CN107663534A

CN107663534A - A kind of PCR method for identifying Bungarus Parvus

Info

Publication number: CN107663534A
Application number: CN201610613807.1A
Authority: CN
Inventors: 周亚伟
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-07-29
Filing date: 2016-07-29
Publication date: 2018-02-06

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 Bungarus Parvus.Using the method differentiate Bungarus Parvus 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 Bungarus Parvus extract under the conditions of Bungarus Parvus medicinal material, Bungarus Parvus water extract, Bungarus Parvus 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

A kind of PCR method for identifying Bungarus Parvus

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 Bungarus Parvus medicinal material, Bungarus Parvus extract, the limit The Bungarus Parvus preparation Rapid identification of condition extract, the preparation containing Bungarus Parvus and maximum conditions processing.

Background technology

Bungarus multicinctus (Bungarus multicinctus), it was commonly called as Ji Xia, Bai Jiehei, Bungarus Parvus, silver-colored first band, silver Iron clad etc..Toxicity is extremely strong, is the fourth-largest poisonous snake in land.The Bungarus multicinctus whole body body back of the body has that white ring and black ring are spaced, and white ring is narrower, Tail is elongated, and body grows 10~18cm, has the poisonous snake of proteroglyphic tooth.Back side black is black-and-blue, has 30~50 whites or milk yellow is narrow Band；The dirty white of the outside of belly.The head back of the body is dark brown, and the young pillow back of the body has light inverted " v " spot.Ridge is higher, and cross section is triangular in shape, tail end End is sharper.Head ellipse, it is less obvious with neck region point, close the back of the body and have typical 9 pieces of big scales, no cheek squama, back of the body center a line ridge squama Expansion is in hexagon；Urostege single file.

Bungarus Parvus is《Chinese Pharmacopoeia》A kind of conventional Chinese medicine that version one in 2015 is recorded, is moved from Elapidae Thing Bungarus Parvus Bungarus multicinctus Blyth hirudo leech, there are wind-dispelling, only dredging collateral, convulsion.Money is white Flower snake is definite as animal drug clinical efficacy, but merchandise resources is complicated, configuration of medicinal materials differentiates difficulty, and special according only to form Sign is difficult to be differentiated exactly.Therefore require it is quick, stably, accurate authentication method.

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 Bungarus Parvus medicinal material in the country, the medicine after the processing of identification limit condition The PCR method of Bungarus Parvus is not reported so far in preparation.Due to crushing, mixing etc. in the production process of preparation, be present Link, water boiling and extraction, alcohol decoct the process procedures such as extraction, and even the condition such as HTHP is handled, otherwise these conditions make Bungarus Parvus powder is changed into mixture, or making Bungarus Parvus be deteriorated a large amount of organic principles because of extraction process, makes Obtaining the identification of Bungarus Parvus in preparation becomes difficult.And some preparations use a variety of class medicinal materials, or use a variety of class With non-class animal drug so that other a few taste class medicinal materials or non-class animal drug there is also the possibility of interference identification, because And the identification of Bungarus Parvus in preparation is set to become very difficult.

The content of the invention

The invention provides one kind be used for identify Bungarus Parvus medicinal material, Bungarus Parvus extract, limit extract with And the primer and authentication method of the preparation after the processing of preparation containing Bungarus Parvus, maximum conditions, PCR authentication methods operation letter It is single, it can fast and accurately identify Bungarus Parvus powder, extract, limit extract, mix preparation, even locate under maximum conditions The preparation of reason.

The special primer of a pair of identification Bungarus Parvus：Sense primer, anti-sense primer, its sequence are：

Sense primer：5′-GACTTAGCTTTCTCATCTGTGATCCATATTA-3′

Anti-sense primer：5′-GCCTGCAGCCCCTCAGATGATATTTGTCCTCA-3′

A kind of PCR authentication methods of Bungarus Parvus, comprise the following steps：It is divided into following four step：

1st, sample is prepared：Prepare Bungarus Parvus powder, Bungarus Parvus water extraction is prepared using heating condensation reflux unit Thing, alcohol extract and high pressure prepare maximum conditions extract, are mixed to get several similar medicinal powders and using heating condensing unit Mixing water extract, alcohol extract are prepared, 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 Bungarus Parvus true and false is identified with this, passes through the steady of methods of experiments It is qualitative.Method：1.5% gel, 130V electrophoresis 25min, there is single band at 230bp, it is determined that institute's sample material is that money is white Flower snake.

Brief description of the drawings

Fig. 1 is Bungarus Parvus powder and the PCR qualification results of other class medicinal powders, as a result as shown in figure 1, money Long-noded pit viper has homogeneous band at 230bp, and other confusion varieties do not have band to illustrate that this method can distinguish Bungarus Parvus powder End and confusion varieties.M is Marker, be followed successively by from top to bottom 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp, 1~3 is Bungarus Parvus powder, and 4~6 be zaocys dhumnade powder, and 7~9 be long-nosed pit viper powder.

