CN107779512A - A kind of PCR method for identifying earthworm - Google Patents
A kind of PCR method for identifying earthworm Download PDFInfo
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- CN107779512A CN107779512A CN201610723540.1A CN201610723540A CN107779512A CN 107779512 A CN107779512 A CN 107779512A CN 201610723540 A CN201610723540 A CN 201610723540A CN 107779512 A CN107779512 A CN 107779512A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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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 earthworm.Using the method differentiate earthworm 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 earthworm medicinal material, aqueous extract of earthworm, Lumbricus ethanol extract, the limit (high temperature, high pressure, long-time) condition Pheretima 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 earthworm medicinal material, Pheretima extract, maximum conditions extract, contains
There are the preparation of earthworm and the earthworm preparation Rapid identification of maximum conditions processing.
Background technology
Earthworm is medicinal earthworm, is had, and mainly has decompression, antithrombotic, anti-arrhythmia, anticancer, the immune, Kang Cheongju of enhancing
Trace, antipyretic-antalgic, calmness, relieving asthma, the effect such as antibacterial.In addition, also found from wide dragon it is a variety of with pharmacological activity into
Point as earthworm fibrinolysin, Lumbrokinase, lumbricin, lumbrifebrine, terrestro-lumbrolysin, basic amino acid, secondary yellow he purine, yellow he purine,
Amber claps acid and aliphatic acid and calmodulin is earthworm Neo Calcium binding protein etc., and these compositions are in the regulation a variety of physiology work(of cell
Important regulative is played in energy.Wide dragon is in industry acknowledged as genunie medicinal materials best in quality, is not only home sale
In easy-to-sell goods, but also be foreign exchange earning specified kind, though its as medicine or nutrient and healthcare products utilization,
There are huge benefit potentiality in terms of economic, society and ecology.
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 earthworm medicinal material in the country, in the pharmaceutical preparation after the processing of identification limit condition
The PCR method of earthworm 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 Earthworm be changed into mixed
Compound, otherwise earthworm is deteriorated a large amount of organic principles because of extraction process so that and the identification of earthworm 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 earthworm in preparation is set to become very difficult.
The content of the invention
The invention provides one kind be used for identify earthworm medicinal material, Pheretima extract, limit extract and preparation containing earthworm,
The primer and authentication method of preparation after maximum conditions processing, the PCR authentication methods are simple to operate, fast and accurately can identify ground
Imperial powder, extract, limit extract, mix preparation, the preparation even handled under maximum conditions.
The special primer of a pair of identification earthworms：Sense primer, anti-sense primer, its sequence are：
Sense primer：5’-AAAGCTTCCAACTGGGATTAGATACCCCACTAT-3’
Anti-sense primer：5’-TGACTGCAGAGGGTGACGGGCGGTGTGT-3’
A kind of PCR authentication methods of earthworm, comprise the following steps：It is divided into following four step：
1st, sample is prepared：Prepare Earthworm, aqueous extract of earthworm, alcohol extract and height are prepared using heating condensation reflux unit
The standby maximum conditions extract of compacting, is mixed to get earthworm preparation and high pressure prepares the earthworm 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 earthworm 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 earthworm.
Brief description of the drawings
Fig. 1 is that earthworm identifies electrophoretogram, shows the PCR qualification results of Earthworm and other Powdered animal drugs, as a result
As shown in figure 1, earthworm has homogeneous band at 230bp, and other confusion varieties do not have band to illustrate that this method is separably imperial
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 Earthworm, and 4~5 be scorpion, and 6~7 be Centipede, and 8~9 be Ground Beetle.Bar
Part：95 DEG C of 5min, 38 circulations (95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 5min.
Fig. 2 is that earthworm identifies electrophoretogram, shows the PCR qualification results of aqueous extract of earthworm and alcohol extract.Aqueous extract of earthworm, alcohol
Extract extract has homogeneous band at 230bp, illustrates that this method can identify the extract of earthworm.Note：M is Marker, from
Top to bottm is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp, and 1~2 is aqueous extract of earthworm, and 3~4
It is scorpio water extract for Lumbricus ethanol extract, 5,6 be scorpio alcohol extract, and 7 be centipede water extract, and 8 be centipede alcohol extract, and 9 be ground beetle
Worm water extract, 10 be ground bettle alcohol extract.Condition：95 DEG C of 5min, 35 circulations (95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s), 72
℃5min。
Fig. 3 is that earthworm identifies electrophoretogram, shows the PCR mirror of Pheretima extract under the conditions of the limit (high temperature, high pressure, long-time)
Determine result.Earthworm limit extract has homogeneous band at 230bp, illustrates that this method can identify the extract of earthworm.Note：M
For Marker, 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp are followed successively by from top to bottom, and 1~2 is earthworm
Limit water extract, 3~4 be earthworm limit alcohol extract, and 5 be scorpio maximum conditions water extract, and 6 be scorpio maximum conditions alcohol extract, 7
It is centipede maximum conditions alcohol extract for centipede maximum conditions water extract, 8,9 be ground bettle maximum conditions water extract, and 10 be ground bettle
Maximum conditions alcohol extract.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.
