CN103131752A - Molecular biological method for rapidly identifying trichosanthin medicinal material - Google Patents
Molecular biological method for rapidly identifying trichosanthin medicinal material Download PDFInfo
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- CN103131752A CN103131752A CN 201110373203 CN201110373203A CN103131752A CN 103131752 A CN103131752 A CN 103131752A CN 201110373203 CN201110373203 CN 201110373203 CN 201110373203 A CN201110373203 A CN 201110373203A CN 103131752 A CN103131752 A CN 103131752A
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Abstract
The invention relates to a new molecular identification method for identifying trichosanthin and its similar products; adventurous innovation is made in two key links of primer design and annealing temperature. Primer design is performed on a known ITS of trichosanthin medicinal materials; the length is about 20 bp; and general primer design principles are followed. Meanwhile, the annealing temperature is screened and determined according to the number of polymorphism strips. The characteristic identification strips of trichosanthin are determined to be 560, 960 bp strips, and 1930, 1400, 839, 715 bp strips are considered as auxiliary identification strips of trichosanthin. The invention has the following advantages of: (1) simplicity and practicality, wherein although the difficulty for primer design is increased slightly, the specificity is greatly improved, an ideal identification primer can be obtained after design of 2-3 primers, and thus the problem of large screening of random primers is avoided; (2) stability and repeatability, wherein since the specificity and annealing temperature of the primer are improved, the amplification strips are generally only 1-5 strips, and certified products with different producing areas and different storage time have no effect on PCR results; (3) large provided information content, wherein both certified products and most fake products can be amplified simultaneously, and thus standard identification electrophoretograms of the certified products and fake products can be established to realize accurate identification of the certified products and fake products.
Description
Technical field
The present invention relates to a kind of molecule novel identification method of differentiating Snakegourd Root and similar product thereof, belong to technical field of traditional Chinese medicines.
Background technology
There is 84 kind of 8 mutation in the snake gourd plant whole world, in state-owned 37 kind of 6 mutation.This genus has multiple medicinal plant, and pharmacopeia version in 2005 has been recorded 2 kinds, and " Chinese medicinal herbal " recorded 7 kinds, snakegourd T.kirilowii Maxim. for example, and its fruit (Snakegourd Fruit), root (Snakegourd Root) they are all conventional Chinese medicines.The Trichosanthin that proposes from Snakegourd Root has been widely used in the early and middle portion labor induction, and the diseases such as treatment ectopic pregnancy, hydatidiform mole are used for again the treatment of acquired immune deficiency syndrome (AIDS) in recent years.Investigation finds that Snakegourd Root used source, various places is complicated, the Snakegourd Root of different plant origins, and its curative effect is also not quite identical, and some adulterant such as Hubei snakegourd, Semen Momordicae root etc. also have certain untoward reaction.
Accurately differential plant Ji Yuan is correct the exploitation and basis safe and effective for medication, has the exploration that the methods such as randomly amplified polymorphic DNA and protein immunization detection are differentiated Snakegourd Root and adulterant thereof.Repeatability is not good enough as a result but exist, and is vulnerable to the shortcoming of the medicinal material place of production and storage time impact, and the application in Chinese medicinal materials Molecular Identification field is subject to certain limitation.For the shortcoming of RAPD, the present invention has carried out bold innovation, is included in the single primer of design 20bp on the snake gourd known dna sequence, improves PCR annealing temperature etc.By the screening to 3 primers, obtain and to the primer of Snakegourd Root and adulterant DNA generation polymorphism collection of illustrative plates thereof, to have determined the feature electrophoretic band of Snakegourd Root and various adulterants thereof, for Snakegourd Root class medicinal material is provided by the method accurately that provides fast.
Summary of the invention
The present invention has realized bold innovation on design of primers and 2 the key links of annealing temperature.Design of primers is carried out on the known ITS of Snakegourd Root class medicinal material, and length is the 20bp left and right, follows the principle of design of general primer.Simultaneously, annealing temperature is also definite according to how much screening of polymorphic bands.
Embodiment
According to 3 single primer: TkS1-64F of ITS sequences Design in the Snakegourd Root ribosomal RNA gene, Tks2-1 12F, Tks2-130R.The PCR reaction system is 25 μ L, wherein Tris-HCI (pH 9.0) 10mmolL
-1, KC1 50mmolL
-1, Mg
2+1.5 mmolL
-1, each 0.15 mmolL of dNTP
-1, Taq 1U (promega), Primer 0.15 μ molL
-1, template DNA 50 ng.Amplification is carried out on ABI9700, and amplification program is 95 ℃ of denaturation 4 min, then carries out 40 circulations: 95 ℃ of sex change 30s, and 50 ℃ of annealing 45s, 72 ℃ are extended 60s, and after loop ends, 72 ℃ are extended 5min.
