CN102251026A - Detection primer for medicinal plant liriope muscari (decn.) bailey molecule and detection method thereof - Google Patents
Detection primer for medicinal plant liriope muscari (decn.) bailey molecule and detection method thereof Download PDFInfo
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- CN102251026A CN102251026A CN2011101216851A CN201110121685A CN102251026A CN 102251026 A CN102251026 A CN 102251026A CN 2011101216851 A CN2011101216851 A CN 2011101216851A CN 201110121685 A CN201110121685 A CN 201110121685A CN 102251026 A CN102251026 A CN 102251026A
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Abstract
The invention discloses a detection primer for a medicinal plant liriope muscari (decn,) bailey molecule and a detection method thereof. The detection primer comprises a pair of PCR (Polymerase Chain Reaction) detection primers for amplifying a chloroplast trnL-F sequence and a pair of PCR detection primers for amplifying a ribosome ITS (Internal Transcribed Spacer) sequence, wherein each reaction primer consists of forward and reverse primers. The detection method can be used for quickly and accurately judging whether a sample is the medicinal plant liriope muscari (decn.) bailey molecule, and has the characteristic of capability of quickly and effectively identifying liriope muscari (decn.) bailey and a hybrid strain and intraspecific variation thereof.
Description
Technical field
The present invention relates to a kind of detection primer and detection method thereof of medicinal plant liriope muscari Baily molecule.The quality authenticate technology field that belongs to the medicinal material germplasm.
Background technology
Liriope muscari Baily (Liriope Muscari (Decn.) Bailey) is a liliaceous plant Radix Liriopes platymiscium, is used as medicine with dried root.Nineteen ninety-five, " Chinese pharmacopoeia was recorded so far with the name of " Radix Liriopes ", has the effect that nourishing Yin and promoting production of body fluid, moistening lung clear away heart-fire, and is used for diseases such as dryness of the lung dry cough, cough due to consumptive disease, thirsty, the vexed insomnia of Tianjin wound, the dry constipation of intestines.Modern pharmacological research shows, contains compositions such as steroid saponin, polyose in the liriope muscari Baily medicinal material, and the function of improving cardiovascular, immunomodulatory, Respiratory Regulation is arranged, but lowering blood glucose, antitumor, anti-inflammatory, anti-arrhythmia and antifatigue etc.
It is investigated that Ophiopogon 18 kinds (mutation) and Radix Liriopes belong to 16 kinds of tool piece roots and valuable in 8 kinds (mutation), very easily obscure.Chinese medicinal materials is differentiated and methods such as adopting basic source phytomorph, medicinal material proterties, micro-and physics and chemistry usually to be had certain drawback.The tuber of dwarf lilyturf class medicinal material, its plant forms type is abundant, the kind boundary is very undistinct, identifies disputable according to the morphological classification method.Phenotypic character alters a great deal in the L.Muscari kind, has to be mainly used in the Root of Broadlaf liriope of viewing and admiring, and main medicinal liriope muscari Baily is arranged.Molecular biology research also shows and has higher genetic diversity in its kind.It is the genuinest that liriope muscari Baily produces medicinal material with Quanzhou, Fujian and two areas, Putian again.Institute thinks the assurance drug safety, liriope muscari Baily is effectively differentiated seem particularly important.
Summary of the invention
A kind of detection primer and detection method thereof of medicinal plant liriope muscari Baily molecule have been the object of the present invention is to provide.Whether detection method of the present invention judgement sample rapidly and accurately is medicinal plant liriope muscari Baily molecule, has can differentiate liriope muscari Baily fast and effectively and obscure the characteristics of planting and planting interior variation.
