CN107779511A - A kind of PCR method for identifying centipede - Google Patents
A kind of PCR method for identifying centipede Download PDFInfo
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- CN107779511A CN107779511A CN201610723539.9A CN201610723539A CN107779511A CN 107779511 A CN107779511 A CN 107779511A CN 201610723539 A CN201610723539 A CN 201610723539A CN 107779511 A CN107779511 A CN 107779511A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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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 centipede.Using the method differentiate centipede 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 centipede medicinal material, centipede water extract, centipede alcohol extract, the limit (high temperature, high pressure, long-time) condition Scolopendra extract, Chinese medicine preparation (a variety of species medicinal materials), mixing water extract, mixing alcohol extract and the Chinese medicine preparation of the limit (high temperature, high pressure, long-time) condition processing, mixing water extract, the Rapid identification for mixing alcohol extract.
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 centipede medicinal material, Scolopendra extract, maximum conditions extract, contains
There are the preparation of centipede and the centipede preparation Rapid identification of maximum conditions processing.
Background technology
Centipede is China's tradition rare traditional Chinese medicine, have antispastic cramp and relieves dizziness, remove obstruction in channels to relieve pain, dispersing pathogen accumulation the effect of, for liver
In wind move, spasm twitch, child convulsion, middle air port, hemiplegia, lockjaw, rheumatoid arthritis stubborn, migraine and general headache, sore, scrofula,
Snake bite and insect sting etc..According to《Chinese Pharmacopoeia》It is Scolopendridae animal Scolopendpa Subspinipes Mutilans L. KOCH that version one in 2010, which records its source,
The hirudo leech of (Scolopendra subspinipes mutilans L.Koch), main product in China Hunan, Hubei, Guangxi with
And the ground such as Yunnan.
PCR method：PCR is the abbreviation of PCR, is a kind of enzymatic chemical reaction, be able to will be treated in test tube
The target gene of survey 100,000 times or even up to a million times of amplified matter in a short period of time, substantially increase the sensitive of gene diagnosis
Degree, reduce the difficulty of analysis.PCR methods are most commonly used methods in current gene diagnosis.
Though the report of the existing PCR method identification centipede medicinal material in the country, in the pharmaceutical preparation after the processing of identification limit condition
The PCR method of centipede is not reported so far.Due to the links such as crushing, mixing in the production process of preparation, be present, decocting, which is boiled, to be carried
Take, alcohol decoct extraction etc. process procedure, even the condition such as HTHP handle, or these conditions make Centipede be changed into mixed
Compound, otherwise centipede is deteriorated a large amount of organic principles because of extraction process so that and the identification of centipede becomes difficult in preparation.
And some preparations use many animals class medicinal material, or use many animals class medicinal material so that other a few taste animal medicinal materials
In the presence of the possibility of interference identification, thus the identification of centipede in preparation is set to become very difficult.
The content of the invention
The invention provides one kind be used for identify centipede medicinal material, Scolopendra extract, limit extract and preparation containing centipede,
The primer and authentication method of preparation after maximum conditions processing, the PCR authentication methods are simple to operate, can fast and accurately identify Wu
Centipede powder, extract, limit extract, mix preparation, the preparation even handled under maximum conditions.
The special primer of a pair of identification centipedes：Sense primer, anti-sense primer, its sequence are：
Sense primer：5’-GGTATGGAATGGAAGGTGGGT-3’
Anti-sense primer：5’-TCATCACACATACACGGGACG-3’
A kind of PCR authentication methods of centipede, comprise the following steps：It is divided into following four step：
1st, sample is prepared：Prepare Centipede, centipede water extract, alcohol extract and height are prepared using heating condensation reflux unit
The standby maximum conditions extract of compacting, is mixed to get centipede preparation and high pressure prepares the centipede preparation after maximum conditions processing.
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, 35 circulations (95 DEG C of 30s, 58 DEG C of 30s, 72
DEG C 30s), 72 DEG C of 5min, obtain amplified production.
4th, with electroresis appraisal amplified production size, the centipede 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 210bp, it is determined that institute's sample material is centipede.