In 3 primers, the TkS1-64F expanding effect is good, shows as Snakegourd Root and all can increase, and the adulterant great majority can increase, polymorphic bands 1-5 bar, and band is clear, and good reproducibility can be used for the discriminating of Snakegourd Root medicinal material.The characteristic differentiation band of Snakegourd Root is defined as 560,960 bp bands, and the bands such as 1930,1400,839,715 bp can be used as the auxiliary discriminating band of Snakegourd Root.
Claims (3)
1. a molecule novel identification method of differentiating Snakegourd Root and similar product thereof, be according to 3 single primers of ITS sequences Design in the Snakegourd Root ribosomal RNA gene, and its PCR reaction system is 25 μ L, wherein Tris-HCI (pH 9.0) 10mmolL
-1, KC1 50mmolL
-1, Mg
2+1.5 mmolL
-1, each 0.15 mmolL of dNTP
-1, Taq 1U (promega), Primer 0.15 μ molL
-1, template DNA 50 ng, amplification is carried out on ABI9700, and amplification program is 95 ℃ of denaturation 4 min, then carries out 40 circulations: 95 ℃ of sex change 30s, 50 ℃ of annealing 45s, 72 ℃ are extended 60s, and after loop ends, 72 ℃ are extended 5min.
2. require described a kind of molecule novel identification method of differentiating Snakegourd Root and similar product thereof according to right 1, it is characterized in that: amplification is carried out on ABI9700, amplification program is 95 ℃ of denaturation 4 min, then carry out 40 circulations: 95 ℃ of sex change 30s, 50 ℃ of annealing 45s, 72 ℃ are extended 60s, and after loop ends, 72 ℃ are extended 5min.
3. the described method of right 1 or 2, it is characterized in that: the characteristic differentiation band of Snakegourd Root is defined as 560,960 bp bands, and the bands such as 1930,1400,839,715 bp can be used as the auxiliary discriminating band of Snakegourd Root.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110373203 CN103131752A (en) | 2011-11-22 | 2011-11-22 | Molecular biological method for rapidly identifying trichosanthin medicinal material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110373203 CN103131752A (en) | 2011-11-22 | 2011-11-22 | Molecular biological method for rapidly identifying trichosanthin medicinal material |
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| Publication Number | Publication Date |
|---|---|
| CN103131752A true CN103131752A (en) | 2013-06-05 |
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| CN 201110373203 Pending CN103131752A (en) | 2011-11-22 | 2011-11-22 | Molecular biological method for rapidly identifying trichosanthin medicinal material |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104181269A (en) * | 2014-08-15 | 2014-12-03 | 中国农业科学院蜜蜂研究所 | Method for identifying bee pollen based on kaempferol 3-O-beta-D-glucose-(2-1)-beta-D-glucoside |
| CN108680418A (en) * | 2018-06-01 | 2018-10-19 | 广东金作农业科技有限公司 | A kind of rapid fluorescence colouring method of crop in cruciferae pollen |
| JP2022528668A (en) * | 2018-03-23 | 2022-06-15 | 中国医学科学院北京協和医院 | Humulus pollen allergen soaked extract, leachate and its preparation method |
-
2011
- 2011-11-22 CN CN 201110373203 patent/CN103131752A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104181269A (en) * | 2014-08-15 | 2014-12-03 | 中国农业科学院蜜蜂研究所 | Method for identifying bee pollen based on kaempferol 3-O-beta-D-glucose-(2-1)-beta-D-glucoside |
| CN104181269B (en) * | 2014-08-15 | 2015-11-25 | 中国农业科学院蜜蜂研究所 | The method of Bee Pollen is differentiated based on Kaempferol 3-O-β-D-Glucose-(2 → 1)-β-D-Glucose glycosides |
| JP2022528668A (en) * | 2018-03-23 | 2022-06-15 | 中国医学科学院北京協和医院 | Humulus pollen allergen soaked extract, leachate and its preparation method |
| JP7394866B2 (en) | 2018-03-23 | 2023-12-08 | 中国医学科学院北京協和医院 | Cucumber pollen allergen immersion extract, infusion solution and its preparation method |
| CN108680418A (en) * | 2018-06-01 | 2018-10-19 | 广东金作农业科技有限公司 | A kind of rapid fluorescence colouring method of crop in cruciferae pollen |
| CN108680418B (en) * | 2018-06-01 | 2021-03-23 | 广东金作农业科技有限公司 | Dyeing liquid and method for quickly dyeing cruciferous crop pollen |
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Application publication date: 20130605 |