CN1227358C

CN1227358C - Identification method of PCR-restriction map for fritillary bulb and similar medicine material

Info

Publication number: CN1227358C
Application number: CN 03131798
Authority: CN
Inventors: 李萍; 王冲之
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2003-07-30
Filing date: 2003-07-30
Publication date: 2005-11-16
Anticipated expiration: 2023-07-30
Also published as: CN1487091A

Abstract

The present invention belongs to the technical field of the identification of germ plasms of Chinese medicinal materials and aims to provide a polymerase chain reaction-restriction map molecular identification method (PCR-RFLP) for accurately identifying medicinal materials of tendrilleaf fritillary bulb and non-tendrilleaf fritillary bulb. The sequences of primers for PCR amplification are: P1:5'-CGT AAC AAG GTT TCC GTA GGTGAA-3' and P2: 5'-GCT ACG TTC ATC GAT-3'; the tendrilleaf fritillary bulb has the characteristic DNA base sequence: 5'-TGCCCGCCCT GCCCGGGACC TCGC-3'; a restriction enzyme Sma I and an isoschizomer thereof can identify and cut the sequence. The identification process comprises the following steps: extracting DNA of the medicinal materials; using primers P1 and P2 for PCR amplification; using the restriction enzyme Sma I or the isoschizomer thereof to digest PCR products; analyzing a result by agarose gel electrophoresis. Whether the medicinal material is the tendrilleaf fritillary bulb can be judged by comparing the result with a PCR-RFLP characterist result of the tendrilleaf fritillary bulb.

Description

Unibract Fritillary Bulb class medicinal material polymerase chain reaction-restriction map authentication method

One, technical field:

The present invention relates to the quality authenticate technology field of Chinese medicinal materials germplasm.

Two, background technology:

The Chinese medicine Unibract Fritillary Bulb is the dry bulb of liliaceous plant Unibract Fritillary Bulb Fritillaria cirrhosa D.Don, dark violet bulb of fritillary Fritillaria unibracteata Hsiao et K.C.Hsia, Gansu bulb of fritillary Fritillaria przewalskii Maxim or Bulbus Fritillariae cirrhosae Fritillaria delavayi Franch., Unibract Fritillary Bulb has clearing heat and moistening lung, the effect of preventing phlegm from forming and stopping coughing, be used for lung-heat type cough, the few phlegm of dry cough, deficiency of Yin phthisical cough is coughed up the treatment of diseases such as sputum streaked with blood.Because the good effect of Unibract Fritillary Bulb, toxicity is low, and natural resources is limited, and therefore the artificial culture difficulty is the famousst and precious kind in the bulb of fritillary class medicinal material, and market value is very high.The phenomenon of as seen pretending to be Unibract Fritillary Bulb on the market with the bulblet of other kind of plant of Fritillaria.Yet, be difficult to Unibract Fritillary Bulb and its pseudo-product difference are come with common kenel and authentication method such as micro-, seek therefore that to be used for the method that Unibract Fritillary Bulb identifies reliably be a major issue of bulb of fritillary medicinal material evaluation.The Unibract Fritillary Bulb main product is in China southwest, because its sibship is nearer, regional distribution is comparatively concentrated, thereby has similar genetic background.By more various bulb of fritillary nrDNAITS district base sequence, found the difference of Unibract Fritillary Bulb and other kind bulb of fritillary dna sequence dna, this difference can be distinguished by the polymerase chain reaction (PCR) and the segment polymorphism (RFLP) of restriction enzyme reaction.But this discriminating is for different species, and the kind and the discrimination method of its used primer, restriction enzyme are all inequality.

Three, summary of the invention:

The PCR-RFLP molecular assay method that the purpose of this invention is to provide a kind of accurate discriminating Unibract Fritillary Bulb comprises characteristic DNA base sequence, one couple of PCR primers, a kind of restriction enzyme digestion sites and the authentication method thereof of Unibract Fritillary Bulb.

