CN1438235A

CN1438235A - Pure honeysuckle polymerase chain reaction-restriction map identification method

Info

Publication number: CN1438235A
Application number: CN 03113003
Authority: CN
Inventors: 李萍; 王冲之; 周开亚
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2003-03-20
Filing date: 2003-03-20
Publication date: 2003-08-27
Anticipated expiration: 2023-03-20
Also published as: CN1238369C

Abstract

The invention provides a polymerase chain reactino-restrictive enzyme-cut chart molecule (PCR-RFLP) method accurately identifying honeysuckle in genuine producing areas (Henan, Shandong) and nongenuine producing areas (other producing areas). The characteristic DNA alkali sequence of honeysuckle in genuine: 5'-GCCCCCCCGC CCCGCCTCCC ACAGGGTCGC G-3'; restrictive inner cut enayme EcoNi and its same cut-enzyme can identify and cut this sequence. The identifying course: extracting DNA of medical materials; using a pair of guide matters to make PCR increase; using restrictive inner cut enayme EcoNI and its same cut enayme to digest PCR product; analyzing the result by using gelation and electrophoresis of gelose; in contrast with the characteristic result of PCR-RFLP of genuine honeysuckle, judging if the tested product is genuine honeysuckle.

Description

Genuineness Japanese Honeysuckle 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:

Japanese Honeysuckle is the dry flower of caprifoliaceae plant honeysuckle Lonicera japonica Thunb. or the flower of just opening, and stronger anti-microbial effect is arranged, and multiple acute and chronic inflammation etc. is had curative effect preferably.Traditional Chinese medicine honeysuckle has tangible genuineness, and as by us the rough determination of chlorogenic acid content being shown, the chlorogenic acid content of Henan, genuine producing region and Shandong Japanese Honeysuckle generally is higher than other non-genuine producing region, shows that quality of medicinal material and region, habitat etc. are closely related.Yet the Japanese Honeysuckle in non-genuine producing region and genuine producing region can't be distinguished by form, feature such as micro-.Owing to the otherness of the mutual isolation of areal distribution between Henan, genuine producing region, Shandong two population and each population of non-genuine producing region, natural condition causes existing between the different population fine difference on evolving, thereby on dna sequence dna, demonstrating the part difference, this difference can be distinguished by the polymerase chain reaction (PCR) and the segment polymorphism (RFLP) of restriction enzyme reaction.The discriminating that PCR-RFLP is used for sibling species has had many successful examples, as abroad four kinds of Bartonella pathogenic bacterias being differentiated fast.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 purpose of this invention is to provide the PCR-RFLP molecular assay method of a kind of accurate discriminating genuine (Henan, Shandong) and non-genuine (other producing regions) producing region Japanese Honeysuckle, comprise the characteristic DNA base sequence of genuineness Japanese Honeysuckle, a kind of restriction enzyme digestion sites and authentication method thereof.

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 genuine producing region Japanese Honeysuckle is: 5 '-GCCCCCCCGC CCCGCCTCCCACAGGGTCGC G-3 ', difference at genuine and non-genuine producing region Japanese Honeysuckle dna sequence dna, select suitable DNA restriction enzyme digestion sites, by analyzing the restriction endonuclease map difference of polymerase chain reaction product, reach quick, accurately differentiate the purpose of genuine and non-genuine producing region Japanese Honeysuckle.Japanese Honeysuckle sample to needs are identified extracts its DNA, under given PCR condition, with a pair of primer (LjP1, LjP2; TakaiwaF, Oono K, Sugiura M.Nucleotide sequence of the 17S-25S spacer region from ricerDNA.Plant Mol Biol, 1985,4:355-364), trial-product can amplify the section of DNA fragment; With restriction enzyme EcoN I or its isoenzyme the pcr amplification product of trial-product is carried out after enzyme cuts, detect by agarose gel electrophoresis, will occur 708,461, the band of 247bp, the band of genuine producing region 461bp significantly bright in or closely equal the band of 708bp, the band of non-genuine producing region 708bp is significantly bright in the band of 461bp.When for test agent through carrying out pcr amplification with this law, and after the PCR product carried out digestion with restriction enzyme, observe the size and the brightness of dna fragmentation through agarose gel electrophoresis, just can identify the Japanese Honeysuckle in genuine and non-genuine producing region exactly.Producing region, the discriminator track ground Japanese Honeysuckle PCR-RFLP molecular assay method that is used for of the present invention's design is to adopt following steps:

The extraction of a, medicinal material DNA: carry out routinely, will be with deionized water for DNA concentration adjustment to the 0.1～0.3 μ g/ μ l of test agent;

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

LjP1：5’-CGTAAC?AAG?GTT?TCC?GTA?GGT?GAA-3’

LjP2：5’-TTATTG?ATA?TGC?TTA?AAC?TCA?GCG?GG-3’

Add restriction enzyme EcoN I or its isoenzyme in c, the pcr amplification product and carry out enzyme and cut, the restriction enzyme site of restriction enzyme EcoN I or its isoenzyme is: CCTNN^NNNAGG;

D, agarose gel electrophoresis analysis;

The judgement of e, qualification result, if the band of 461bp significantly bright in or the band that closely equals 708bp then trial-product be the Japanese Honeysuckle in genuine producing region, if the significantly bright band in 461bp of the band of 708bp then trial-product be the Japanese Honeysuckle in non-genuine producing region.

