CN103923909A - Specific molecular marker for panax ginseng and panax quinquefolius and identification method thereof - Google Patents

Abstract

Belonging to the technical field of traditional Chinese medicine, the invention relates to identification methods of Chinese herbal medicines, in particular to a specific molecular marker for panax ginseng and panax quinquefolius and an identification method thereof. The invention provides a DNA molecular marker for specific identification of panax ginseng and panax quinquefolius. The DNA molecular marker is located on the (SSR) loci of DNA sequences Seq.No.1-5 respectively. 5 pairs of primers are designed according to the sequences to perform PCR (polymerase chain reaction) amplification on the DNA templates of panax ginseng and panax quinquefolius. PCR products for specific identification of panax ginseng and panax quinquefolius can be amplified respectively by each pair of the primers, the products with different lengths are detected by polyacrylamide gel electrophoresis and agarose gel electrophoresis, so that panax ginseng and panax quinquefolius can be rapidly identified. The method needs a small amount of samples, is not restricted by product shapes, and has low requirement for sample's DNA length. The PCR is stable and has good repeatability.

Description

People participates in specific molecular marker and the discrimination method thereof of Radix Panacis Quinquefolii

Technical field

The invention belongs to technical field of traditional Chinese medicines, relate to the discrimination method of Chinese medicinal materials.Be specifically related to people and participate in specific molecular marker and the discrimination method thereof of Radix Panacis Quinquefolii.

Background technology

Ginseng (Panax ginseng C.A.Meyer) and Radix Panacis Quinquefolii (Panax quinquefolius L.) are the most widely used important Chinese medicine material of current China, belong to perennial root herbaceous plant Araliaceae (Araliaceae) ginseng.Studies show that, ginseng is distributed in the ground such as China northeast, Far-east Area of Russia and Korea; Radix Panacis Quinquefolii originates in low mountainous region, North America, after China's successful introduction northeastward, all there is plantation in North China, Si great cultivation area, He Kang Yunnan, Central China.Prior art discloses ginseng and the Radix Panacis Quinquefolii property of medicine differs greatly, but plesiomorphism, the people of especially domestic cultivation participates in cultivating Radix Panacis Quinquefolii and is difficult to accurately differentiate according to morphological specificity, and both powder are difficult to difference especially.Therefore a kind of discriminating people who does not rely on morphological specificity of the market requirement participates in the method for Radix Panacis Quinquefolii.

Summary of the invention

The object of the present invention is to provide the discrimination method of important Chinese medicine material, be specifically related to people and participate in the specific molecular marker of Radix Panacis Quinquefolii, and discrimination method, especially a kind of discriminating people who does not rely on morphological specificity participates in the method for Radix Panacis Quinquefolii.

The invention provides the DNA molecular marker that can specific recognition people participates in Radix Panacis Quinquefolii, described DNA molecular marker lays respectively on (SSR) site in the DNA sequence dna of Seq.No.1～5, described Seq.No.1, Seq.No.2, Seq.No.3, in Seq.No.4 and Seq.No.5DNA sequence, there is respectively a simple repeated sequence (SSR) site, can identify specifically ginseng and Radix Panacis Quinquefolii by this sequence site;

Further, the invention provides the method that participates in Radix Panacis Quinquefolii based on the quick discriminating people of above-mentioned DNA molecular marker, wherein, according to above-mentioned sequences Design 5 to primer, the DNA profiling of ginseng and Radix Panacis Quinquefolii is carried out to pcr amplification, every pair of primers can amplify respectively the PCR product of specific recognition ginseng and Radix Panacis Quinquefolii, detects the product that these length differ with polyacrylamide gel electrophoresis and agarose gel electrophoresis, thereby differentiates that fast people participates in Radix Panacis Quinquefolii.

More specifically, quick discriminating people of the present invention participates in the method for Radix Panacis Quinquefolii, it is characterized in that, it comprises,

1, specific amplification people participates in the primer sequence in simple repeated sequence (simple repeat sequence the is called for short SSR) site of Radix Panacis Quinquefolii; The present invention filters out 5 DNA sequence dnas that contain SSR site from contain in a large number the ginseng sequence in SSR site, is respectively Seq.No.1, Seq.No.2, and Seq.No.3, Seq.No.4 and Seq.No.5, these SSR sites participate in Radix Panacis Quinquefolii for distinguishing people specifically;

2, utilize above-mentioned primer to differentiate that people participates in Radix Panacis Quinquefolii:

According to above-mentioned 5 Ginseng DNA sequences, design 5 couples of polymerase chain reaction (polymerase chainreaction, be called for short PCR) primer, its sequence is respectively Seq.No.6 and Seq.No.7, Seq.No.8 and Seq.No.9, Seq.No.10 and Seq.No.11, Seq.No.12 and Seq.No.13, Seq.No.14 and Seq.No.15;

