CN1438326A - Chinese-medicine Fritillaria-thunbergii ploymerase chain reaction appraising trigger and appraising method - Google Patents
Chinese-medicine Fritillaria-thunbergii ploymerase chain reaction appraising trigger and appraising method Download PDFInfo
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- CN1438326A CN1438326A CN 03113004 CN03113004A CN1438326A CN 1438326 A CN1438326 A CN 1438326A CN 03113004 CN03113004 CN 03113004 CN 03113004 A CN03113004 A CN 03113004A CN 1438326 A CN1438326 A CN 1438326A
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- primer
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- fritillary bulb
- thunberg fritillary
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Abstract
The invention belongs to traditional Chinese medicine appraisal field, it is priming for appraising the Chinese Fritillaria taipaiensis. The priming DNA sequence is: the priming 1:5'-CTAAATAGGCGGGCGAGCTA ATC-3'; the priming 2:5'-TATTAGCATT AATCGATCGAATTC-3'. The appraising method is: extracts the total DNA of medicinal materials; uses a couple of priming AS, AS-1 to expand the DNA in order to check its quality, if there is positive expansion, the model DNA is eligible; uses the appraising priming to expand it; uses electrophoresis to check the result, if gets the positive expansion, it is Chinese Fritillaria taipaiensis, if not, it isn't zhebeimu.
Description
One, technical field:
The present invention is a kind of primer and authentication method thereof that is used to identify the Chinese medicine Thunberg Fritillary Bulb, relates to that Chinese medicinal materials identifies
Technical field.
Two, background technology:
The bulb of fritillary is the important cough-relieving apophlegmatic medicine of a class, and its effective constituent is bulb of fritillary alkaloid.The version Chinese Pharmacopoeia recorded 9 kinds of the bulbs of fritillary in 2000, and its alkaloidal kind of the bulb of fritillary not of the same race and content have nothing in common with each other, and its toxicity and drug effect also there are differences.Thunberg Fritillary Bulb is to use more a kind ofly in the market, but its toxicity is also bigger, should control its consumption during use, thereby Thunberg Fritillary Bulb is accurately differentiated just very important.Yet, utilize form and physico-chemical method that its evaluation is needed rich experience, and the result is reliable inadequately, the especially more difficult discriminating of the medicinal material of fragmentation or pulverulence.And be suitable for the discriminating of species based on the molecular genetic marker technique of DNA of plants sequence, therefore, be badly in need of setting up the method that a kind of molecular genetic marker technique is identified Thunberg Fritillary Bulb.At present, molecular genetic marker is being widely used aspect the plant and animal species discriminating.Aspect the medicinal material evaluation, set up distinctive polymerase chain reaction (PCR) technology, and be successfully used to the discriminating of Chinese medicines such as tortoise plastron, deer class.But this method is not that all Chinese medicine is all had unified method and primer, for different Chinese medicinal materialss, and the method for its evaluation, primer is all inequality.
Three, summary of the invention:
The purpose of this invention is to provide a kind of primers designed and the authentication method thereof that can accurately differentiate the Chinese medicinal materials Thunberg Fritillary Bulb distinctive polymerase chain reaction of its true and false to the Thunberg Fritillary Bulb medicinal material.
The primers designed dna sequence dna of the Thunberg Fritillary Bulb polymerase chain reaction of the present invention's design is:
Primer 1:5 '-CTAAATAGGCGG GCGAGCTAATC-3 '
Primer 2: 5 '-TATTAGCATTAATCGATCGAATTC-3 '
Synthesize with the dna sequence dna of full-automatic dna synthesizer, can obtain the white powder crystallization of primers designed by above-mentioned primers designed.
The method that Chinese medicinal materials Thunberg Fritillary Bulb polymerase chain reaction is identified is:
(1) medicinal material DNA extraction: carry out routinely, with deionized water with trial-product dna profiling concentration adjustment to 0.2～0.5 μ g/ μ L.