Brief description of the drawings
Fig. 1 is that centipede identifies electrophoretogram, shows the PCR qualification results of Centipede and other Powdered animal drugs, as a result
As shown in figure 1, centipede has homogeneous band at 210bp, and other confusion varieties do not have band to illustrate that this method can distinguish centipede
Powder and confusion varieties.Note：M is Marker, be followed successively by from top to bottom 1000bp, 700bp, 500bp, 400bp, 300bp,
200bp, 100bp, 1~3 is Centipede, and 4~5 be scorpion, and 6~7 be Earthworm, and 8~9 be Ground Beetle.Bar
Part is：95 DEG C of 5min, 40 circulations (95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 5min.
Fig. 2 is that centipede identifies electrophoretogram, shows the PCR qualification results of centipede water extract and alcohol extract.Centipede water extract, alcohol
Extract extract has homogeneous band at 210bp, illustrates that this method can identify the extract of centipede.Note：M is Marker, from
Top to bottm is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp, and 1~2 is centipede water extract, and 3~4
It is scorpio water extract for centipede alcohol extract, 5,6 be scorpio alcohol extract, and 7 be aqueous extract of earthworm, and 8 be Lumbricus ethanol extract, and 9 be ground beetle
Worm water extract, 10 be ground bettle alcohol extract.Condition is：95 DEG C of 5min, 40 circulations (95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s),
72℃5min。
Fig. 3 is that centipede identifies electrophoretogram, shows the PCR mirror of Scolopendra extract under the conditions of the limit (high temperature, high pressure, long-time)
Determine result.Centipede limit extract has homogeneous band at 210bp, illustrates that this method can identify the extract of centipede.Note：M
For Marker, 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp are followed successively by from top to bottom, and 1~2 is centipede
Limit water extract, 3~4 be centipede limit alcohol extract, and 5 be scorpio maximum conditions water extract, and 6 be scorpio maximum conditions alcohol extract, 7
It is earthworm maximum conditions alcohol extract for earthworm maximum conditions water extract, 8,9 be ground bettle maximum conditions water extract, and 10 be ground bettle
Maximum conditions alcohol extract.Condition is：95 DEG C of 5min, 40 circulations (95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 5min.
Fig. 4 is that centipede identifies electrophoretogram, shows a variety of species class medicinal material mix preparations, mixing water extract, mixed alcohol
Extract, the mix preparation of maximum conditions processing, mixing water extract, the qualification result for mixing alcohol extract.Preparation containing centipede has
Band, illustrate the true and false that can distinguish centipede.Note：M is Marker, be followed successively by from top to bottom 1000bp, 700bp, 500bp,
400bp, 300bp, 200bp, 100bp, 1 is a variety of medicinal material mixed-powders, and 2 be mixing water extract, and 3 be mixing alcohol extract, and 4 be pole
Limit condition handles mixed-powder, and 5 be that water extract is mixed under maximum conditions, and 6 be that alcohol extract is mixed under maximum conditions, and 7 for scorpio
Imperial Ground Beetle, 8 be that scorpio earthworm ground bettle mixes water extract, and 9 be that scorpio earthworm ground bettle mixes alcohol extract, and 10 be the limit
Under the conditions of scorpio earthworm ground bettle be mixed-powder, 11 be that scorpio earthworm ground bettle mixes water extract under maximum conditions, and 12 be pole
Scorpio earthworm ground bettle mixes alcohol extract under the conditions of limit.Condition is：95 DEG C of 5min, 40 circulation (95 DEG C of 30s, 55 DEG C of 30s, 72
DEG C 30s), 72 DEG C of 5min.
Specific implementation method：
1 material
Scorpion, Centipede, Earthworm, Ground Beetle provide by Tonghua JinMa Co., Ltd；
2 special primers
Sense primer：5’-GGTATGGAATGGAAGGTGGGT-3’
Anti-sense primer：5’-TCATCACACATACACGGGACG-3’
3 sample DNAs extract
The DNA extraction method that the present invention uses for：Centipede 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, 35 circulations (95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s), 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 210bp.
Claims (8)
1. establish a kind of method for identifying centipede：The DNA of centipede sample is extracted, is expanded by PCR, the process such as electrophoresis detection, mirror
Determine the true and false of centipede and its extract, maximum conditions extract and preparation.