Carry out respectively pcr amplification with the DNA profiling that Seq.No.6+Seq.No.7 participates in Radix Panacis Quinquefolii to people, 52 DEG C of PCR annealing temperatures, result shows, ginseng can produce the long PCR product of 181bp, and Radix Panacis Quinquefolii can not produce PCR product;

Carry out respectively pcr amplification with the DNA profiling that Seq.No.8+Seq.No.9 participates in Radix Panacis Quinquefolii to people, 55 DEG C of annealing temperatures, result shows, and ginseng can only produce the long PCR product of a 203bp, and Radix Panacis Quinquefolii can produce 191bp and two long PCR products of 203bp;

Carry out respectively pcr amplification with the DNA profiling that Seq.No.10+Seq.No.11 participates in Radix Panacis Quinquefolii to people, 57 DEG C of annealing temperatures, result shows, and ginseng can produce the long PCR product of a 243bp, and Radix Panacis Quinquefolii produces the long PCR product of 235bp;

Carry out respectively pcr amplification with the DNA profiling that Seq.No.12+Seq.No.13 participates in Radix Panacis Quinquefolii to people, 57 DEG C of annealing temperatures, result demonstration, ginseng can only produce the long PCR product of a 184bp, and Radix Panacis Quinquefolii can produce 166bp and two long PCR products of 157bp;

Carry out respectively pcr amplification with the DNA profiling that Seq.No.14+Seq.No.15 participates in Radix Panacis Quinquefolii to people, 57 DEG C of annealing temperatures, result shows, and ginseng can produce 251bp and two PCR products of 273bp, and Radix Panacis Quinquefolii can not produce PCR product.

In embodiments of the invention, people is participated in to Radix Panacis Quinquefolii and identifies:

1) get 0.05 gram, sample to be identified, extract total DNA of sample by the low PH method of high salt;

2) get the DNA of 25 ~ 50ng sample to be checked, from 5 pairs of above-mentioned primers optional 3 pairs, press

Each the specific annealing temperature of primer is carried out to pcr amplification, every pair of primers arranges 3 times and repeats and blank;

3) size of polyacrylamide gel electrophoresis inspection PCR product;

4) size of the PCR product producing according to selected primer pair and the standard that table 1 proposes, judge the attribute of sample.

In above-mentioned qualification of the present invention,

Select primer pair (Seq.No.6+Seq.No.7), primer pair (Seq.No.14+Seq.No.15) and other any one primer pair to carry out pcr amplification, can use agarose gel electrophoresis detected result, result shows, adopt the DNA of the first two primer pair amplification Radix Panacis Quinquefolii, can not obtain spawn, and the DNA of amplification ginseng will obtain the product shown in table 1; All product not of uniform size as shown in table 1 will be obtained with increase the respectively DNA of ginseng and Radix Panacis Quinquefolii of other 3 pairs of primers;

Table 1. is for the 5 pairs of PCR primers and the product size of the qualification of ginseng, Radix Panacis Quinquefolii

The result of selected 3 pairs of primer amplifications all meets above-mentioned standard, could belong to ginseng or Radix Panacis Quinquefolii by judgement sample.

Advantage of the present invention has:

Required sample is few, no more than 0.005 gram, is not also subject to the restriction of product form, no matter to be form intact or pulverized, or add other material for sample, as long as DNA all can not identified by method of the present invention by havoc; In addition, primer pair sequence of the present invention is longer, and the product of amplification is no more than 280bp, therefore not high to the DNA length requirement of sample, and PCR is stable and reproducible.

Brief description of the drawings

The poly-propionic acid amide gel electrophoresis figure of Fig. 1 primer pair (Seq.No.6+Seq.No.7) amplification ginseng and Radix Panacis Quinquefolii individuality, totally 35 samples and a blank (being designated as-), M is the large tick marks of DNA molecular (mark); Wherein, sample title and source are: 1-4: new guest cultivates Radix Panacis Quinquefolii; 5: Fusong Da-maya; 6: Tonghua Da-maya; 7: Tonghua two gingivals cyst of mucous gland in the newborn; 8: Jian two gingivals cyst of mucous gland in the newborn; 9: two gingivals cyst of mucous gland in the newborn (new guest); 10-11: the long neck in Jian; 12-13: Jian circle reed; 14-15: Jian ring reed; 16-17: Jian Ginseng under Forest; 18-19: Huan's benevolence Radix Ginseng; 20-24: Canada's cultivation Radix Panacis Quinquefolii (dragon is precious); 25-29: U.S.'s cultivation Radix Panacis Quinquefolii; 30-31: Canada's cultivation Radix Panacis Quinquefolii (god resembles); 32-33: wide pasture Radix Ginseng; 34-35: Korea S's Radix Ginseng.