In order to reach above-mentioned purpose, solution of the present invention is:
A kind of detection primer of medicinal plant liriope muscari Baily molecule, comprise the one couple of PCR reaction detection primer of the chloroplast(id) trnL-F sequence that is used to increase and the one couple of PCR reaction detection primer of the rrna ITS sequence that is used to increase, described reaction primer is formed by forward and reverse primer, and the dna sequence dna of the one couple of PCR reaction detection primer of the chloroplast(id) trnL-F sequence that wherein is used to increase is:
SEQ?ID?No.1：5’-CGAAATCGGTAGACGCTACG-3’；
SEQ?ID?No.2：5’-ATTTGAACTGGTGACACGAG-3’；
The dna sequence dna of one couple of PCR reaction detection primer of rrna ITS sequence of being used to increase is: SEQ IDNo.3:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ';
SEQ?ID?No.4：5’-TCCTCCGCTTATTGATATGC-3’。
A kind of detection method of medicinal plant liriope muscari Baily molecule, it is characterized in that comprising the steps: the collection of liriope muscari Baily blade, utilize existing modified CTAB method to extract genomic dna, comprise the TrnL-F characteristic DNA sequence and the ITS characteristic DNA sequence of liriope muscari Baily; Be template with the TrnL-F characteristic DNA sequence and the ITS characteristic DNA sequence of liriope muscari Baily respectively then, utilize above-mentioned primer, carry out pcr amplification respectively, reaction finishes the back and detects amplified production with agarose gel electrophoresis, according to the location determination result of amplification of DNA fragments, when amplification trnL-F sequence, amplify a 400bp band limpid in sight; Just be accredited as the medicinal plant liriope muscari Baily when when amplification ITS sequence, then being a 690bp band.
Described PCR reaction system is:
PCR reaction system cumulative volume 25 μ L, (following reagent is all available from Sangon Biotech (Shanghai) Co., Ltd.)
10×buffer 2.5μL；
MgCl
2(25mmol/L) 1.5μL；
dNTP?mixture(10mmol/L) 0.5μL；
Each 1.5 μ L of primer (10 μ mol/L);
Dna profiling (10mg/L) 5.0 μ L;
Taq DNA enzyme (5U/ μ L) 0.3 μ L;
Sterilization redistilled water 12.2 μ L.
Described PCR response procedures is: 95 ℃ of pre-sex change 4min, and 95 ℃ of sex change 50sec, 50 ℃ of annealing 50sec, 72 ℃ are extended 90sec, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations.
Beneficial effect of the present invention is: whether detection method of the present invention judgement sample rapidly and accurately is medicinal plant liriope muscari Baily molecule, its result is not affected by environment, result's repeatability and accuracy are all higher, can be its former plant discriminating the molecule foundation is provided, have and to differentiate liriope muscari Baily fast and effectively and obscure the characteristics of planting and planting interior variation.
Embodiment
Embodiment 1
A kind of detection primer of medicinal plant liriope muscari Baily molecule, comprise the one couple of PCR reaction detection primer of the chloroplast(id) trnL-F sequence that is used to increase and the one couple of PCR reaction detection primer of the rrna ITS sequence that is used to increase, described reaction primer is formed by forward and reverse primer, and the dna sequence dna of the one couple of PCR reaction detection primer of the chloroplast(id) trnL-F sequence that wherein is used to increase is:
SEQ?ID?No.1：5’-CGAAATCGGTAGACGCTACG-3’；
SEQ?ID?No.2：5’-ATTTGAACTGGTGACACGAG-3’；
The dna sequence dna of one couple of PCR reaction detection primer of rrna ITS sequence of being used to increase is: SEQ IDNo.3:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ';
SEQ?ID?No.4：5’-TCCTCCGCTTATTGATATGC-3’。
A kind of detection method of medicinal plant liriope muscari Baily molecule comprises the steps: the collection of liriope muscari Baily blade, utilizes existing modified CTAB method to extract genomic dna, comprises the TrnL-F characteristic DNA sequence and the ITS characteristic DNA sequence of liriope muscari Baily; Be template with the TrnL-F characteristic DNA sequence and the ITS characteristic DNA sequence of liriope muscari Baily respectively then, utilize above-mentioned primer, carry out pcr amplification respectively, reaction finishes the back and detects amplified production with agarose gel electrophoresis, according to the location determination result of amplification of DNA fragments, when amplification trnL-F sequence, amplify a 400bp band limpid in sight; Just be accredited as the medicinal plant liriope muscari Baily when when amplification ITS sequence, then being a 690bp band.
