CN104818337A - Method for identifying Fenyuan No.2 Yuanzhi by using specific primers - Google Patents
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Abstract
The invention provides a method for identifying Fenyuan No.2 Yuanzhi by using specific primers, which comprises the following steps: designing 8 pairs of specific primers capable of quickly detecting Fenyuan No.2 Yuanzhi on the basis of the ITS sequence of the Fenyuan No.2 Yuanzhi, and establishing a PCR (polymerase chain reaction) detection system on such basis. The method can respectively amplify the 367bp or 463bp PCR amplification product, thereby providing molecular identification references for drug identification of the Fenyuan No.2 Yuanzhi. The method can be utilized to construct a detection kit, is simple to operate, has the advantages of favorable specificity, high sensitivity, no need of sequencing and the like, and can identify the Fenyuan No.2 Yuanzhi.
Description
Technical field
The present invention relates to the molecular Biological Detection technology of polygala root, specifically a kind of method adopting Auele Specific Primer to differentiate Fen No. 2 polygala roots far away.
Background technology
Polygala root derives from the dry root of milk wort polygala root Polygala tenuifolia Willd. and ovum leaf polygala root P.sibirica L., is conventional Chinese medicine, is recorded in Shennong's Herbal the earliest, be listed in top grade, and is regarded as supporting life key medicine.Warm in nature, bitter, pungent, through modern study, the function of have intelligence development of calming the nerves, eliminate the phlegm, subsiding a swelling, finds that polygala root has antibacterial, anti-inflammatory, improves cognition, Improving memory ability, anti-senile dementia, antidepressant, antitumor, the anti-ageing effect of waiting for a long time.The principle active component of polygala root is saponin(e, mouth mountain ketone, oligosaccharides ester and alkaloid etc., wherein: polygalic acid is expelling phlegm for arresting cough in polygala root, calm main active ingredient; In polygala root, sugar esters composition 3,6 ＇-two mustard acyl sucrose has antidepressant effect; Polygala root sugar ester A, C are the basic substances of polygala root sedative action.In recent years, because the continuous increase of Polygala tenuifolia usage quantity and the unreasonable of wild resource thereof are excavated, the wild storage capacity of Polygala tenuifolia declines just year by year, cannot meet the growing demand of people.From the eighties in last century, just start domestication and the artificial growth of Wild Polygala tenuifolia, formed multiple large-scale production planting bases of polygala root at present.But the production of traditional Chinese medicine material plantation is due to without commercial seed source; primitive stage that be still in an extensive style at present, that certainly pick up from kind; therefore; be necessary at present and be badly in need of carrying out fine-variety breeding to existing goods polygala root; and then for its large-scale production plantation fine germplasm resources is provided; Polygala tenuifolia is produced towards high-quality, high yield, steady quality, controlled future development, realizes the Sustainable development of resource.
Shanxi No. 1 and Fen No. 2 polygala roots (P.tenuifolia Willd.) far away far away are two polygala root kinds of Academy of Agricultural Sciences of Shanxi Province Fenyang cash crop institute (hereinafter referred to as Fenyang through doing) seed selection.Shanxi far away No. 1 and Fen far away No. 2 more close in morphology, Shanxi is far away No. 1: this strain plant height 35-45cm, and leaf look dark green, fresh round plant type, long 25-30cm, diameter 0.4-0.8cm, yellow-white, thickness is comparatively even, and anti-root rot, resistance to stain, drought tolerance are comparatively strong, 2.5 years breeding times; Fen is far away No. 2: this strain plant height 25-35cm, and leaf color jade green, fresh cylindrical, and flowers and fruits all concentrate on top.Fresh long 28-32cm, diameter 0.6-0.8cm, even thickness, root body is close with nearly reed head position root skin color, yellow-white, 2.5 years breeding times, following proterties comparatively parent (fresh cylindrical branch is more for Hongdong Local variety: plant height 30-35cm, dark green leaf color, flowers and fruits concentrate on overground part complete stool, fresh long 25cm-30cm) there is obvious difference.But the dry root of the just Polygala tenuifolia circulated in medicinal material market at present, and there are rattlesnaker oot and the different commercial grades (as length, thickness etc.) of cylinder, therefore only rely on the profile of medicinal material to distinguish that No. 1 far away of Shanxi also exists larger difficulty with No. 2 far away of Fen.