Fig. 2 is the poly-propionic acid amide gel electrophoresis figure of primer pair (Seq.No.8+Seq.No.9) amplification ginseng and Radix Panacis Quinquefolii individuality, sample number into spectrum is identical with Fig. 1, wherein the PCR reaction system of No. 12 samples is polluted by Radix Panacis Quinquefolii DNA, repeat the product that 191bp no longer appears in experiment, prove not exception of No. 12 samples.

Fig. 3 is the poly-propionic acid amide gel electrophoresis figure of primer pair (Seq.No.10+Seq.No.11) amplification ginseng and Radix Panacis Quinquefolii individuality, and sample number into spectrum is identical with Fig. 1.

Fig. 4 is the poly-propionic acid amide gel electrophoresis figure of primer pair (Seq.No.12+Seq.No.13) amplification ginseng and Radix Panacis Quinquefolii individuality, and sample number into spectrum is identical with Fig. 1.

Fig. 5 is the poly-propionic acid amide gel electrophoresis figure of primer pair (Seq.No.14+Seq.No.15) amplification ginseng and Radix Panacis Quinquefolii individuality, and sample number into spectrum is identical with Fig. 1.

Embodiment

Embodiment 1

Collect 51 parts, Da-maya, two gingivals cyst of mucous gland in the newborn, long neck, circle reed and 5 kinds of common ginseng farm variety samples of ring reed and Radix Ginseng, Ginseng under Forest, Radix Ginseng and Radix Ginseng sample from the different places of production and market, from the U.S., Canada and LiaoNing, China collection 16 parts, sample of Radix Panacis Quinquefolii (material is as shown in table 2);

Adopt the high salt SDS method of improvement to extract total DNA of sample, from less material, obtain high-quality DNA;

Pcr amplification, PCR reaction system is as follows: cumulative volume 20 μ L, containing having an appointment 20ng genomic dna, the each 0.25 μ mol/L of forward primer and reverse primer, 1 × reaction buffer, 1.0mmol/L MgCl2,0.1mmol/L dNTP, 1U Taq enzyme, processes water with DEPC and supplements volume to 20 μ L, separately replaces masterplate DNA as negative control with deionized water;

Pcr amplification program is as follows: PCR reacts at Applied Biosystem ^tMon 2720 Thermal Cycler amplification instruments, carry out, reaction parameter is: 94 DEG C of denaturation 1min, and according to each Ta temperature 40s to the concrete setting of primer, 72 DEG C of 40s, 35 circulations, 72 DEG C of 5min fill, and reaction product is preserved at 4 DEG C;

Carry out the detection of PCR product by following program: PCR reaction product is splined on 4% poly-propionic acid amide gel, and (every plate glue is containing 3.6g urea, 17mL deionized water, 3mL10 × TBE solution, 4.5mL40%Arc-Bis solution, 0.3mL10% (NH3) 2S2O2 solution, 14 μ L TEMED), applied sample amount is 5 μ L, gel is placed in the electrophoresis chamber that contains 0.5 × tbe buffer liquid and carries out electrophoresis, voltage 500V, and electrophoresis time is 100min.Gel by 0.1%AgNO3 solution-dyed 10min after, with 1.5%NaOH and 0.4% formaldehyde mixed solution colour developing 15min.With digital camera Taking Pictures recording, use Smart View ^tMthe biological electrophoresis image analysis software of Plus (Shanhai Furi Science and Technology Co., Ltd.) is calculated clip size;

Result shows: 51 parts of samples of Ginseng of every a pair of primer amplification, all there is without exception the distinctive PCR product of the ginseng shown in table 1 (wherein, when increase a garden reed sample with Seq.No.8+Seq.No.9, there is the distinctive product of Radix Panacis Quinquefolii, repeat experiment and repeatedly all no longer occur this product, prove that this time experiment is polluted by Radix Panacis Quinquefolii DNA, this sample is exception not); The 16 parts of Radix Panacis Quinquefolii samples that increase, all there is the distinctive PCR product of Radix Panacis Quinquefolii in all samples.