Technical scheme of the present invention is as follows: at first extract total DNA from each medicinal material, utilize a pair of primer, with PCR method amplification section of DNA fragment, after purified, this fragment is carried out determined dna sequence, set up the dna sequence data storehouse of each sample, on the basis of comparison database dna sequence dna, the characteristic DNA base sequence that obtains Unibract Fritillary Bulb is: 5 '-TGCCCGCCCT GCCCGGGACC TCGC-3 ', difference at Unibract Fritillary Bulb and other kind bulb of fritillary dna sequence dna, select suitable DNA restriction enzyme digestion sites,, reach quick by analyzing the restriction map difference of polymerase chain reaction product, accurately differentiate the purpose of Unibract Fritillary Bulb.Bulb of fritillary sample to needs are identified extracts its DNA, and under given PCR condition, with a pair of primer (P1, P2), trial-product can amplify the section of DNA fragment; With restriction endonuclease sma I or its isoenzyme the pcr amplification product of trial-product is carried out detecting by agarose gel electrophoresis after enzyme cuts, Unibract Fritillary Bulb can occur 119, the band of 188bp, and above-mentioned two bands do not appear in the bulb of fritillary of other kind.When for test agent through carrying out pcr amplification with this law, and after the PCR product carried out digestion with restriction enzyme,, just can identify Unibract Fritillary Bulb or non-Unibract Fritillary Bulb medicinal material exactly through the size that agarose gel electrophoresis is observed dna fragmentation.Being used to of the present invention design differentiates that Unibract Fritillary Bulb PCR-RFLP molecular assay method is to adopt following steps:

1, the extraction of medicinal material DNA: carry out routinely, will be with deionized water for DNA concentration adjustment to the 0.2～0.5 μ g/ μ l of test agent;

2, amplification of DNA fragments promptly carries out polymerase chain reaction, and the dna sequence dna that is used for a pair of primer of polymerase chain reaction is:

P1：5’-CGT?AAC?AAG?GTT?TCC?GTA?GGT?GAA-3’

P2：5’-GCT?ACG?TTC?TTC?ATC?GAT-3’

3, add restriction endonuclease sma I or its isoenzyme in the pcr amplification product and carry out enzyme and cut, the restriction enzyme site of restriction endonuclease sma I or its isoenzyme is: CCC^GGG;

4, agarose gel electrophoresis analysis;

5, the judgement of qualification result, if occur 119,188bp band then trial-product is a Unibract Fritillary Bulb.

Effect of the present invention is: can solve Unibract Fritillary Bulb and other kind bulb of fritillary phase region and else identify a difficult problem, provide and identify required one couple of PCR primers, PCR reaction conditions, the characteristic DNA base sequence of Unibract Fritillary Bulb, a kind of restriction enzyme.The Unibract Fritillary Bulb effective constituent contained than the bulb of fritillary of other kind has difference, and toxicity is very little, and is difficult for cultivation, so the price of Unibract Fritillary Bulb is significantly higher than other kind bulb of fritillary on the market.Yet Unibract Fritillary Bulb and other kind bulb of fritillary are difficult to be distinguished by form, feature such as micro-, therefore, are badly in need of finding a kind of reliable discrimination method.The present invention utilizes the difference of Unibract Fritillary Bulb and other kind bulb of fritillary medicinal material dna sequence dna, has set up quick, convenient, reliable PCR-RFLP discrimination method, and for the quality of medicinal material that guarantees Unibract Fritillary Bulb, the market behavior of hitting the personation Unibract Fritillary Bulb has significant values.

Four, embodiment:

1, the extraction of DNA: carry out routinely, will be with deionized water for DNA concentration adjustment to the 0.2～0.5 μ g/ μ l of test agent;

2, polymerase chain reaction:

(1) polymerase chain reaction is reference with 30 μ l, and the consumption of various article is respectively:

10 * PCR damping fluid, 3 μ l

MgCl ₂(25mM) 2.4μl

DNTP (each 10mM) 0.6 μ l

Each 0.5 μ l of primer P1, P2 (30pmol/ μ l)

Trial-product dna profiling 1～2 μ l

TaqDNA polysaccharase 1U (0.2 μ l)

Deionized water polishing to 30 μ l

(2) PCR reaction conditions: be reflected on the PCR instrument and carry out, reaction conditions is:

95 ℃ are given sex change 3min, carry out 30 circulations by following condition then:

95 ℃ of sex change 20～40 seconds

53 ℃ of renaturation 20～40 seconds

Extend 72 ℃ 20～40 seconds

Keep 2～4 minutes polishings at 72 ℃ after the loop ends, reaction finishes the back 4 ℃ of preservations.