2. right 1 is characterised by identifying the true and false of centipede in mix preparation, process comprises the following steps：
1) sample is prepared：Centipede, centipede water extract and alcohol extract, the Wu of the limit (high temperature, high pressure, long-time) condition extraction
Centipede water extract and alcohol extract, centipede and the mix preparation and the limit (high temperature, high pressure, long-time) condition of other class medicinal materials are handled
Centipede and other animal 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 centipede true and false is identified.
3. sample in right 2：Centipede, centipede water extract and alcohol extract, the centipede water extract of maximum conditions extraction and alcohol extracting
Thing, centipede mix preparation, the centipede preparation of maximum conditions processing：
1) centipede crushes, and obtains centipede 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 centipede three times
Water extract and alcohol extract.
3) by centipede water extract and alcohol extract, 121 DEG C of high pressure 30min, maximum conditions extraction centipede water extract and alcohol extract are obtained.
4) several animal medicinal materials are mixed to get mix preparation, by mix preparation using the method extraction of heating condensing reflux, obtained
Water extract and mixing alcohol extract are mixed, the preparation of maximum conditions processing is obtained using 121 DEG C of high pressure 30min, mixes water extract, mixed
Close alcohol extract.
4. centipede 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’-GGTATGGAATGGAAGGTGGGT-3’
Anti-sense primer：5’-TCATCACACATACACGGGACG-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 5min, 40 circulations (95 DEG C of 30s, 55 DEG C of 30s, 72
DEG C 30s), 72 DEG C of 5min, obtain amplified production.
8. electrophoresis in right 2：There is single band at 1.5% gel, 130V electrophoresis 30min, 210bp, it is determined that institute's sample material is
Centipede.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610723539.9A CN107779511A (en) | 2016-08-24 | 2016-08-24 | A kind of PCR method for identifying centipede |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610723539.9A CN107779511A (en) | 2016-08-24 | 2016-08-24 | A kind of PCR method for identifying centipede |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001572A (en) * | 1996-07-26 | 1999-12-14 | Univera Pharmaceuticals, Inc. | Method of identifying Aloe using PCR |
| CN104531869A (en) * | 2014-12-25 | 2015-04-22 | 中国中医科学院中药研究所 | Primer pair for identifying Scolopendra subspinipes multilans(L.)Koch and application of primer pair |
| CN105112525A (en) * | 2015-08-27 | 2015-12-02 | 中国医学科学院药用植物研究所 | Method and PCR (polymerase chain reaction) reagent kit for identifying DNA (deoxyribonucleic acid) barcodes of animal medicinal materials |
-
2016
- 2016-08-24 CN CN201610723539.9A patent/CN107779511A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001572A (en) * | 1996-07-26 | 1999-12-14 | Univera Pharmaceuticals, Inc. | Method of identifying Aloe using PCR |
| CN104531869A (en) * | 2014-12-25 | 2015-04-22 | 中国中医科学院中药研究所 | Primer pair for identifying Scolopendra subspinipes multilans(L.)Koch and application of primer pair |
| CN105112525A (en) * | 2015-08-27 | 2015-12-02 | 中国医学科学院药用植物研究所 | Method and PCR (polymerase chain reaction) reagent kit for identifying DNA (deoxyribonucleic acid) barcodes of animal medicinal materials |
Non-Patent Citations (6)
| Title |
|---|
| 刘中权等: "从高温高压蒸煮过的中华鳖甲和骨骼中提取及扩增DNA", 《中草药》 * |
| 周莉莉等: "蜈蚣醇提取物和水提取物部分药理作用比较", 《时珍国医国药》 * |
| 崔占虎 等: "DNA分子标记技术在中成药鉴定中的应用与展望", 《中药材》 * |
| 张红印 等: "中药材蜈蚣及其混伪品DNA条形码鉴别研究", 《中国中药杂志》 * |
| 黄璐琦: "《中药分子鉴定操作指南》", 30 April 2014, 上海科学技术出版社 * |
| 黄璐琦: "《分子生药学》", 30 June 2000, 中国协和医科大学联合出版社 * |
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