Table 2 the present invention ginseng used and Radix Panacis Quinquefolii material

SEQUENCE LISTING

<110> Fudan University

<120> people participates in specific molecular marker and the discrimination method thereof of Radix Panacis Quinquefolii

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 185

<212> DNA

<213> Panax ginseng

<400> 1

tacatttgct tgcttgattt ctcctcctcc ttcttccttc cttccatgga tggatcttgg 60

tttcatgaaa gtcaaaatcc cttctccctc ttctcttgaa gtggccgaac tcctctctct 120

ctctccctct ctctcttttc ctcttagcaa cttggaaggg taaaagagat gaaagaaaga 180

agaaa 185

<210> 2

<211> 203

<212> DNA

<213> Panax ginseng

<400> 2

tgcccccttt tgttttctca atgctttgat attgtgacaa agcctttttt atttttttgt 60

cctttttttg ggtactgtat taatgttacc gctgttcttt aggaggaaaa aagtagaaac 120

taagtcatac taaaattgat tcgacagttg gctggaccat catgcaaaat tcaggattca 180

tcagcgacta aagaaaatat gga 203

<210> 3

<211> 231

<212> DNA

<213> Panax ginseng

<400> 3

atcaaacgaa ttcacggaca gaaatttgat ttttactatt cattattgtg caatgaaata 60

ttctaataaa acaaaaacct taaataaaat ataatatatt ctatttataa tctaataaaa 120

aaaacataat atatatatat atatacatat atatatatat ataacaacac atgttatatt 180

aaaaaaatca cccgtgacaa ccttcagact ttccttcccc catattcatt t 231

<210> 4

<211> 180

<212> DNA

<213> Panax ginseng

<400> 4

aagtttggca gaaggtaagg gagggatgga taaatgtaat gacccaatat tattattatt 60

attattatta ttattattac atgttgctct acacgtggcg gatgatgatc tttaactgac 120

tggactggac tacactagac tagactagac attgtccccg aacccaaaca gtgccgtctt 180

<210> 5

<211> 240

<212> DNA

<213> Panax ginseng

<400> 5

ctaagcagtg gaagtcaaac acatcttcta aatgaagatg aacttacatg ctgaaatcta 60

tatgtgcagg tatatatata tatatgtgtg tgtgtgtgtg tgtgcgtgtg tggactatat 120

atacacaact gtgtagtaca cagtcactta tggcgatatt tttttggaga tatagcgacg 180

gttcaaccgt cgccatagac cagaatagct atagcgacag tttacccatc gctatagccc 240

<210> 6

<211> 21

<212> DNA

<213> primer

<400> 6

tacatttgct tgatttctcc t 21

<210> 7

<211> 22

<212> DNA

<213> primer

<400> 7

tttcttcttt ctttcatctc tt 22

<210> 8

<211> 19

<212> DNA

<213> primer

<400> 8

tgcccccttt tgttttctc 19

<210> 9

<211> 24

<212> DNA

<213> primer

<400> 9

tccatatttt ctttagtcgc tgat 24

<210> 10

<211> 22

<212> DNA

<213> primer

<400> 10

atcaaacgaa ttcacggaca ga 22

<210> 11

<211> 24

<212> DNA

<213> primer

<400> 11

aaatgaatat gggggaagga aagt 24

<210> 12

<211> 20

<212> DNA

<213> primer

<400> 12

aagtttggca gaaggtaagg 20

<210> 13

<211> 20

<212> DNA

<213> primer

<400> 13

aagacggcac tgtttgggtt 20

<210> 14

<211> 21

<212> DNA

<213> primer

<400> 14

ctaagcagtg gaagtcaaac a 21

<210> 15

<211> 20

<212> DNA

<213> primer

<400> 15

gggctatagc gatgggtaaa 20

Claims (4)

1. contain people participate in Radix Panacis Quinquefolii specific molecular marker site as Seq.No.1, Seq.No.2, Seq.No.3, the DNA sequence dna shown in Seq.No.4 and Seq.No.5.

2. the PCR primer pair based on DNA sequence dna claimed in claim 1, its combined sequence is: Seq.No.6 and Seq.No.7, Seq.No.8 and Seq.No.9, Seq.No.10 and Seq.No.11, Seq.No.12 and Seq.No.13, Seq.No.14 and Seq.No.15.

3. differentiate that people participates in a method for Radix Panacis Quinquefolii, it is characterized in that, comprising: specific amplification people participates in the primer sequence as claimed in claim 2 in the simple repeated sequence site of Radix Panacis Quinquefolii, utilize described primer to differentiate that people participates in Radix Panacis Quinquefolii, it comprises step:

1) get sample to be identified, extract total DNA of sample by the low PH method of high salt;

2) get the DNA of sample to be checked, from described primer pair optionally wherein 3 pairs, by each specific to primer

Annealing temperature is carried out pcr amplification, and every pair of primers arranges 3 times and repeats and blank;

3) according to the PCR product size of selected primer pair amplification, the attribute of sample is judged.

4. by method claimed in claim 3, it is characterized in that, the judging criterion of described each primer pair is as shown in table 1:

Table 1. is for the 5 pairs of PCR primers and the product size of the qualification of ginseng, Radix Panacis Quinquefolii

When the result of selected 3 pairs of primer amplifications all meets above-mentioned standard, judgement sample belongs to ginseng or Radix Panacis Quinquefolii.