CN109439660A - One species-specific primer and the PCR discrimination method of Chinese medicine zaocys dhumnade - Google Patents
One species-specific primer and the PCR discrimination method of Chinese medicine zaocys dhumnade Download PDFInfo
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- CN109439660A CN109439660A CN201811523650.9A CN201811523650A CN109439660A CN 109439660 A CN109439660 A CN 109439660A CN 201811523650 A CN201811523650 A CN 201811523650A CN 109439660 A CN109439660 A CN 109439660A
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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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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a species-specific primer and the PCR discrimination method of Chinese medicine zaocys dhumnade, which is cyt b-05-F and cyt b--05-R, and sequence is respectively as shown in SEQ.ID.NO.1 and SEQ.ID.NO.2.A kind of PCR discrimination method of Chinese medicine zaocys dhumnade is the following steps are included: the genomic DNA for extracting Chinese medicine is added primer described in claim 1 and carries out PCR reaction as template；After reaction, for PCR product through agarose gel electrophoresis, having single band at 200bp is zaocys dhumnade certified products.This method is easy to operate, is easy to grasp, accuracy height, can the precise Identification snake drugs true and false.
Description
Technical field
The invention belongs to the identifications of Chinese medicine to identify field, and in particular to Molecular Identification, in particular to a species specificity are drawn
Object and the PCR discrimination method of Chinese medicine zaocys dhumnade.
Background technique
Visited according to market and literature survey it is found that the adulterant of Chinese medicine zaocys dhumnade have Ptyas korras (Ptyas korros),
Dinodon rufozonatum snake (Dinodon rufozonatum), water snake (Hurria rhynchops), indian cobra (Naja naja), Meng Jia
It draws cobra (Naja kaouthia), dodge squama snake (Xenopeltis unicolor), Natrix piscater (Xenochrophis
Piscator), Bungarus multicinctus (Bungarus multicinctus), Malaysia krait (Bungarus candidus), long-nosed pit viper
(Agkistrodon acutus) etc..
In current authentication technique, " Chinese Pharmacopoeia " 2015 edition owner will be identified using properties and characteristics and microscopic features,
Foundation are as follows: surface dark brown or green black, Density Diamond scale: back squama line number is in pairs, carries on the back the strong barring of center 2-4 row scale,
It forms two and passes through all black lines.For cephalic disc in centre, oblate, eye lower recess greatly is glossy.8 pieces of upper labial, the 4th, 5
Piece enter socket of the eye, 1 piece of cheek squama, descends 1 piece of squama at the moment, smaller, 2 pieces of squama after eye.Spine is towering at ridge.Abdomen splits edge to curls inward
Song, ridge muscle is thick, yellow-white or light brown, it is seen that the rib cage of marshalling.Tail portion is tapered and grows, urostege duplicate rows.Skinner
Only let the hair grow the skin squama of tail, middle section is more smooth.Gas raw meat, it is lightly seasoned.Scutellum is close colourless or faint yellow, and surface has longitudinal stripe.Table
Gather brown or brown-black pigment particle are seen in epidermis face, and it is agglomerating to be often linked to be netted, branched or aggregation.Rhabdium is faint yellow
Or it is close colourless.There is light and dark fine and closely woven band.The nearly colourless or light gray of bone chip, is in irregular fragment, bone lacuna spindle shape,
Most equidirectional arrangement, bone canalicules are close and relatively thick.These identify main points and identification person are required to have identification of experience quite abundant, no
The identification between zaocys dhumnade and mixed adulterant is not can be effectively used to then.
The polymerase chain reaction recorded in 2015 editions " Chinese Pharmacopoeias " is a kind of relatively effective molecular identification method, no
It crosses the method and does not have specificity in the identification of zaocys dhumnade and certain class medicinal materials, a lot of other class medicinal materials are in 300-
The position 400bp also will appear band, be not effectively achieve the purpose for distinguishing zaocys dhumnade and its mixed adulterant.
Summary of the invention
The primary purpose of the present invention is that being directed to the cyt b gene of zaocys dhumnade, design provides a species-specific primer.
Another object of the present invention is to provide the PCR discrimination methods of Chinese medicine zaocys dhumnade.
The purpose of the invention is achieved by the following technical solution:
A kind of primer pair, cyt b-05-F and cyt b--05-R are designed for the characterizing gene cyt b of zaocys dhumnade,
Its sequence is respectively as shown in SEQ.ID.NO.1 and SEQ.ID.NO.2.
Cyt b-05-F:GCTCTACAAATCACAACCG (SEQ.ID.NO.1)；
Cyt b-05-R:TTTTGTTTAGGTAAGATCCGTAG (SEQ.ID.NO.2).
For zaocys dhumnade and its mixed adulterant cyt b gene heterogeneity, higher, length is about 200bp to above-mentioned primer pair
Segment and design.Cyt b gene segment overall length 307bp, primer cyt b-05-F length is 19bp, positioned at the 9 of cyt b gene
~27 sites；Primer cyt b-05-F length is 23bp, positioned at 9~27 site, 194~216 site of cyt b gene.