(2) evaluation of template DNA quality: with another universal primer is differentiated the quality of template DNA, this is to the primer region sequence between the 5S rRNA of each kind of plant that can increase.The dna sequence dna of primer is: AS:5 '-GGATCC GTG CTT GGG CGA GAG TAG TA-3 '; AS-1:5 '-GGA TCC TTA GTG CTGGTA TGA TCG CA-3 ' (Cai ZH, Li P, Dong TX et, al.The molecular diversity of5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis.PlantaMedica, 1999,65:360-364.); Primer concentration is 30pmol/ μ l, and except that primer in the reaction system 1, primer 2 are changed into AS, the AS-1, other each article consumption is with (3); Except that the renaturation temperature changes into 53 ℃, amplification reaction condition is with (4); Electrophoretic analysis is with (5).If obtain positive amplification, show that then template is qualified, can carry out the check of subsequent step; If no amplified band, then need improve the DNA extraction condition, wait to obtain to carry out again after the qualified template check of subsequent step.
(3) distinctive PCR: primer is with the deionized water dissolving and be diluted to 30pmol/ μ L.Being 30 μ L with reaction system is reference point, and the consumption of each article is:
10 * TaqDNA polymerase buffer, 3 μ l
MgCl
2(25mM) 2.4μl
dNTP(10mM) 0.6μl
Primer 1, each 0.5 μ l of primer 2
Taq archaeal dna polymerase 1.0～2.0 units
Trial-product dna profiling 1.0～2.0 μ l
Deionized water is added to 30 μ l
(4) PCR circulation: on the PCR instrument, 95 ℃ are given sex change 4min, enter 30 circulations by following parameter then:
95 ℃ of sex change 10～50 seconds
60～65 ℃ of renaturation 20～50 seconds
Extend 72 ℃ 20～60 seconds
Keep 2～10 minutes polishings at 72 ℃ after the loop ends.
(5) electrophoresis observation: take out PCR reaction solution 4 μ l and mix with sample loading buffer 1 μ l, 2% agarose gel electrophoresis (containing 0.5% ethidium bromide, i.e. EB) detects amplification.
As total reaction volume is not 30 μ l, and the consumption of reaction article is a reference point with the ratio of 30 μ l amounts of reactants, correspondingly increases or reduces.
If positive amplification is arranged, then trial-product is a Thunberg Fritillary Bulb; If negative, promptly there is not amplified band, then trial-product is not a Thunberg Fritillary Bulb.
Effect of the present invention is:
The present invention solves the discriminating problem of bulb of fritillary class medicinal material Thunberg Fritillary Bulb.We are in the discriminating to Thunberg Fritillary Bulb, designed a pair of high specific primer of differentiating Thunberg Fritillary Bulb, this primers designed can only be differentiated Thunberg Fritillary Bulb specifically under given PCR condition, and all can not amplify this gene fragment for the bulb of fritillary of any other kind or total DNA of biomaterial extraction.After carrying out pcr amplification with this primers designed for test agent, observe the appearance that has or not amplified band through agarose gel electrophoresis, can judge the true and false of medicinal material exactly.Can make the test kit that is used for the Thunberg Fritillary Bulb discriminating with this to primer.The present invention differentiates the Thunberg Fritillary Bulb medicinal material quickly and accurately for the Chinese medicine department that produces and sells, and does the quality control of medicinal material well, is very necessary, hits fake and forged commodity for prefectures and cities' medicine inspection department, purifies medicinal material market and has crucial value.
Four, embodiment:
At first from bulb of fritillary class medicinal material former plant not of the same race, extract total DNA, with PCR method increase a plurality of gene fragments and purifying respectively, each purifying fragment is carried out dna sequence analysis, set up Thunberg Fritillary Bulb and sibling species thereof, obscure the dna sequence data storehouse of planting, on the basis of comparison database, select suitable dna fragmentation, the situation at medicinal material puppet, mixed product occur, design a pair of distinctive PCR primer:
Primer 1:5 '-CTAAATAGGCGG GCGAGCTAATC-3 '
Primer 2: 5 '-TATTAGCATTAATCGATCGAATTC-3 '
Synthesize with the dna sequence dna of full-automatic dna synthesizer, can obtain the white powder crystallization of primers designed by above-mentioned primers designed.
Adopt above-mentioned primer that the true and false of Thunberg Fritillary Bulb is differentiated, 30 μ l are example with reaction system, adopt following steps to carry out the distinctive polymerase chain reaction of Thunberg Fritillary Bulb:
(1) medicinal material DNA extraction: carry out routinely, with deionized water with trial-product dna profiling concentration adjustment to 0.2～0.5 μ g/ μ L.