Fig. 4 is that earthworm 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 earthworm has
Band, illustrate the true and false that can distinguish earthworm.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 to mix alcohol extract under maximum conditions, and 7 be scorpio Wu
Centipede ground bettle mixed-powder, 8 be that scorpio centipede ground bettle mixes water extract, and 9 be that scorpio centipede ground bettle mixes alcohol extract, and 10 are
Scorpio centipede ground bettle is mixed-powder under maximum conditions, and 11 be that scorpio centipede ground bettle mixes water extract under maximum conditions, 12
Alcohol extract is mixed for scorpio centipede ground bettle under maximum conditions.Condition：95 DEG C of 5min, 35 circulation (95 DEG C of 30s, 58 DEG C of 30s,
72 DEG C of 30s), 72 DEG C of 5min.
Specific implementation method：
1 material
Earthworm, Centipede, scorpion, Ground Beetle provide by Tonghua JinMa Co., Ltd
2 special primers
Sense primer：5’-AAAGCTTCCAACTGGGATTAGATACCCCACTAT-3’
Anti-sense primer：5’-TGACTGCAGAGGGTGACGGGCGGTGTGT-3’
3 sample DNAs extract
The DNA extraction method that the present invention uses for：Earthworm 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 observed in gel imager, product
There should be single band at 230bp.
Claims (8)
1. establish a kind of method for identifying earthworm：The DNA of earthworm sample is extracted, is expanded by PCR, the process such as electrophoresis detection, mirror
Determine the true and false of earthworm and its extract, maximum conditions extract and preparation.
2. right 1 is characterised by identifying the true and false of earthworm in mix preparation, process comprises the following steps：
1) sample is prepared：Earthworm, aqueous extract of earthworm and alcohol extract, the ground of the limit (high temperature, high pressure, long-time) condition extraction
Imperial water extract and alcohol extract, earthworm and the mix preparation and the limit (high temperature, high pressure, long-time) condition of other class medicinal materials are handled
Earthworm 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 earthworm true and false is identified.
3. sample in right 2：Earthworm, aqueous extract of earthworm and alcohol extract, the aqueous extract of earthworm of maximum conditions extraction and alcohol extracting
Thing, earthworm mix preparation, the earthworm preparation of maximum conditions processing：
1) earthworm crushes, and obtains earthworm 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 earthworm three times
Water extract and alcohol extract.
3) by aqueous extract of earthworm and alcohol extract, 121 DEG C of high pressure 30min, maximum conditions extraction aqueous extract of earthworm 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. earthworm 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’-AAAGCTTCCAACTGGGATTAGATACCCCACTAT-3’
Anti-sense primer：5’-TGACTGCAGAGGGTGACGGGCGGTGTGT-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, 35 circulations (95 DEG C of denaturation 30s,
58 DEG C of 30s, 72 DEG C of 30s), 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
Earthworm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610723540.1A CN107779512A (en) | 2016-08-24 | 2016-08-24 | A kind of PCR method for identifying earthworm |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610723540.1A CN107779512A (en) | 2016-08-24 | 2016-08-24 | A kind of PCR method for identifying earthworm |
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| CN107779512A true CN107779512A (en) | 2018-03-09 |
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| CN201610723540.1A Pending CN107779512A (en) | 2016-08-24 | 2016-08-24 | A kind of PCR method for identifying earthworm |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676294A (en) * | 2020-05-22 | 2020-09-18 | 华南理工大学 | Relative quantification method of tubificidae in sludge reduction technology and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6309840B1 (en) * | 1997-01-03 | 2001-10-30 | The Chinese Univerisity Of Hong Kong | Polymerase chain reaction-restriction fragment length polymorphism test for the authentication of herbal Chinese medicines |
| CN103898234A (en) * | 2014-04-21 | 2014-07-02 | 牡丹江友搏药业股份有限公司 | Method for identifying DNA bar code molecule of earthworm |
-
2016
- 2016-08-24 CN CN201610723540.1A patent/CN107779512A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6309840B1 (en) * | 1997-01-03 | 2001-10-30 | The Chinese Univerisity Of Hong Kong | Polymerase chain reaction-restriction fragment length polymorphism test for the authentication of herbal Chinese medicines |
| CN103898234A (en) * | 2014-04-21 | 2014-07-02 | 牡丹江友搏药业股份有限公司 | Method for identifying DNA bar code molecule of earthworm |
Non-Patent Citations (2)
| Title |
|---|
| 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》 * |
| 陈维明 等: "广地龙特异性PCR分子鉴定", 《广州中医药大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676294A (en) * | 2020-05-22 | 2020-09-18 | 华南理工大学 | Relative quantification method of tubificidae in sludge reduction technology and application thereof |
| CN111676294B (en) * | 2020-05-22 | 2022-04-22 | 华南理工大学 | Relative quantification method of tubificidae in sludge reduction technology and application thereof |
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