A kind of PCR discrimination method of Chinese medicine zaocys dhumnade, comprising the following steps:
The genomic DNA of Chinese medicine is extracted as template, above-mentioned primer is added and carries out PCR reaction；After reaction, PCR
For product through agarose gel electrophoresis, having single band at 200bp is zaocys dhumnade certified products；
The Chinese medicine includes zaocys dhumnade certified products and its mixed adulterant；
The mixed adulterant is Ptyas korras, dinodon rufozonatum snake, water snake, indian cobra, Naja kaouthia, dodges squama snake, fishing trip
One or more of snake, Bungarus multicinctus, Malaysia krait or long-nosed pit viper；
The genomic DNA for extracting Chinese medicine, is using Animal genome DNA extraction kitDNA
Mini Kit (QIAGEN company, Germany) chooses without the medicinal material sample 30mg for going mouldy and damaging by worms, is cut with what is sterilized
Knife is cut into fragment, is placed in 2mL centrifuge tube；180 μ L Buffer ATL and 20 μ L proteinase K, oscillation treatment is added
20s, 56 DEG C of water-baths to tissue cracking completely；Of short duration centrifugation, to remove in centrifuge tube lid along liquid；200 μ L Buffer are added
AL vibrates 15s, 70 DEG C of water-bath 10min, of short duration centrifugation；200 μ L dehydrated alcohols are added, vibrate 15s, of short duration centrifugation；By the above institute
It obtains mixed liquor to be carefully added into centrifugal column, centrifugal column is put into 2mL collecting pipe, 1min is centrifuged with 6000 × g, by centrifugal column
It takes out, is put into a clean 2mL collecting pipe；500 μ L Buffer AW1 are added into centrifugal column, with 6000 × g centrifugation
1min takes out centrifuge tube, is put into a clean 2mL collecting pipe；500 μ L Buffer AW2 are added into centrifugal column, with
20000 × g is centrifuged 3min, and centrifuge tube is taken out, is put into a clean 2mL collecting pipe；50 μ L Buffer AE, room is added
Temperature is lower to stand 1min, and 6000 × g is centrifuged 1min, collecting pipe is covered, -20 DEG C spare；
The described PCR reaction, it includes 1 μ L of template, each 1 μ L of forward primer, reverse primer that reaction system, which includes: 25 μ L,
12.5 μ L of archaeal dna polymerase, 9.5 μ L of aqua sterilisa；
The described PCR reaction, response procedures are as follows: 95 DEG C of initial denaturation 4min, 30 circulations (95 DEG C of denaturation 30s, 58.8 DEG C
Renaturation 10s, 72 DEG C of extension 1min), then 72 DEG C of extension 5min.
The present invention has the following advantages and effects with respect to the prior art:
Compared to other discrimination methods of zaocys dhumnade, operation of the present invention is simple, has experience abundant without practitioner
Discrimination process can be completed, and obtain precise Identification result；It is high specificity, high sensitivity, reproducible, it is applied widely,
Certain application prospect is all had in terms of the precise Identification of snake drugs, medicine materical crude slice and the various product containing zaocys dhumnade.
Detailed description of the invention
Fig. 1 is zaocys dhumnade and its mixed adulterant genome dna electrophoresis figure；
Fig. 2 is the PCR reaction product electrophoretogram of zaocys dhumnade Yu other 10 kinds of mixed adulterants；
Wherein, 1 zaocys dhumnade, 2 indian cobras, 3 Malaysia kraits, 4 long-nosed pit vipers, 5 dinodon rufozonatum snakes, 6 Ptyas korras, 7 Bungarus multicinctus, 8 dodge
Squama snake, 9 Naja kaouthias, 10 Natrix piscaters, 11 water snakes, N negative control；
M:DNA ladder most bright band is 500bp, is followed successively by 400bp, 300bp, 200bp, 100bp downwards.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment
A kind of PCR discrimination method of Chinese medicine zaocys dhumnade, comprising the following steps:
(1) it extracts genomic DNA: choosing without the medicinal material sample about 30mg for going mouldy and damaging by worms, be cut into the scissors sterilized
Fragment is placed in 2mL centrifuge tube.It is added 180 μ L Buffer ATL and 20 μ L proteinase K, oscillation treatment 20s, 56 DEG C
Water-bath cracks completely to tissue.Of short duration centrifugation, to remove in centrifuge tube lid along liquid.200 μ L Buffer AL are added, vibrate
15s, 70 DEG C of water-bath 10min, of short duration centrifugation.200 μ L dehydrated alcohols are added, vibrate 15s, of short duration centrifugation.By mixing obtained as above
Liquid is carefully added into centrifugal column, and centrifugal column is put into 2mL collecting pipe, is centrifuged 1min with 6000 × g, centrifugal column is taken out,
It is put into a clean 2mL collecting pipe.500 μ L Buffer AW1 are added into centrifugal column, 1min is centrifuged with 6000 × g, it will
Centrifuge tube takes out, and is put into a clean 2mL collecting pipe.500 μ L Buffer AW2 are added into centrifugal column, with 20000 ×
G is centrifuged 3min, and centrifuge tube is taken out, is put into a clean 2mL collecting pipe.50 μ L Buffer AE are added, it is quiet at room temperature
1min is set, 6000 × g is centrifuged 1min, collecting pipe is covered, -20 DEG C spare.