(2) evaluation of template DNA quality: with another universal primer is differentiated the quality of template DNA, this is to can the increase 5S rRNA intergenic region sequence of each kind of plant of primer.The dna sequence dna of primer is: AS:5 '-GGA TCC GTG CTT GGG CGA GAG TAG TA-3 '; AS-1:5 '-GGA TCC TTAGTG CTG GTA TGA TCG CA-3 '; Primer concentration is 30pmol/l, and except that primer in the reaction system 1, primer 2 are changed into AS, the AS-1, other each article consumption is with (3); Except that the renaturation temperature is 53 ℃, amplification reaction condition is with (4); Electrophoretic analysis is with (5).If obtain positive amplification, show that then template is qualified, can carry out the check of subsequent step; If no amplified band, then need improve the DNA extraction condition, wait to obtain to carry out again after the qualified template check of subsequent step.
(3) distinctive PCR: primer is with the deionized water dissolving and be diluted to 30pmol/ μ L.Being 30 μ L with reaction system is reference point, and the consumption of each article is:
10 * TaqDNA polymerase buffer, 3 μ l
MgCl
2(25mM) 2.4μl
dNTP(10mM) 0.6μl
Primer 1, each 0.5 μ l of primer 2
Taq archaeal dna polymerase 1.0～2.0 units
Trial-product dna profiling 1.0～2.0 μ l
Deionized water is added to 30 μ l
(4) PCR circulation: on the PCR instrument, 95 ℃ are given sex change 4min, enter 30 circulations by following parameter then:
95 ℃ of sex change 10～50 seconds
60～65 ℃ of renaturation 20～50 seconds
Extend 72 ℃ 20～60 seconds
Keep 2～10 minutes polishings at 72 ℃ after the loop ends.
(5) electrophoresis observation: taking-up PCR reacts 4 μ l to be mixed with sample loading buffer 1 μ l, and 2% agarose gel electrophoresis (containing 0.5% ethidium bromide, i.e. EB) detects amplification.
If positive amplification is arranged, then trial-product is a Thunberg Fritillary Bulb; If negative, promptly there is not amplified band, then trial-product is not a Thunberg Fritillary Bulb.Chinese medicine Thunberg Fritillary Bulb polymerase chain reaction identification primers and authentication method＜110〉China Medicine University＜120〉Chinese medicine Thunberg Fritillary Bulb polymerase chain reaction identification primers and authentication method＜160〉2＜210〉1＜211〉23＜212〉DNA＜2 13〉Thunberg Fritillary Bulb, (Fritillaria thunbergii Miq.)＜400〉1ctaaataggc gggcgagcta atc＜210〉2＜211〉24＜212〉DNA＜213〉Thunberg Fritillary Bulb, (Fritillaria thunbergii Miq.)＜400〉1tattagcatt aatcgatcga attc
Claims (3)
1, a kind of Chinese medicine Thunberg Fritillary Bulb polymerase chain reaction identification primers, it is characterized in that: the primers designed dna sequence dna is: primer 1:5 '-CTAAATAGGC GGGCGAGCTA ATC-3 '; Primer 2: 5 '-TATTAGCATTAATCGATCGAATTC-3 ' synthesizes with the dna sequence dna of full-automatic dna synthesizer by above-mentioned primers designed, promptly obtains the white powder crystallization of primers designed.
2, identify the method for Chinese medicine Thunberg Fritillary Bulb according to the described primers designed of claim 1, it is characterized in that Chinese medicinal materials Thunberg Fritillary Bulb polymerase chain reaction (PCR) authentication step is:
(1) medicinal material DNA extraction: carry out routinely, with deionized water with trial-product dna profiling concentration adjustment to 0.2～0.5 μ g/ μ L.
