CN102363817A - Method for identifying truth and false of bear bile powder by polymerase chain reaction - Google Patents
Method for identifying truth and false of bear bile powder by polymerase chain reaction Download PDFInfo
- Publication number
- CN102363817A CN102363817A CN2011103896277A CN201110389627A CN102363817A CN 102363817 A CN102363817 A CN 102363817A CN 2011103896277 A CN2011103896277 A CN 2011103896277A CN 201110389627 A CN201110389627 A CN 201110389627A CN 102363817 A CN102363817 A CN 102363817A
- Authority
- CN
- China
- Prior art keywords
- polymerase chain
- chain reaction
- bases
- sample
- false
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a method for identifying the truth and false of bear bile powder by polymerase chain reaction. In the method, the truth and false of a bear bile powder sample is identified by the extraction of a genome of the sample, polymerase chain reaction primers, which are used for amplification, of the sample, an amplified polymerase chain reaction system, an amplification condition, an amplification result and a detection system for an amplification product of the polymerase chain reaction. The method is simple and reliable, and negative effects caused by forged and fake bear bile serving as a Chinese medicinal material and the bear bile powder on patients are reduced, so the method has a great significance for improving the quality of Chinese medicines, ensuring the administration safety of people, better developing markets of the Chinese medicines at home and abroad and the like.
Description
Technical field
The present invention is the method that the Fel Ursi powder true and false is differentiated in a kind of polymerase chain reaction.
Technical background
Bear gall is because of rare famous and precious, and market value is expensive, and market often has is processed into powder with chicken, ox, sheep, Fel Sus domestica drying and palms Fel Ursi powder off as and sell phenomenon, and adulteration is very serious.Fel Ursi powder is mingled except that influencing the quality of medicine own, the more important thing is the curative effect that influences Chinese medicine and compatibility and causes adverse consequences to the patient, so the discriminating of the Fel Ursi powder and the preparation true and false thereof has important meaning in reality.Because the composition and the complicated component of Chinese patent medicine, interfering factors is many, identifies that with traditional pharmacognosy method its true and false and purity have bigger difficulty.And identify on the dna level and broken the conventional boundary of identifying of Chinese medicine; Can or plant following level and identify exactly from the kind of biology; Do not organized or of the influence of medicinal material position identifying, thus significant for accurate, reasonable, safe medication, guarantee patient's rights and interests.
Summary of the invention
The objective of the invention is to solve above-mentioned existing existing problem, provide that a kind of resolving power is strong, the method for the Fel Ursi powder true and false is differentiated in a kind of polymerase chain reaction of favorable reproducibility.
Concrete grammar of the present invention is following:
The genome process for extracting of a, sample (Fel Ursi powder and pseudo-article)
Get Fel Ursi powder 2 grams, pseudo-article chicken, sheep, ox, each 2 gram of Fel Sus domestica dry product are put into test tube separately, adopt phenol-chloroform-primary isoamyl alcohol method for extracting that its genomic dna is extracted.
The polymerase chain reaction primer of b, the sample that is used to increase
Upstream primer: 5'CGCCATGAACCCTCCCTCTA 3'
Downstream primer: 5'CCTGGGCAGTGAATCCCTCTT 3'
C, the polymerase chain reaction system and the amplification condition that are used to increase
In reaction system: the dna profiling 2 μ l that get 50ng/ μ l sample; 10 times of damping fluid 2 μ l; 2.5mM deoxyribonucleoside triphosphate (dNTP) 2 μ l, upstream primer: 5'CGCCATGAACCCTCCCTCTA 3'1 μ l, downstream primer: 5'CCTGGGCAGTGAATCCCTCTT 3'1 μ l; Polysaccharase 0.2 μ l puts into test tube, is supplemented to 25 μ l with the sterilization deionized water.
Its amplification condition: 94.0 ℃ 8 minutes; 35 circulations afterwards comprise: 94.0 ℃ 1 minute, 58.6 ℃ 1 minute, 72.0 ℃ 2 minutes, last 72.0 ℃ of 35 circulations in 10 minutes; Obtain the PCR amplification product.
The detection method of d, PCR amplification product
Its concentration 1.5% sepharose of preparation in the horizontal strip electrophoresis groove; Above-mentioned PCR amplification product 5 μ l are added the point sample hole, in the contrast hole, add 5 μ l molecular weight standards, 2000 types as a comparison; Voltage 120 V form images in the figure spectrum imaging system behind the electrophoresis 40min.
E, judge the true and false of Samples of Bear Bile through electrophoretogram.
The invention has the beneficial effects as follows
This method not only can be distinguished originate close certified products and adulterant, and can be processed into the powder Fel Ursi powder to drying and differentiate.
Fel Ursi powder is a bear secretory product, is difficult to pushed away by medicinal material kind and the quality of contrary former animal, and originate close certified products and adulterant then more are not easily distinguishable.Therefore, a lot of traditional Chinese medicines authentication methods can't solve the discriminating problem of present many animal tcms.Carry out the Chinese medicinal materials dna molecular marker with present method and differentiate, only need get the sample of minute quantity and can carry to such an extent that enough be used for the dna profiling that polymerase chain reaction,PCR (PCR) increases, differentiate judgement then.This method speed is fast, resolving power strong, favorable reproducibility, can clarify the true and false and the quality (relating to dosage, ratio) of bear gall and pulvis thereof, remedies the weakness that animal tcm is differentiated in the traditional Chinese medicine evaluation, for clinical rational drug use provides scientific basis.