Scholar is had once to carry out deep research for the Ji Yuan of polygala root and medicinal material, traditional authentication method comprises: base chiller, macroscopical identification, microscopical identification and physics and chemistry qualification etc., and modern discrimination method comprises: chromatography, mass spectroscopy, nuclear magnetic resonance method, differential thermal analysis, scanning electron-microscopy, electrophoresis etc.And along with modern molecular biology technique research deeply and development, some molecular marking techniques such as restriction fragment length polymorphism (RFLP) analysis, randomly amplified polymorphic DNA (RAPD) analysis, the technology such as simple sequence repeats (ISSR) mark, amplified fragment length polymorphism (AFLP) analysis of being interrupted are widely used, but they also also exist a lot of deficiencies.RFLP technical result is stablized, and is that codominance is expressed, can distinguishes heterozygosis, pure and mild, but to DNA demand comparatively large and somewhat expensive, complicated operation; RAPD has popularity and versatility, have lower to the specification of quality of DNA, technology is easy, it is rapid to detect, the advantage such as highly sensitive, workable, its shortcoming is mainly that it is a dominant marker, can not differentiate heterozygote and the problem such as homozygote and common migration, poor repeatability.ISSR mark has quick, easy, polymorphism advantages of higher, but its primer poor universality, amplification condition etc. need to design targetedly; AFLP technology has the reliability of RFLP technology and the high efficiency of round pcr, but to genomic dna purity and restriction endonuclease specification of quality higher, running cost is also relatively high, and the molecule marker obtained mostly is dominant marker.Internal Transcribed Spacer (ITS) sequence is present in the rrna highly repeated, and rate of evolution is fast and length is little, adds that coevolution makes this fragment very consistent between genome different units, is thus applicable to carrying out various molecule manipulation.Because this region is less by the impact of outside environmental elements, rate of evolution is fast, thus having intraspecific variablity little and to make a variation large feature between planting, is the important molecular markers of Identification of Species and Phylogenetic Analysis, can be used for the research of biological classification, phylogeny and genetic diversity.
The present invention is in conjunction with the advantage of above Method and Technology, and according to principle and the principle of molecular marking technique, design 8 pairs of specific PCR diagnostic primerses, object is the molecular method that proposition one species specificity differentiates Fen No. 2 polygala roots far away, and wishes to obtain practical application.
Summary of the invention
The object of this invention is to provide a kind of Auele Specific Primer pair can differentiating Fen No. 2 polygala roots far away quickly and accurately, and adopt this Auele Specific Primer to differentiate the method for Fen No. 2 polygala roots far away.
For realizing object of the present invention, a kind of Auele Specific Primer pair for differentiating Fen No. 2 polygala roots far away provided by the invention, described Auele Specific Primer is to being in following (1) to (8) primer pair any pair:
(1) upstream primer F1:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R1:5 ＇ TCGCCCCAAGAGATGTGA 3 ＇
(2) upstream primer F2:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R2:5 ＇ TCGCCCCAAGAGATCAGA 3 ＇
(3) upstream primer F3:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R3:5 ＇ TCGCCCCAAGAGATGTGA 3 ＇
(4) upstream primer F4:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R4:5 ＇ TCGCCCCAAGAGATCAGA 3 ＇
(5) upstream primer F5:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R5:5 ＇ ACCAAGTATCGCATTTCGC 3 ＇
(6) upstream primer F6:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R6:5 ＇ ACCAAGTATCGCATTTCGC 3 ＇
(7) upstream primer F7:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R7:5 ＇ TCGCCCCAAGAGATGAGA 3 ＇
(8) upstream primer F8:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R8:5 ＇ TCGCCCCAAGAGATGAGA 3 ＇
Above primer sequence number consecutively in sequence table is: SEQ ID NO:1 to SEQ ID NO:18.
