CN107663535A - A kind of PCR method for identifying long-nosed pit viper - Google Patents
A kind of PCR method for identifying long-nosed pit viper Download PDFInfo
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- CN107663535A CN107663535A CN201610613809.0A CN201610613809A CN107663535A CN 107663535 A CN107663535 A CN 107663535A CN 201610613809 A CN201610613809 A CN 201610613809A CN 107663535 A CN107663535 A CN 107663535A
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention belongs to Chinese medicine and Materia Medica Identification technical field, is related specifically to a kind of PCR method and its special primer for being capable of unique identification long-nosed pit viper.Using the method differentiate long-nosed pit viper the true and false, only by simple DNA extractions, PCR specific amplifications, electrophoresis detection can the complete paired samples true and false discriminating.It is mainly used in the Rapid identification of Agkistrodon extract under the conditions of long-nosed pit viper medicinal material, long-nosed pit viper water extract, long-nosed pit viper alcohol extract, the limit (high temperature, high pressure, long-time), Chinese medicine preparation (a variety of similar extracts) and the limit (high temperature, high pressure, long-time) condition processing Chinese medicine preparation (a variety of similar extracts).
Description
Technical field
The present invention relates to Chinese medicine and Chinese medicine, particularly Chinese medical extract identification technology field, one kind specific to reflect
The PCR method and its special primer of deposit money white flower.It is mainly used in long-nosed pit viper medicinal material, Agkistrodon extract, maximum conditions extract, contains
There are the preparation of long-nosed pit viper and the long-nosed pit viper preparation Rapid identification of maximum conditions processing.
Background technology
Long-nosed pit viper is a kind of conventional Chinese medicine recorded of Chinese Pharmacopoeia (version in 2015), from Boidae animal long-noded pit viper
Agkistrodon acutus Guenther hirudo leech.There are wind-dispelling, dredging collateral, only the effect of convulsion.It is numb for rheumatoid arthritis stubborn
Contraction, hemiplegia, spasm of twitching, lockjaw, leprosy, mange.
Long-nosed pit viper is definite as the clinical efficacy of valuable animal drug, but merchandise resources is complicated, configuration of medicinal materials differentiates difficult, city
In the long-nosed pit viper medicinal material to be circulated on field, the sale of certified products long-nosed pit viper is mixed up after processing with intermediary pallas pit viper, mountain solder horn, boa etc. more.
These kinds with long-nosed pit viper is equal does not belong to together, because its source is close, cause identification difficult.In addition, medicinal material is cut away in working process
After internal organ, processing is dried, the relief features, color on skin thicken so that distinguish difficult, such as Serpentis, jade
Its size such as spot rat snake, mucosal rat snake, cobra, morphological feature and long-nosed pit viper have similarity, and this is also cause identification difficult another
One major reason.Though the report of the existing PCR method identification long-nosed pit viper medicinal material in the country, the pharmaceutical preparation after the processing of identification limit condition
The PCR method of middle long-nosed pit viper is not reported so far.Due to the links such as crushing, mixing in the production process of preparation, be present, decocting is boiled
Extraction, alcohol decoct the process procedures such as extraction, and even the condition such as HTHP is handled, otherwise these conditions are changed into long-nosed pit viper powder
Mixture, otherwise long-nosed pit viper is deteriorated a large amount of organic principles because of extraction process so that and the identification of long-nosed pit viper becomes tired in preparation
It is difficult.And some preparations use a variety of class medicinal materials, or use a variety of class and non-class animal drug so that other a few taste snakes
Class medicinal material or non-class animal drug make the identification of long-nosed pit viper in preparation become very tired there is also the possibility of interference identification
It is difficult.
The content of the invention
The invention provides one kind be used for identify long-nosed pit viper medicinal material, Agkistrodon extract, limit extract and preparation containing long-nosed pit viper,
The primer and authentication method of preparation after maximum conditions processing, the PCR authentication methods are simple to operate, can fast and accurately identify and beg
Snake powder, extract, limit extract, mix preparation, the preparation even handled under maximum conditions.
The special primer of a pair of identification long-nosed pit vipers：Sense primer, anti-sense primer, its sequence are：
Sense primer：5’-TCCAAATCATAACCGGCTTCTTCTTG-3’
Anti-sense primer：5’-AATTAAAAGGGTGGTGCCTGATAGTCAA-3’
A kind of PCR authentication methods of long-nosed pit viper, comprise the following steps：It is divided into following four step：
1st, sample is prepared：Prepare long-nosed pit viper powder, long-nosed pit viper water extract, alcohol extract and height are prepared using heating condensation reflux unit
The standby maximum conditions extract of compacting, it is mixed to get several similar medicinal powders and mixing water extraction is prepared using heating condensing unit
Thing, alcohol extract, mixed-powder under maximum conditions, mixing water extract, mixing alcohol extract are prepared under condition of high voltage.
2nd, sample DNA extracts：DNA is extracted from above sample.
3rd, PCR reacts：PCR primer is obtained using PCR method, 95 DEG C of 5min, 30 circulate (95 DEG C of 30s, 64 DEG C of 45s),
72 DEG C of 5min, obtain amplified production.
4th, with electroresis appraisal amplified production size, the long-nosed pit viper true and false is identified with this, passes through the stability of methods of experiments.Side
Method：1.5% gel, 130V electrophoresis 25min, there is single band at 230bp, it is determined that institute's sample material is long-nosed pit viper.
Brief description of the drawings
Fig. 1 is long-nosed pit viper powder and the PCR qualification results of other class medicinal powders, as a result as shown in figure 1, long-nosed pit viper exists
There is homogeneous band at 230bp, and other confusion varieties do not have band to illustrate that this method can distinguish long-nosed pit viper powder and confusion varieties.M
For Marker, 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp are followed successively by from top to bottom, and 1~3 is long-nosed pit viper
Powder, 4 be zaocys dhumnade powder, and 5 be Bungarus Parvus powder, and 6 be water.
Fig. 2 is the PCR qualification results of long-nosed pit viper water extract and alcohol extract.Long-nosed pit viper water extract, alcohol extract extract are in 230bp
There is homogeneous band at place, illustrates that this method can identify the extract of long-nosed pit viper.M is Marker, be followed successively by from top to bottom 1000bp,
700bp, 500bp, 400bp, 300bp, 200bp, 100bp, 1 is long-nosed pit viper powder, and 2 be long-nosed pit viper water extract, and 3 be long-nosed pit viper alcohol extract, 4
It is zaocys dhumnade alcohol extract for zaocys dhumnade water extract, 5,6 Bungarus Parvus water extracts, 7 be Bungarus Parvus alcohol extract.
Fig. 3 is the PCR qualification results of long-nosed pit viper water extract and alcohol extract under the conditions of the limit (high temperature, high pressure, long-time).Money
Long-noded pit viper limit extract has homogeneous band at 230bp, illustrates that this method can identify the extract of Bungarus Parvus.M is
Marker, 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp are followed successively by from top to bottom, 1 is long-nosed pit viper limit bar
Part powder, 2 be long-nosed pit viper maximum conditions water extract, and 3 be long-nosed pit viper maximum conditions alcohol extract, and 4 be zaocys dhumnade maximum conditions water extract, 5
It is Bungarus Parvus maximum conditions water extract for zaocys dhumnade maximum conditions alcohol extracting extract, 6,7 be Bungarus Parvus maximum conditions alcohol
Extract.
Fig. 4 is mixed-powder under several similar medicinal material mixed-powders, mixing water extract, mixing alcohol extract, maximum conditions, mixed
Heshui extract, mixing alcohol extract qualification result.Preparation containing Bungarus Parvus has band, illustrates that the true of long-nosed pit viper can be distinguished
It is pseudo-.M is Marker, is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp from top to bottom, and 1 is a variety of
Similar medicinal material mixed-powder, 2 be mixing water extract, and 3 be mixing alcohol extract, and 4 be that maximum conditions handle mixed-powder, and 5 be the limit
Condition mixes water extract, and 6 be that maximum conditions mix alcohol extract, and 7 be zaocys dhumnade and Bungarus Parvus mixed-powder, and 8 be zaocys dhumnade
Mix water extract with Bungarus Parvus, 9 be that zaocys dhumnade and Bungarus Parvus mix alcohol extract, 10 be under maximum conditions zaocys dhumnade and
Bungarus Parvus mixed-powder, 11 be that maximum conditions zaocys dhumnade and Bungarus Parvus mix water extract, and 12 be the maximum conditions crow tip
Snake and Bungarus Parvus mixing alcohol extract.
Specific implementation method：
1 material
Bungarus Parvus powder, zaocys dhumnade powder, long-nosed pit viper powder provide by Tonghua JinMa Co., Ltd
2 special primers
Sense primer：5’-TCCAAATCATAACCGGCTTCTTCTTG-3’
Anti-sense primer：5’-AATTAAAAGGGTGGTGCCTGATAGTCAA-3’
3 sample DNAs extract
The DNA extraction method that the present invention uses for：Long-nosed pit viper sample about 0.1g is taken into 1.5mL centrifuge tubes, 275 μ L is added and disappears
Change liquid (200 μ L, 0.5mol/L EDTA of nucleus lysate 50 μ L, the μ L of Proteinase K (20mg/mL) 20, the μ L of RNase solution 5),
55 DEG C of incubation 1h, add 250 μ L Wizard SV Lysis Buffer and mix, and are added in centrifugation tubing string, 10000r/min centrifugations
2 minutes；Discard filtered fluid, add 800 μ L eluents (potassium acetate 162.8mmol/L, Tris-HCl (pH7.5) 22.6mmol/L,
EDTA (pH8.0) 0.019mmol/L, 60% ethanol), 10000r/min centrifugations 1min；Filtered fluid is discarded, is washed repeatedly with eluent
De- 3 times, each 10000r/min is centrifuged 1 minute；2min is centrifuged again after discarding last time filtered fluid, and Filter column is transferred to newly
Centrifuge tube in, add 100 μ L ddH2O, after room temperature places 2min, 10000r/min centrifuges 2min, and -20 DEG C of preservations of filtrate are standby
With.
4 PCR react
The PCR reaction systems of table 1 (25 μ L)
Reaction condition：95 DEG C of 5min, 30 circulations (95 DEG C of 30s, 64 DEG C of 45s), 72 DEG C of 5min.
5 electrophoresis detections
1.5% Ago-Gel of the Red containing Gel is prepared, 130V electrophoresis 25min, is seen in gel on ultraviolet transilluminator
Examine, product there should be single band at 230bp.
Claims (8)
1. establish a kind of method for identifying long-nosed pit viper：The DNA of long-nosed pit viper sample is extracted, is expanded by PCR, the process such as electrophoresis detection, mirror
Determine the true and false of long-nosed pit viper and its extract, maximum conditions extract and preparation.
2. right 1 is characterised by identifying the true and false of long-nosed pit viper in mix preparation, process comprises the following steps：
1) sample is prepared：What long-nosed pit viper powder, long-nosed pit viper water extract and alcohol extract, the limit (high temperature, high pressure, long-time) condition were extracted begs
Snake water extract and alcohol extract, long-nosed pit viper and the mix preparation and the limit (high temperature, high pressure, long-time) condition of other class medicinal materials are handled
Long-nosed pit viper and other class medicinal materials mix preparation.
2) sample DNA extracts：DNA is extracted from sample.