The utilization of this technology is the quality that improves Chinese medicinal materials, ensures the safety of millions upon millions of people's health and life, the basis of guarantee traditional Chinese medical science curative effect; Phenomenon such as can effectively contain forgery, mingle, abuse, misapply, use with improves the prestige of tcm-, ensures people's drug safety, and its economic benefit is self-evident.
Subordinate list: the result after Fel Ursi powder increases with pseudo-article
| The genuine piece Samples of Bear Bile | The chicken sample | The sheep sample | The ox sample | The pig sample |
| 344 bases | 163 bases | 109 bases | 109 bases | 109 bases |
| 375 bases | 250 bases | 173 bases | 173 bases | 280 bases |
| ? | 350 bases | 359 bases | 359 bases | 360 bases |
| ? | 406 bases | 530 bases | 422 bases | 375 bases |
| ? | 626 bases | 80 bases | 565 bases | 390 bases |
| ? | ? | ? | 790 bases | 405 bases |
| ? | ? | ? | ? | 531 bases |
| ? | ? | ? | ? | 675 bases |
| ? | ? | ? | ? | 785 bases |
| ? | ? | ? | ? | 1000 bases |
Can find out in the table that Fel Ursi powder and pseudo-article and adulterant there are differences on banding pattern that increases and segmental size: the result of genuine piece bear gall amplification mainly contains 2 stripe size and is respectively: 375 bases, 344 bases; Main puppet article chicken sample amplification result mainly contains 5 bands and is respectively: 626 bases, 406 bases, 350 bases, 250 bases, 163 bases; Sheep sample amplification result mainly contains 5 bands: 800 bases, 530 bases, 359 bases, 173 bases, 109 bases; Ox sample amplification result mainly contains 6 bands: 790 bases, 565 bases, 422 bases, 359 bases, 173 bases, 109 bases; Pig sample amplification result mainly contains 1000 bases of 10 bands, 785 bases, 675 bases, 531 bases, 405 bases, 390 bases, 375 bases, 360 bases, 280 bases, 109 bases; No matter on banding pattern and segmental size; All exist difference between the sample, this is a polymorphum foundation of identifying the sample true and false and kind.
Claims (1)
1. the method for a polymerase chain reaction discriminating Fel Ursi powder true and false is characterized in that,
The genome process for extracting of a, sample
Get Fel Ursi powder 2 grams, pseudo-article chicken, sheep, ox, each 2 gram of Fel Sus domestica dry product are put into test tube separately, adopt phenol-chloroform-primary isoamyl alcohol method for extracting that its genomic dna is extracted;
The polymerase chain reaction primer of b, the sample that is used to increase
Upstream primer: 5'CGCCATGAACCCTCCCTCTA 3'
Downstream primer: 5'CCTGGGCAGTGAATCCCTCTT 3'
C, the polymerase chain reaction system and the amplification condition that are used to increase
In reaction system: the dna profiling 2 μ l that get 50ng/ μ l sample; 10 times of damping fluid 2 μ l; 2.5mM deoxyribonucleoside triphosphate 2 μ l, upstream primer: 5'CGCCATGAACCCTCCCTCTA 3'1 μ l, downstream primer: 5'CCTGGGCAGTGAATCCCTCTT 3'1 μ l; Polysaccharase 0.2 μ l puts into test tube, is supplemented to 25 μ l with the sterilization deionized water;
Its amplification condition: 94.0 ℃ 8 minutes; 35 circulations afterwards comprise: 94.0 ℃ 1 minute, 58.6 ℃ 1 minute, 72.0 ℃ 2 minutes, last 72.0 ℃ 10 minutes; Obtain the PCR amplification product;
The detection method of d, PCR amplification product
Its concentration 1.5% sepharose of preparation in the horizontal strip electrophoresis groove; Above-mentioned PCR amplification product 5 μ l are added the point sample hole, in the contrast hole, add 5 μ l molecular weight standards, 2000 types as a comparison; Voltage 120 V form images in the figure spectrum imaging system behind the electrophoresis 40min;
E, judge the true and false of Samples of Bear Bile through electrophoretogram.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103896277A CN102363817B (en) | 2011-11-30 | 2011-11-30 | Method for identifying truth and false of bear bile powder by polymerase chain reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103896277A CN102363817B (en) | 2011-11-30 | 2011-11-30 | Method for identifying truth and false of bear bile powder by polymerase chain reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102363817A true CN102363817A (en) | 2012-02-29 |
| CN102363817B CN102363817B (en) | 2013-02-13 |
Family
ID=45690410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103896277A Expired - Fee Related CN102363817B (en) | 2011-11-30 | 2011-11-30 | Method for identifying truth and false of bear bile powder by polymerase chain reaction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102363817B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106682919A (en) * | 2017-01-05 | 2017-05-17 | 哈尔滨市食品药品检验检测中心 | Commercially available Ranae oviductus medicinal material inspection method based on DNA bar code technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560272A (en) * | 2004-03-01 | 2005-01-05 | 中国中医研究院中药研究所 | Process for identifing real and flase of chinese herbal medicine black-striped snake by high specificity polyase chain type reaction technology |