A kind of method adopting Auele Specific Primer to differentiate Fen No. 2 polygala roots far away provided by the invention, is characterized in that, comprise the following steps:
1) DNA in polygala root sample is extracted;
2) with step 1) in extract DNA be template, use any pair primer in the primer pair of above-mentioned (1) to (8) to carry out pcr amplification reaction;
3) analyze PCR primer with agarose gel electrophoresis, have pcr amplification band person to be Fen No. 2 polygala roots far away, without pcr amplification band person then non-Fen No. 2 polygala roots far away.
Described pcr amplification reaction system is:
Described Auele Specific Primer is to being in the primer pair of above-mentioned (1) to (8) any pair.
The PCR reaction conditions of described primer pair:
(1) to (4) is the PCR reaction conditions of primer pair: 93 DEG C of 5min; 93 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations; 72 DEG C of 5min, 10 DEG C of 10min;
(5) to (8) to the PCR reaction conditions of primer pair is: 93 DEG C of 5min; 93 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 30s, totally 30 circulations; 72 DEG C of 5min, 10 DEG C of 10min.
The present invention is based on the ITS sequence of Fen No. 2 polygala roots far away, designing can the Auele Specific Primer of rapid detection Fen No. 2 polygala roots far away, and sets up PCR detection system on this basis, for the discriminating of Fen No. 2 polygala roots far away provides Molecular Identification foundation.Easy and simple to handle according to the detection kit that the method builds, specificity is good, highly sensitive, can realize the detection to Fen No. 2 polygala roots far away, is suitable for promoting the use of widely.
Accompanying drawing explanation
Fig. 1 utilizes the Auele Specific Primer of Fen No. 2 polygala roots far away to the agarose gel electrophoresis figure of 1-4 to Shanxi No. 1 polygala root far away and Fen No. 2 polygala root pcr amplification products far away, wherein the DNA profiling of 1-1,2-1,3-1,4-1 is Shanxi No. 1 polygala root far away, the DNA profiling of 1-2,2-2,3-2,4-2 is Fen No. 2 polygala roots far away, and product band is all 463bp; M is DL 2000DNA Marker, from top to bottom as 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.
Fig. 2 utilizes the Auele Specific Primer of Fen No. 2 polygala roots far away to the agarose gel electrophoresis figure of 5-8 to Shanxi No. 1 polygala root far away and Fen No. 2 polygala root pcr amplification products far away, wherein the DNA profiling of 5-1,6-1,7-1,8-1 is Shanxi No. 1 polygala root far away, the DNA profiling of 5-2,6-2,7-2,8-2 is Fen No. 2 polygala roots far away, the band of 5-2,6-2 product is 367bp, 7-2, the band of 8-2 product is 463bp; M is DL 2000DNA Marker.
Fig. 3 utilizes primer pair 1 to increase the agarose gel electrophoresis figure of Fen No. 2 polygala roots far away of different concns, Fen No. 2 polygala root PCR primer bands far away that wherein No. 1-5 is Fen No. 2 polygala root DNA weaker concns far away when being 0.01715ng/ μ l, 0.0143ng/ μ l, 0.0122ng/ μ l, 0.01068ng/ μ l, 0.0095ng/ μ l, 463bp; M is DL 2000DNA Marker.
Embodiment
Following examples for illustration of present method, but are not used for limiting the scope of the invention.
If do not specialize, following examples are experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (NewYork:Gold Spring Harbor Laboratory Press, 1989) the operative technique code described in, or according to the experiment condition that manufacturer advises; Raw materials usedly be commercial goods.