Described PCR reaction system is:
PCR reaction system cumulative volume 25 μ L,
10×buffer 2.5μL；
MgCl
2(25mmol/L) 1.5μL；
dNTP?mixture(10mmol/L) 0.5μL；
Each 1.5 μ L of primer (10 μ mol/L);
Dna profiling (10mg/L) 5.0 μ L;
Taq DNA enzyme (5U/ μ L) 0.3 μ L;
Sterilization redistilled water 12.2 μ L.
The PCR response procedures is: 95 ℃ of pre-sex change 4min, and 95 ℃ of sex change 50sec, 50 ℃ of annealing 50sec, 72 ℃ are extended 90sec, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations.
After amplified production is purified, use forward, reverse two primers to check order.Sequencing result uses sequence software for editing and sequence alignment software, with reference to kindred plant sequence among the GenBank, determines rDNA internal transcribed spacer district ITS1 and ITS2 and 3 coding region 18S, the boundary of 5.8S and 26S.Determine the border sequence of chloroplast(id) trnL-F and gene trnL and trnF.Wherein liriope muscari Baily TrnL-F sequence is seen SEQ ID No.5; Liriope muscari Baily ITS sequence is seen SEQ ID No.6.In all materials for examination, the TrnL-F sequence height unanimity of liriope muscari Baily, with the tuber of dwarf lilyturf key distinction on 3 base sites, be respectively 113 by A-G, 303 by A-T, 327 by C-T.Root of Broadlaf liriope is at the 35th disappearance base T, and 78 disappearance A, 156 and 233 are by G-A, 338 T-G.
In the specimen material of liriope muscari Baily, sample sequence is in full accord, long 624bp.The ITS1 district is 1-240bp, and the 5.8S district is 241-402bp, and ITS2 is 403-624bp.Have 19 of variant sites, wherein 10 is the information site, is distributed on 4 sites of 100,118,211 and 212 in ITS1 district, on 1 point that the 5.8S district is 247, on 5 sites of 431,480,494,561 and 578 in ITS2 district.In addition, find in the liriope muscari Baily kind, to have 2 samples in one section GCCTCGGCG sequence of 200 to 208 disappearances.
Edit and compare, draw comparison result.If meeting the liriope muscari Baily trnL-F sequence and the ITS sequence of foregoing description, the result (amplifies a 400bp band limpid in sight during amplification trnL-F sequence; When amplification ITS sequence, then being a 690bp band), just be accredited as the medicinal plant liriope muscari Baily.
Claims (4)
1. the detection primer of a medicinal plant liriope muscari Baily molecule, it is characterized in that comprising and be used to increase the increase one couple of PCR reaction detection primer of rrna ITS sequence of the one couple of PCR reaction detection primer and being used to of chloroplast(id) trnL-F sequence, described reaction primer is formed by forward and reverse primer, and the dna sequence dna of the one couple of PCR reaction detection primer of the chloroplast(id) trnL-F sequence that wherein is used to increase is:
SEQ?ID?No.1：5’-CGAAATCGGTAGACGCTACG-3’；
SEQ?ID?No.2：5’-ATTTGAACTGGTGACACGAG-3’；
The dna sequence dna of one couple of PCR reaction detection primer of rrna ITS sequence of being used to increase is: SEQ IDNo.3:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ';
SEQ?ID?No.4：5’-TCCTCCGCTTATTGATATGC-3’。
2. the detection method of a medicinal plant liriope muscari Baily molecule as claimed in claim 1, it is characterized in that comprising the steps: the collection of liriope muscari Baily blade, utilize modified CTAB method to extract genomic dna, comprise the TrnL-F characteristic DNA sequence and the ITS characteristic DNA sequence of liriope muscari Baily; Be template with the TrnL-F characteristic DNA sequence and the ITS characteristic DNA sequence of liriope muscari Baily respectively then, utilize above-mentioned primer, carry out pcr amplification respectively, reaction finishes the back and detects amplified production with agarose gel electrophoresis, according to the location determination result of amplification of DNA fragments, when amplification trnL-F sequence, amplify a 400bp band limpid in sight; Just be accredited as the medicinal plant liriope muscari Baily when when amplification ITS sequence, then being a 690bp band.