| CN101270357A (en) * | 2008-03-11 | 2008-09-24 | 华东理工大学 | Method for extracting DNA from deep process type traditional Chinese medicine or traditional Chinese medicinal materials |
-
2011
- 2011-11-30 CN CN2011103896277A patent/CN102363817B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560272A (en) * | 2004-03-01 | 2005-01-05 | 中国中医研究院中药研究所 | Process for identifing real and flase of chinese herbal medicine black-striped snake by high specificity polyase chain type reaction technology |
| CN101270357A (en) * | 2008-03-11 | 2008-09-24 | 华东理工大学 | Method for extracting DNA from deep process type traditional Chinese medicine or traditional Chinese medicinal materials |
Non-Patent Citations (4)
| Title |
|---|
| 刘萍等: "熊胆与猴胆、鸡胆汁的高效毛细管电泳法鉴别", 《中国药物应用与监制》 * |
| 李国华: "动物药鉴别研究进展", 《药物评价研究》 * |
| 李锋等: "天然熊胆、熊胆粉及其伪品的凝胶电泳鉴别研究", 《时珍国药研究》 * |
| 杨光明等: "分子生物学技术在中药鉴定中的应用", 《世界科学技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106682919A (en) * | 2017-01-05 | 2017-05-17 | 哈尔滨市食品药品检验检测中心 | Commercially available Ranae oviductus medicinal material inspection method based on DNA bar code technology |
| CN106682919B (en) * | 2017-01-05 | 2021-04-06 | 哈尔滨市食品药品检验检测中心 | Method for inspecting commercial oviductus ranae medicinal materials based on DNA bar code technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102363817B (en) | 2013-02-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cai et al. | Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus | |
| Camelo‐Castillo et al. | Relationship between periodontitis‐associated subgingival microbiota and clinical inflammation by 16S pyrosequencing | |
| CN104846101B (en) | A kind of special primer pair and method for identifying pseudo-ginseng | |
| CN106498050A (en) | A kind of Chinese patent drug living species constituent monitoring method based on SMRT sequencing technologies | |
| CN104388598A (en) | Digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and application of digital PCR quantitative detecting kit for HBV cccDNA | |
| CN102732520A (en) | Preparation method for serum miRNAs specific to active pulmonary tuberculosis | |
| CN104946730A (en) | PCR (Polymerase Chain Reaction) specific primers and detection method for authenticating antlers | |
| CN109486972A (en) | A kind of CPA primer sets for detecting pseudomonas aeruginosa, CPA nucleic acid test strip kit and its application | |
| Cao et al. | Xihuang Pill enhances anticancer effect of anlotinib by regulating gut microbiota composition and tumor angiogenesis pathway | |
| Cui et al. | Effect of dual infection with Eimeria tenella and subgroup J avian leukosis virus on the cecal microbiome in specific-pathogen-free chicks | |
| Medeiros et al. | Oral yeast colonization in patients with primary and secondary Sjögren's syndrome | |
| CN104830969A (en) | Panax notoginseng molecule ID and identification method | |
| CN102363817B (en) | Method for identifying truth and false of bear bile powder by polymerase chain reaction | |
| CN105002160A (en) | Method for extracting DNA of Chinese patent medicines or Chinese herbal medicine-containing health care products | |
| CN104630360A (en) | Rapid HPS (haemophilus parasuis) isothermal amplification detection primers, kit and detecting method | |
| CN101831494A (en) | Molecular biology authentication method of bulbus fritillariae cirrhosae in Chinese patent medicine containing fritillariae | |
| CN101613759A (en) | Identify PCR method and the special primer thereof of mone snake | |
| CN104342495A (en) | Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus | |
| CN105274245B (en) | A kind of method and its special primer pair for identifying David's-harp | |
| Cho et al. | Zerumbone restores gut microbiota composition in ETBF colonized AOM/DSS mice | |
| CN103614484A (en) | Specific PCR (Polymerase Chain Reaction) identification method of paecilomyces hepiali powder | |
| CN103451312A (en) | Method for quickly identifying scorpion medicinal materials in field of molecular biology | |
| CN103882095A (en) | Application of small molecule RNA as tuberculosis marker | |
| Liu et al. | Precise species detection in traditional herbal patent medicine, Qingguo wan, using shotgun metabarcoding | |
| CN109837339A (en) | Primer sets, probe groups, kit and method for the detection of children's safety medication related gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20120229 Assignee: CHANGCHUN PUHUA PHARMACEUTICAL Co.,Ltd. Assignor: Liaoning Medical University Contract record no.: 2013210000140 Denomination of invention: Method for identifying truth and false of bear bile powder by polymerase chain reaction Granted publication date: 20130213 License type: Exclusive License Record date: 20131218 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130213 Termination date: 20211130 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |