseudo-mixed product thereof
The PCR-RFLP qualification of medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) and pseudo-mixed product thereof is carried out by the qualification program schema of Fig. 4, separation and Extraction DNA from plant tissue to be measured, with this DNA for template pair of primers: forward primer has SEQ ID NO.1 sequence, reverse primer has SEQ ID NO.2 sequence, add each reactive component by table one, go out ITS by pcr amplification
2gene, amplification program is: 95 DEG C of denaturation 5min; Then enter circulation, 95 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 40sec, 35 circulations; Finally extend 72 DEG C of 10min.
Table one, PCR application of sample table
Then get appropriate above-mentioned pcr amplification product 2% sepharose and carry out electrophoretic separation, observe under ultraviolet lamp after ethidium bromide staining, according to the size result of determination of amplified production.If the band amplified is near 500bp, and the assorted band of the single nothing of band and primer band, then illustrate and increase successfully, reclaim amplified production;
In pcr amplification product, add restriction enzyme NCO I carry out endonuclease reaction, endonuclease reaction condition is: 10 μ L PCR primer, 2 μ L 10 × Dilution Buffer (10mM Tris-HCL, 100mM KCL, 1mM EDTA, 1mM DTT, 0.2mg/mL BSA, 50% glycerine), 18 μ L sterilizing ultrapure waters, 20U NCO I, 37 DEG C temperature bath 4h;
Get appropriate step endonuclease reaction product 2% agarose electrophoresis to be separated, if sample has two bands, size is about 335bp and 200bp respectively, then test sample is Radix Apioris Fortunei (Radix Lespedezae Buergeri).If enzyme still has the am-plified fragments of about 500bp size after cutting, then this medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) is mixed with pseudo-mixed product.
<110> Zhejiang Pharmaceutical College
<120> differentiates the PCR-RFLP method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) and pseudo-mixed product thereof fast
<160> 3
<170> PatentIn Version 2.1
<210> 1
<211> 20
<212> DNA
<213> primer sequence
<400> 1
1 atgcgatact tggtgtgaat
<210> 2
<211> 21
<212> DNA
<213> primer sequence
<400> 2
1 gacgcttctc cagactacaa t
<210> 3
<211> 492
<212> DNA
<213> nature
<220>
<221> misc_feature
<222> (80,100,112)
<223> n=a or g or c or t
<220>
<221> ITS2
<222> (1)...(492)
<400> 3
1 cggggggaag ccgattctca gctgggcttt tcccggttcg ctcgccgtta
51 ctaagggaat ccttgtaagt ttcttttcct ccgcttattg atatgcttaa
101 actcagcggg tgttcccgcc tgacctgggg tcgctgtcga ggttctcggg
151 cctttcgtgg tgccccgttg cgaagtgccg cgcccgacat ggagattctc
201 ctccttcctc ctctggacga gggggaaagg atcaggggtt cgtttcaaac
251 caccgcttgt cgtggcgtgc atcgccgcgg gacagatttt taaccaacca
301 cggatgcgat gttccatggg aggccaatgt ccgccccaac tccaagcccc
351 tgactcggta ggggtggagg gggcgacgcg tgcgtgacgc ccaggcaggc
401 gtgccctcga cctaatggcg tcgggcgcaa cttgcgttca aagactcgat
451 gattcacggg attctgcaat tcacaccagt aattcgcata ta
Claims (9)
1. differentiate that Radix Apioris Fortunei (Radix Lespedezae Buergeri) and multiple puppet thereof mix the PCR-RFLP method of product fast for one kind, comprise and extract medicinal material DNA and agarose gel electrophoresis analysis, it is characterized in that, carry out pcr amplification with forward and reverse primer of the Internal Transcribed Spacer (ITS2) sequence of pair for amplification rDNA and use digestion with restriction enzyme PCR primer, judge the true and false of medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) according to enzymic digestion product agarose gel electrophoresis analytical results and whether be mixed with pseudo-mixed product.
2. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, described pcr amplification gene is the Internal Transcribed Spacer (ITS2) of rDNA, has the gene order described in SEQID NO.1.
3. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, pcr amplification primer has SEQID NO.2 and the nucleotide sequence described in SEQID NO.3.
4. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, the restriction enzyme of use is NCO I, and its recognition sequence is CCATGG.
5. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, the gene segment size of pcr amplification is 400 ~ 600bp.
6. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, the gene segment size of preferred pcr amplification is 480 ~ 550bp.
7. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, after enzymic digestion there is 335bp and 200bp band in the analysis of medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) sample electrophoresis, and DNA cloning product being not the limited property endonuclease digestion of its pseudo-mixed product.
8. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, described PCR reaction conditions is: 95 DEG C of denaturation 5min; Then enter circulation, 95 DEG C of sex change 30 seconds, 45 ~ 55 DEG C of annealing 30 seconds, 72 DEG C extend 40 seconds, 25 ~ 35 circulations; Last 72 DEG C extend 10 minutes.
9. a kind of quick discriminating Radix Apioris Fortunei (Radix Lespedezae Buergeri) as claimed in claim 1 and multiple puppet thereof mix the PCR-RFLP method of product, it is characterized in that, the digestion with restriction enzyme reaction conditions of pcr amplification product is: 10 μ L PCR primer, 2 μ L 10 × enzyme cutting buffering liquids, 8 μ L sterilizing ultrapure waters, 5 ~ 25U restriction enzyme, 20 ~ 37 DEG C of constant temperature 2 ~ 8 hours.
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| CN107630014A (en) * | 2017-10-19 | 2018-01-26 | 中国科学院昆明植物研究所 | A kind of authentication method of Aconitum transsectum DNA bar code and Aconitum transsectum based on big data |
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