3. the detection method of a kind of medicinal plant liriope muscari Baily molecule as claimed in claim 2 is characterized in that: described PCR reaction system is:
PCR reaction system cumulative volume 25 μ L,
10×buffer 2.5μL；
MgCl
2(25mmol/L) 1.5μL；
dNTP?mixture(10mmol/L) 0.5μL；
Each 1.5 μ L of primer (10 μ mol/L);
Dna profiling (10mg/L) 5.0 μ L;
Taq DNA enzyme (5U/ μ L) 0.3 μ L;
Sterilization redistilled water 12.2 μ L.
4. the detection method of a kind of medicinal plant liriope muscari Baily molecule as claimed in claim 2 is characterized in that: described PCR response procedures is: 95 ℃ of pre-sex change 4min, 95 ℃ of sex change 50sec, 50 ℃ of annealing 50sec, 72 ℃ are extended 90sec, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations.
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Cited By (3)
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| CN102952878A (en) * | 2012-09-28 | 2013-03-06 | 中国科学院过程工程研究所 | ITS (Internal Transcribed Spacer) sequence and method for identifying certified lepidium meyenii walp and counterfeit lepidium meyenii walp as well as doped lepidium meyenii walp |
| CN105087564A (en) * | 2015-08-21 | 2015-11-25 | 福建中医药大学 | Specific molecular marking primer and method for identifying sarcandra glabra and three adulterants |
| CN108315467A (en) * | 2018-04-04 | 2018-07-24 | 杭州师范大学 | The specific molecular marker LPMI001 differentiated for Root of Broadlaf liriope |
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| CN1357635A (en) * | 2000-12-05 | 2002-07-10 | 中国科学院植物研究所 | Certified rhubarb product identifying method and reagent kit |
| CN1970751A (en) * | 2006-08-09 | 2007-05-30 | 南京师范大学 | Method for extracting whole genome DNA from Apocymum venetum L. leaves |
| CN101191144A (en) * | 2006-11-22 | 2008-06-04 | 中国科学院大连化学物理研究所 | Method for identifying and distinguishing useful yew correlated with taxane content |
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2011
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Patent Citations (3)
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| CN1357635A (en) * | 2000-12-05 | 2002-07-10 | 中国科学院植物研究所 | Certified rhubarb product identifying method and reagent kit |
| CN1970751A (en) * | 2006-08-09 | 2007-05-30 | 南京师范大学 | Method for extracting whole genome DNA from Apocymum venetum L. leaves |
| CN101191144A (en) * | 2006-11-22 | 2008-06-04 | 中国科学院大连化学物理研究所 | Method for identifying and distinguishing useful yew correlated with taxane content |
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| 赵海光: "基于ITS和trnL-F序列碱基差异的繁缕及其近缘种的亲缘关系分析", 《植物资源与环境学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952878A (en) * | 2012-09-28 | 2013-03-06 | 中国科学院过程工程研究所 | ITS (Internal Transcribed Spacer) sequence and method for identifying certified lepidium meyenii walp and counterfeit lepidium meyenii walp as well as doped lepidium meyenii walp |
| CN102952878B (en) * | 2012-09-28 | 2014-07-02 | 中国科学院过程工程研究所 | ITS (Internal Transcribed Spacer) sequence and method for identifying certified lepidium meyenii walp and counterfeit lepidium meyenii walp as well as doped lepidium meyenii walp |
| CN105087564A (en) * | 2015-08-21 | 2015-11-25 | 福建中医药大学 | Specific molecular marking primer and method for identifying sarcandra glabra and three adulterants |
| CN105087564B (en) * | 2015-08-21 | 2017-11-17 | 福建中医药大学 | Differentiate the molecular specificity labeled primers and method of Chloranthus glaber and 3 kinds of adulterants |
| CN108315467A (en) * | 2018-04-04 | 2018-07-24 | 杭州师范大学 | The specific molecular marker LPMI001 differentiated for Root of Broadlaf liriope |
| CN108315467B (en) * | 2018-04-04 | 2019-02-26 | 杭州师范大学 | The specific molecular marker LPMI001 identified for Root of Broadlaf liriope |
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Application publication date: 20111123 |