Effect of the present invention is: can solve the evaluation difficult problem of genuine and non-genuine producing region Japanese Honeysuckle, provide and identify required one couple of PCR primers, PCR reaction conditions, the characteristic DNA base sequence of genuine producing region Japanese Honeysuckle, a kind of restriction enzyme.Compare with non-genuine Japanese Honeysuckle, the contained effective constituent of genuineness Japanese Honeysuckle has significant difference, and its drug effect is better than non-famous-region drug, so the price of genuineness Japanese Honeysuckle is higher than non-famous-region drug on the market.Yet genuine and non-genuine Japanese Honeysuckle is difficult to be distinguished by form, feature such as micro-, therefore, is badly in need of finding a kind of reliable discrimination method.The present invention utilizes the difference of genuine and non-famous-region drug dna sequence dna, has set up quick, convenient, reliable PCR-RFLP discrimination method, and for the quality of medicinal material of security deposit's honeysuckle flower, the market behavior of hitting the genuine producing region of personation Japanese Honeysuckle has significant values.

Four, description of drawings

Accompanying drawing 1: genuine producing region (Henan, Shandong) and non-genuine producing region (other areas) Japanese Honeysuckle PCR-RFLP product agarose gel electrophoretogram.A1: Fengqiu, Henan, A2: Xinmi City, Henan, A3: Fei County, Shandong, A4: Pingyi, Shandong, B1: Nanjing, B2: Hunan Province, B3: Guangzhou, Guangdong, B4: Kweiyang, Guizhou, B5: Chongqing force is fallen, B6: Kunming, Yunnan, MK:100bp DNA Ladder; 500:500bp position, 700:700bp position.

Five, embodiment:

The extraction of a, DNA: carry out routinely, will be with deionized water for DNA concentration adjustment to the 0.1～0.3 μ g/ μ l of test agent;

A, 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 LjP1, LjP2 (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

The high speed centrifugation several seconds behind the mixing

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

97 ℃ are given sex change 4min, carry out 30 circulations by following condition then:

97 ℃ of sex change 15 seconds

53 ℃ of renaturation 40～50 seconds

Extend 72 ℃ 30～50 seconds

Keep 10 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 1.5% 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 708bp;

The digestion with restriction enzyme reaction of b, polymerase chain reaction product:

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 10 μ l

10 * R ⁺Restriction enzyme damping fluid 2 μ l

Restriction enzyme EcoN I 2～5U

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;

D, electrophoresis observation enzyme are 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 1～2 hour under 80V voltage, observes electrophoresis result;

The judgement of e, measurement result is used restriction enzyme EcoN I or its isoenzyme that amplified product of polymerase chain reaction is carried out enzyme and is cut, and generation is cut dna fragmentation length and band brightness collection of illustrative plates to the enzyme that genuine and non-genuine producing region Japanese Honeysuckle has the distinctive feature.If the band of trial-product 461bp significantly bright in or the band that closely equals 708bp then be the Japanese Honeysuckle in genuine producing region, if the remarkable bright band in 461bp of the band of 708bp is the Japanese Honeysuckle in non-genuine producing region then, see accompanying drawing 1.Genuineness Japanese Honeysuckle polymerase chain reaction one restriction map authentication method＜110〉China Medicine University＜120〉genuineness Japanese Honeysuckle polymerase chain reaction one restriction map authentication method＜160〉1＜210〉1＜211〉31＜212〉DNA＜213〉Japanese Honeysuckle (Lonicera japonica Thunb.)＜400〉1gcccccccgc cccgcctccc acagggtcgc g

Claims

1 one kinds of distinctive DNA base sequences of genuineness Japanese Honeysuckle is characterized in that dna sequence dna is: 5 '-GCCCCCCCGC CCCGCCTCCC ACAGGGTCGC G-3 '.

2 one kinds of genuineness Japanese Honeysuckle polymerase chain reaction-restriction maps is characterized in that discerning the restriction enzyme EcoN I of the peculiar DNA base sequence of genuine producing region Japanese Honeysuckle and isoenzyme thereof the characteristic spectrum to the postdigestive agarose gel electrophoresis of polymerase chain reaction product.Authentication method is:

(1), the extraction of medicinal material DNA: carry out routinely, will be with deionized water for DNA concentration adjustment to the 0.1～0.3 μ g/ μ l of test agent;

(2), amplification of DNA fragments, promptly carry out polymerase chain reaction (PCR), the dna sequence dna that is used for a pair of primer of polymerase chain reaction is:

LjP1：5’-CGT?AAC?AAG?GTT?TCC?GTA?GGT?GAA-3’

LjP2：5’-TTA?TTG?ATA?TGC?TTA?AAC?TCA?GCG?GG-3’

(3), add restriction enzyme EcoN I or its isoenzyme in the pcr amplification product and carry out enzyme and cut, the restriction enzyme site of restriction enzyme EcoN I or its isoenzyme is: CCTNN^NNNAGG;

(4), agarose gel electrophoresis analysis;

(5), the judgement of qualification result: if the band of 461bp significantly bright in or the band that closely equals 708bp then trial-product be the Japanese Honeysuckle in genuine producing region; If the significantly bright band in 461bp of the band of 708bp then trial-product is the Japanese Honeysuckle in non-genuine producing region.