(2) evaluation of template DNA quality: with another quality to universal primer discriminating template DNA, this is to region sequence between the 5S rRNA of each kind of plant of primer amplification.The dna sequence dna of primer is: AS:5 '-GGATCCGTG CTT GGG CGA GAG TAG TA-3 '; AS-1:5 '-GGA TCC TTA GTG CTG GTATGA TCG CA-3 '; Primer concentration is 30pmol/ μ l, and except that primer in the reaction system 1, primer 2 are changed into AS, the AS-1, other each article consumption is with (3); Except that the renaturation temperature changes into 53 ℃, amplification reaction condition is with (4); Electrophoretic analysis is with (5).If obtain positive amplification, show that then template is qualified, can carry out the check of subsequent step; If no amplified band, then need improve the DNA extraction condition, wait to obtain to carry out again after the qualified template check of subsequent step.
(3) distinctive PCR: primer is with the deionized water dissolving and be diluted to 30pmol/ μ L.Being 30 μ L with reaction system is reference point, and the consumption of each article is:
10 * TaqDNA polymerase buffer, 3 μ l
MgCl
2(25mM) 2.4μl
dNTP(10mM) 0.6μl
Primer 1, each 0.5 μ l of primer 2
Taq archaeal dna polymerase 1.0～2.0 units
Trial-product dna profiling 1.0～2.0 μ l
Deionized water is added to 30 μ l
(4) PCR circulation: on the PCR instrument, 95 ℃ are given sex change 4min, enter 30 circulations by following parameter then:
95 ℃ of sex change 10～50 seconds
60～65 ℃ of renaturation 20～50 seconds
Extend 72 ℃ 20～60 seconds
Keep 2～10 minutes polishings at 72 ℃ after the loop ends.
(5) electrophoresis observation: take out PCR reaction solution 4 μ l and mix with sample loading buffer 1 μ l, 2% agarose gel electrophoresis (containing 0.5% ethidium bromide, i.e. EB) detects amplification.
As total reaction volume is not 30 μ l, and the consumption of reaction article is a reference point with the ratio of 30 μ l amounts of reactants, correspondingly increases or reduces.
If positive amplification is arranged, then trial-product is a Thunberg Fritillary Bulb; If negative, promptly there is not amplified band, then trial-product is not a Thunberg Fritillary Bulb.
3, according to the described primers designed of claim 1, it is characterized in that: the primers designed dna sequence dna is: primer 1:5 '-CTAAATAGGC GGGCGAGCTA ATC-3 '; Primer 2: 5 '-TATTAGCATTAATCGATCGA ATTC-3 ', with the distinctive pcr reagent box and the authentication method thereof that are used to differentiate Thunberg Fritillary Bulb of this design of primers.
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|---|---|---|---|
| CNB031130046A CN1177062C (en) | 2003-03-20 | 2003-03-20 | Chinese-medicine Fritillaria-thunbergii ploymerase chain reaction appraising trigger and appraising method |
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| CNB031130046A CN1177062C (en) | 2003-03-20 | 2003-03-20 | Chinese-medicine Fritillaria-thunbergii ploymerase chain reaction appraising trigger and appraising method |
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| CN1177062C CN1177062C (en) | 2004-11-24 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899409A (en) * | 2012-09-20 | 2013-01-30 | 浙江工业大学 | PCR identification kit for Thunberg fritillary bulb and identification method |
| CN107164486A (en) * | 2017-06-07 | 2017-09-15 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for Identification chinese herbs medicine bulbus fritillariae cirrhosae |
| CN107164490A (en) * | 2017-06-07 | 2017-09-15 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for being used to identify fritillaria thunbergii |
| CN109554499A (en) * | 2018-12-21 | 2019-04-02 | 康美华大基因技术有限公司 | A kind of primer sets, the detection method of fritillaria and kit |
-
2003
- 2003-03-20 CN CNB031130046A patent/CN1177062C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899409A (en) * | 2012-09-20 | 2013-01-30 | 浙江工业大学 | PCR identification kit for Thunberg fritillary bulb and identification method |
| CN107164486A (en) * | 2017-06-07 | 2017-09-15 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for Identification chinese herbs medicine bulbus fritillariae cirrhosae |
| CN107164490A (en) * | 2017-06-07 | 2017-09-15 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for being used to identify fritillaria thunbergii |
| CN109554499A (en) * | 2018-12-21 | 2019-04-02 | 康美华大基因技术有限公司 | A kind of primer sets, the detection method of fritillaria and kit |
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| Publication number | Publication date |
|---|---|
| CN1177062C (en) | 2004-11-24 |
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Granted publication date: 20041124 |