(3) electrophoresis detection of PCR product: get above-mentioned reaction solution 4 μ l, mix with 1 μ l load sample damping fluid, with 2% sepharose (containing 0.5 μ g/ μ L ethidium bromide, i.e. EB) electrophoresis detection amplification, various DNA samples all can amplify the DNA band of a treaty 307bp;

3, the digestion with restriction enzyme of polymerase chain reaction product reaction:

The reaction of DNA digestion with restriction enzyme: the reaction solution reference volume is 20 μ l, and wherein the consumption of various article is:

PCR product 5～10 μ L

10 * R ⁺Restriction enzyme damping fluid 2 μ L

Restriction endonuclease sma I 5U

Deionized water polishing to 20 μ L

Reaction solution is put 37 ℃ of insulations 3～4 hours, and reaction finishes to be placed on 65 ℃ of water-baths 10 minutes, makes enzyme deactivation;

4, the electrophoresis observation enzyme is cut the result: enzyme is cut product with 2% sepharose (ethidium bromide that contains 0.5 μ g/ μ l, i.e. EB), and electrophoresis is 30～50 minutes under 4V/cm strength of electric field, observes electrophoresis result;

5, the judgement of measurement result is used restriction endonuclease sma I or its isoenzyme that amplified product of polymerase chain reaction is carried out enzyme and is cut, and produces the enzyme that Unibract Fritillary Bulb and non-Unibract Fritillary Bulb are had a distinctive feature and cuts dna fragmentation length collection of illustrative plates.If trial-product occurs 119, two fragments of 188bp then are Unibract Fritillary Bulb; If above-mentioned two fragments do not occur, then be not Unibract Fritillary Bulb.

＜110〉China Medicine University

＜120〉Unibract Fritillary Bulb class medicinal material polymerase chain reaction-restriction map authentication method

<160>3

<210>1

<211>24

<212>DNA

＜213〉Unibract Fritillary Bulb (Fritillaria cirrhosa D.Don, Fritillaria unibracteata Hsiao et K.C.Hsia, Fritillaria przewalskii Maxim, Fritillaria delavayi Franch.)

<400>1

cgtaacaagg?tttccgtagg?tgaa

<210>2

<211>18

<212>DNA

<400>1

gctacgttct?tcatcgat

<210>3

<211>24

<212>DNA

<400>1

tgcccgccct?gcccgggacc?tcgc

Claims

1, be used to differentiate that the pcr amplification primer of Unibract Fritillary Bulb is right, it is characterized in that: this primer is to being P1:5 '-CGT AACAAG GTT TCC GTA GGT GAA-3 '; P2:5 '-GCT ACG TTC TTC ATC GAT-3 '.

2, a kind of authentication method of Unibract Fritillary Bulb is characterized in that with restriction endonuclease sma I and the isoenzyme thereof that can discern the peculiar DNA base sequence of Unibract Fritillary Bulb polymerase chain reaction product being digested, and produces the characteristic spectrum of agarose gel electrophoresis, wherein

(1) extraction of medicinal material DNA: carry out routinely, will be with deionized water for DNA concentration adjustment to the 0.2～0.5 μ g/ μ l of test agent;

(2) amplification of DNA fragments carries out polymerase chain reaction with the described primer of claim 1 to P1, P2, obtains amplified product of polymerase chain reaction;

(3) add restriction endonuclease sma I or its isoenzyme in the pcr amplification product and carry out enzyme and cut, the restriction enzyme site of restriction endonuclease sma I or its isoenzyme is: CCCGGG;

(4) agarose gel electrophoresis analysis;

(5) judgement of qualification result: if occur 119, two fragments of 188bp, then trial-product is a Unibract Fritillary Bulb, if above-mentioned two fragments do not occur, then trial-product is not a Unibract Fritillary Bulb.