Embodiment 1 is for detecting the synthesis of the PCR primer of Fen No. 2 polygala roots far away
From GenBank, obtain rogation flower ITS sequence 7 altogether, Shanxi No. 1 polygala root far away and Fen No. 2 polygala roots far away checked order simultaneously, utilize the above-mentioned sequence of biological software DNAStar comparison, find out Fen No. 2 polygala roots far away be different from the variant sites of other kind polygala roots.According to the position of variant sites, respectively design the primer of about 20 bases respectively in the upstream and downstream of ITS sequence; Utilize the primer of biological software Primer5.0 to design to analyze, finally determine to be respectively the primer pair that Fen No. 2 polygala roots far away are suitable:
(1) upstream primer F1:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R1:5 ＇ TCGCCCCAAGAGATGTGA 3 ＇
(2) upstream primer F2:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R2:5 ＇ TCGCCCCAAGAGATCAGA 3 ＇
(3) upstream primer F3:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R3:5 ＇ TCGCCCCAAGAGATGTGA 3 ＇
(4) upstream primer F4:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R4:5 ＇ TCGCCCCAAGAGATCAGA 3 ＇
(5) upstream primer F5:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R5:5 ＇ ACCAAGTATCGCATTTCGC 3 ＇
(6) upstream primer F6:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R6:5 ＇ ACCAAGTATCGCATTTCGC 3 ＇
(7) upstream primer F7:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R7:5 ＇ TCGCCCCAAGAGATGAGA 3 ＇
(8) upstream primer F8:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R8:5 ＇ TCGCCCCAAGAGATGAGA 3 ＇
Primer is synthesized by Sani bio tech ltd, Shanghai.
Embodiment 2 utilizes PCR primer to detect specificity and the sensitivity analysis of Fen No. 2 polygala roots far away
1.1 sample source
The medicinal material sample adopted in the present embodiment includes: Shanxi No. 1 polygala root far away, Fen No. 2 polygala roots far away, picks up from Economic Crops Inst., Shanxi Provincial Academy of Agriculture Sciences.
1.2DNA extract
By above-mentioned sample, about 60mg got by each sample, uses liquid nitrogen grinding powdered, utilizes RNA isolation kit to extract DNA.Test kit is purchased from Ai Delai bio tech ltd, Beijing, and model is: CTAB plant genome DNA rapid extraction test kit (article No.: DN14), 50 times.
The gradient dilution of 1.3 sample DNAs
The concentration detecting Fen No. 2 polygala root DNA far away of said extracted is 17.15ng/ μ l.DNA sample is carried out gradient dilution, and the concentration after dilution is respectively 0.01715ng/ μ l, 0.0143ng/ μ l, 0.0122ng/ μ l, 0.01068ng/ μ l, 0.0095ng/ μ l, saves backup in-20 DEG C.
1.4PCR reaction
Reaction system (25 μ l)
The PCR reaction conditions of the 1 to the 4 pair of primer pair is: 93 DEG C of 5min; 93 DEG C of 30s, 60 DEG C of 30s; 72 DEG C of 30s, totally 35 circulations; 72 DEG C of 5min, 10 DEG C of 10min.The PCR reaction conditions of the 5 to the 8 pair of primer pair is: 93 DEG C of 5min; 93 DEG C of 30s, 65 DEG C of 30s; 72 DEG C of 30s, totally 30 circulations; 72 DEG C of 5min, 10 DEG C of 10min.
1.5 result
Get amplified production 5 μ l, 1.5% sepharose carries out electrophoresis, voltage is 138V, electrophoresis result is detected at ZF-258 full automatic gel Image analysis system after 23min, if there is the band of about 463bp in primer pair 1,2,3,4,7,8, there is the band of about 367bp in primer pair 5,6, then proves that institute's sample product are Fen No. 2 polygala roots far away.Electrophoresis detection result as shown in Figure 1, 2.The above results shows that the primer specificity of design in embodiment 1 is strong.
Respectively with the DNA sample of above-mentioned dilution for template carries out pcr amplification reaction.Pcr amplification program and amplified production detection method the same (point sample amount is 5 μ l).As shown in Figure 3, the 0.01715ng/ μ l when Fen No. 2 polygala root DNA profilings far away are diluted to, the band of pcr amplification is not obvious especially to result.Therefore, the detection sensitivity of above-mentioned primer pair to Fen No. 2 polygala roots far away can reach 0.01715ng/ μ l, and can detect Fen No. 2 polygala roots far away of about 0.085ng in sample, detection sensitivity is high.