Fig. 2 is the PCR qualification results of money white flower water extract and alcohol extract.Bungarus Parvus water extract, alcohol extract extraction Thing has homogeneous band at 230bp, illustrates that this method can identify the extract of Bungarus Parvus.M is Marker, from top to bottom It is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp, 1~2 is Bungarus Parvus water extract, 3~4 It is zaocys dhumnade water extract for Bungarus Parvus alcohol extract, 5,6 be zaocys dhumnade alcohol extract, and 7 be long-nosed pit viper water extract, and 8 be long-nosed pit viper alcohol extracting Thing.

Fig. 3 is the PCR of Bungarus Parvus water extract and alcohol extract identification knots under the conditions of the limit (high temperature, high pressure, long-time) Fruit.Bungarus Parvus limit extract has homogeneous band at 230bp, illustrates that this method can identify the extraction of Bungarus Parvus Thing.M is Marker, is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp from top to bottom, and 1~2 is Bungarus Parvus maximum conditions water alcohol extract, 3~4 be Bungarus Parvus maximum conditions alcohol extract, and 5 be zaocys dhumnade maximum conditions water Extract, 6 be zaocys dhumnade maximum conditions alcohol extract, and 7 be long-nosed pit viper maximum conditions water extract, and 8 be long-nosed pit viper 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, and explanation can distinguish Bungarus Parvus The true and false.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-powders, 2 be mixing water extract, and 3 be mixing alcohol extract, and 4 be limit price adjustment processing mixed-powder, and 5 are Maximum conditions mix water extract, and 6 be that maximum conditions mix alcohol extract, and 7 be zaocys dhumnade and long-nosed pit viper mixed-powder, 8 be zaocys dhumnade with Long-nosed pit viper mixes water extract, and 9 be zaocys dhumnade and long-nosed pit viper mixes alcohol extract, and 10 be zaocys dhumnade and long-nosed pit viper mixed-powder under maximum conditions, 11 be maximum conditions zaocys dhumnade and long-nosed pit viper mixes water extract, and 12 be that maximum conditions zaocys dhumnade and long-nosed pit viper mix 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′-GACTTAGCTTTCTCATCTGTGATCCATATTA-3′

Anti-sense primer：5′-GCCTGCAGCCCCTCAGATGATATTTGTCCTCA-3′

3 sample DNAs extract

The DNA extraction method that the present invention uses for：Bungarus Parvus sample about 0.1g is taken into 1.5mL centrifuge tubes, is added (200 μ L, 0.5mol/L EDTA of nucleus lysate 50 μ L, the μ L of Proteinase K (20mg/mL) 20, RNase are molten for 275 μ L digestive juices The μ L of liquid 5), 55 DEG C of incubation 1h, add 250 μ L Wizard SV Lysis Buffer and mix, be added in centrifugation tubing string, 10000r/ Min is centrifuged 2 minutes；Filtered fluid is discarded, adds 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 used Eluent is eluted 3 times repeatedly, and each 10000r/min is centrifuged 1 minute；2min is centrifuged again after discarding last time filtered fluid, incited somebody to action Filter column is transferred in new centrifuge tube, adds 100 μ L ddH₂O, after room temperature places 2min, 10000r/min centrifugation 2min, filter - 20 DEG C of liquid saves backup.

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

1. establish a kind of method for identifying Bungarus Parvus：The DNA of Bungarus Parvus sample is extracted, is expanded by PCR, electrophoresis inspection The processes such as survey, identify the true and false of Bungarus Parvus and its extract, maximum conditions extract and preparation.

2. right 1 is characterised by identifying the true and false of Bungarus Parvus in mix preparation, process comprises the following steps：

1) sample is prepared：Bungarus Parvus powder, Bungarus Parvus water extract and alcohol extract, the limit (high temperature, high pressure, long-time) The Bungarus Parvus water extract and alcohol extract of condition extraction, Bungarus Parvus and the mix preparation and the limit of other class medicinal materials The Bungarus Parvus of (high temperature, high pressure, long-time) condition processing and the mix preparation of other class medicinal materials.

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 Bungarus Parvus true and false is identified.

3. sample in right 2：Bungarus Parvus powder, Bungarus Parvus water extract and alcohol extract, the money of maximum conditions extraction Long-noded pit viper water extract and alcohol extract, Bungarus Parvus mix preparation, the Bungarus Parvus preparation of maximum conditions processing：

1) Bungarus Parvus crushes, and obtains Bungarus Parvus 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 money three times Long-noded pit viper water extract and alcohol extract.

3) by Bungarus Parvus water extract and alcohol extract, 121 DEG C of high pressure 30min, Bungarus Parvus water extract under the conditions of limit is prepared And alcohol extract.

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. Bungarus Parvus 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′-GACTTAGCTTTCTCATCTGTGATCCATATTA-3′

Anti-sense primer：5′-GCCTGCAGCCCCTCAGATGATATTTGTCCTCA-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 Bungarus Parvus.