3) PCR reacts：PCR primer is obtained using PCR method, 93~95 DEG C of 5min, 30~40 circulations (95 DEG C of 30~45s, 55
~65 DEG C of 30~45s, 72 DEG C of 30~45s), 72 DEG C of 5min, obtain amplified production.
4) electroresis appraisal：According to amplified production size, the long-nosed pit viper true and false is identified.
3. sample in right 2：Long-nosed pit viper powder, long-nosed pit viper water extract and alcohol extract, the long-nosed pit viper water extract of maximum conditions extraction and alcohol extracting
Thing, long-nosed pit viper mix preparation, the long-nosed pit viper preparation of maximum conditions processing：
1) long-nosed pit viper crushes, and obtains long-nosed pit viper fine powder.
2) the method extraction of heating condensing reflux is used, temperature is 100 DEG C, material-water ratio 1: 10, time 2h, and extraction obtains long-nosed pit viper three times
Water extract and alcohol extract.
3) by long-nosed pit viper water extract and alcohol extract, 121 DEG C of high pressure 30min, long-nosed pit viper water extract and alcohol extract under the conditions of limit are prepared.
4) several class medicinal materials are mixed to get mix preparation, water extract and alcohol extract are mixed using heating condensing reflux extraction,
121 DEG C of high pressure 30min obtain mixed-powder and extract under maximum conditions.
4. long-nosed pit viper DNA is extracted in right 2：Prepared using the method for digestion, centrifugation, purifying.
5. special primer in right 2, its sequence are：
Sense primer：5’-TCCAAATCATAACCGGCTTCTTCTTG-3’
Anti-sense primer：5’-AATTAAAAGGGTGGTGCCTGATAGTCAA-3’ .
6. PCR reaction systems use 25 μ L in right 1, including：The μ L of template 1,0.5 μ L, PCR Mix of primer 12.5 μ L,
ddH2O10.5μL。
7.PCR reaction conditions：PCR primer is obtained using PCR method, 95 DEG C of pre-degeneration 5min, 30 circulations (95 DEG C of denaturation 30s,
64 DEG C of 45s), 72 DEG C of extension 5min, obtain amplified production.
8. electrophoresis in right 2：There is single band at 1.5% gel, 130V electrophoresis 30min, 230bp, it is determined that institute's sample material is
Long-nosed pit viper.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610613809.0A CN107663535A (en) | 2016-07-29 | 2016-07-29 | A kind of PCR method for identifying long-nosed pit viper |
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| CN201610613809.0A CN107663535A (en) | 2016-07-29 | 2016-07-29 | A kind of PCR method for identifying long-nosed pit viper |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657255A (en) * | 2020-12-23 | 2022-06-24 | 广东一方制药有限公司 | Primer combination for PCR identification of agkistrodon medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613758A (en) * | 2009-06-30 | 2009-12-30 | 中国中医科学院中药研究所 | Identify PCR method and the special primer thereof of long-nosed pit viper |
| CN104013648A (en) * | 2014-04-14 | 2014-09-03 | 浙江中医药大学 | Long-noded pit viper extract and application thereof |
-
2016
- 2016-07-29 CN CN201610613809.0A patent/CN107663535A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613758A (en) * | 2009-06-30 | 2009-12-30 | 中国中医科学院中药研究所 | Identify PCR method and the special primer thereof of long-nosed pit viper |
| CN104013648A (en) * | 2014-04-14 | 2014-09-03 | 浙江中医药大学 | Long-noded pit viper extract and application thereof |
Non-Patent Citations (4)
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| 宋文成等: "中药材蕲蛇及其常见混伪品的特异性PCR鉴别", 《中国中药杂志》 * |
| 崔占虎: "中成药中原料药材的分子鉴别研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657255A (en) * | 2020-12-23 | 2022-06-24 | 广东一方制药有限公司 | Primer combination for PCR identification of agkistrodon medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof |
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