Although above with a general description of the specific embodiments to invention has been detailed description, on basis of the present invention, can make some amendments to it or improve, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (4)
1., for differentiating the Auele Specific Primer pair of Fen No. 2 polygala roots far away, it is characterized in that described Auele Specific Primer is to being in following (1) to (8) primer pair any pair:
(1) upstream primer F1:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R1:5 ＇ TCGCCCCAAGAGATGTGA 3 ＇
(2) upstream primer F2:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R2:5 ＇ TCGCCCCAAGAGATCAGA 3 ＇
(3) upstream primer F3:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R3:5 ＇ TCGCCCCAAGAGATGTGA 3 ＇
(4) upstream primer F4:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R4:5 ＇ TCGCCCCAAGAGATCAGA 3 ＇
(5) upstream primer F5:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R5:5 ＇ ACCAAGTATCGCATTTCGC 3 ＇
(6) upstream primer F6:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R6:5 ＇ ACCAAGTATCGCATTTCGC 3 ＇
(7) upstream primer F7:5 ＇ CGACCGTTGGACACGTATT 3 ＇
Downstream primer R7:5 ＇ TCGCCCCAAGAGATGAGA 3 ＇
(8) upstream primer F8:5 ＇ CGACCGTTGGACACGAATT 3 ＇
Downstream primer R8:5 ＇ TCGCCCCAAGAGATGAGA 3 ＇.
2. adopt Auele Specific Primer to differentiate the method for Fen No. 2 polygala roots far away, it is characterized in that, comprise the following steps:
1) DNA in polygala root sample is extracted;
2) with step 1) in the DNA that extracts be template, use primer pair described in claim 1 to carry out pcr amplification reaction;
3) analyze PCR primer with agarose gel electrophoresis, have pcr amplification band person to be Fen No. 2 polygala roots far away, without pcr amplification band person then non-Fen No. 2 polygala roots far away.
3. differentiate the method for Fen No. 2 polygala roots far away as claimed in claim 2, it is characterized in that, pcr amplification reaction system is:
Described Auele Specific Primer is to being in claim 1 in (1) to (8) primer pair any pair.
4. differentiate the method for Shanxi No. 1 polygala root far away and Fen No. 2 polygala roots far away as claimed in claim 2, it is characterized in that, (1) to (4) is the PCR reaction conditions of primer pair: 93 DEG C of 5min; 93 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations; 72 DEG C of 5min, 10 DEG C of 10min;
(5) to (8) to the PCR reaction conditions of primer pair is: 93 DEG C of 5min; 93 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 30s, totally 30 circulations; 72 DEG C of 5min, 10 DEG C of 10min.
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| CN106701921A (en) * | 2016-12-01 | 2017-05-24 | 辽宁师范大学 | Primer for real-time fluorescence PCR (Polymerase Chain Reaction) specific detection on polygala tenuifolia and detection method |
| CN107254528A (en) * | 2017-06-26 | 2017-10-17 | 山西大学 | Detect sibiricose A in polygala5With sibiricose A6The method of content |
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| CN104107225A (en) * | 2014-07-30 | 2014-10-22 | 山西大学 | Traditional Chinese medicine polygala tenuifolia anticoagulant effective part, and extraction method and application of polygala tenuifolia anticoagulant effective part |
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| CN104107225A (en) * | 2014-07-30 | 2014-10-22 | 山西大学 | Traditional Chinese medicine polygala tenuifolia anticoagulant effective part, and extraction method and application of polygala tenuifolia anticoagulant effective part |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701921A (en) * | 2016-12-01 | 2017-05-24 | 辽宁师范大学 | Primer for real-time fluorescence PCR (Polymerase Chain Reaction) specific detection on polygala tenuifolia and detection method |
| CN107254528A (en) * | 2017-06-26 | 2017-10-17 | 山西大学 | Detect sibiricose A in polygala5With sibiricose A6The method of content |
| CN107254528B (en) * | 2017-06-26 | 2020-06-12 | 山西大学 | Detection of sibicose A in Polygala tenuifolia5With sibicose A6Method of content |
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