The medicinal material is 11 kinds of zaocys dhumnade and other class medicinal materials in the markets such as Guangzhou, Anhui, Jiangxi, Sichuan, totally 13
Item, respectively zaocys dhumnade (Zaocys dhumnades), Ptyas korras (Ptyas korros), dinodon rufozonatum snake (Dinodon
Rufozonatum), water snake (Hurria rhynchops), indian cobra (Naja naja), Naja kaouthia (Naja
Kaouthia), squama snake (Xenopeltis unicolor), Natrix piscater (Xenochrophis piscator), Bungarus multicinctus are dodged
(Bungarus multicinctus), Malaysia krait (Bungarus candidus), long-nosed pit viper (Agkistrodon acutus).
The equal Successful amplification of genomic DNA (Fig. 1) of zaocys dhumnade and other 10 kinds of mixed adulterants, ultramicron UV, visible light are divided light
The purity and concentration of degree meter 11 kinds of class medicinal material sample solutions of detection, A260/A280 value minimum 1.78, up to 1.92,
It can be used for subsequent gene magnification.
(2) PCR reacts:
PCR primer:
Cyt b-05-F sequence is GCTCTACAAATCACAACCG (SEQ.ID.NO.1)；
Cyt b-05-R sequence is TTTTGTTTAGGTAAGATCCGTAG (SEQ.ID.NO.2).
Reaction system: 25 μ L include 1 μ L of template, each 1 μ L of forward primer, reverse primer (60~82ng/ μ L), DNA polymerization
12.5 μ L of enzyme, 9.5 μ L of aqua sterilisa.
Reaction condition: 95 DEG C of initial denaturation 4min, (95 DEG C of denaturation 30s, 58.8 DEG C of renaturation 10s, 72 DEG C extend 30 circulations
1min), then 72 DEG C of extension 5min.
(3) electrophoresis detection: after reaction, whether PCR product observes and records each sample through agarose gel electrophoresis
There is amplified band.
As a result as shown in Fig. 2, only zaocys dhumnade sample in 200bp or so has single band, other 10 kinds mixed adulterant samples
Amplification failure, no electrophoretic band occur.Show zaocys dhumnade cyt b gene fragment-specific primer cyt b-5-F and cyt b-5-R
Reaction can accurately identify zaocys dhumnade and other mixed adulterants, have the characteristics that it is specific it is high, specificity is strong, be in the market zaocys dhumnade and
The identification of other products containing zaocys dhumnade provides foundation.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Ji'nan University
<120>one species-specific primers and the PCR discrimination method of Chinese medicine zaocys dhumnade
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> cyt b-05-F
<400> 1
gctctacaaa tcacaaccg 19
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> cyt b-05-R
<400> 2
ttttgtttag gtaagatccg tag 23
Claims (7)
1. a kind of primer pair, it is characterised in that: be cyt b-05-F and cyt b--05-R, sequence is respectively such as SEQ.ID.NO.1
With shown in SEQ.ID.NO.2.
2. a kind of PCR discrimination method of Chinese medicine zaocys dhumnade, it is characterised in that the following steps are included:
The genomic DNA of Chinese medicine is extracted as template, primer described in claim 1 is added and carries out PCR reaction；Reaction terminates
Afterwards, for PCR product through agarose gel electrophoresis, having single band at 200bp is zaocys dhumnade certified products.
3. discrimination method according to claim 2, it is characterised in that: the Chinese medicine includes zaocys dhumnade certified products and its mixes
Adulterant.
4. discrimination method according to claim 3, it is characterised in that: the mixed adulterant is Ptyas korras, dinodon rufozonatum snake, water
One or more of snake, indian cobra, Naja kaouthia, sudden strain of a muscle squama snake, Natrix piscater, Bungarus multicinctus, Malaysia krait or long-nosed pit viper.
5. discrimination method according to claim 2, it is characterised in that: it is described extract Chinese medicine genomic DNA, be using
Animal genome DNA extraction kitDNA Mini Kit chooses without the medicinal material sample 30mg for going mouldy and damaging by worms,
It is cut into fragment with the scissors sterilized, is placed in 2mL centrifuge tube；180 μ L Buffer ATL and 20 μ L proteinase are added
K, oscillation treatment 20s, 56 DEG C of water-baths to tissue cracking completely；Of short duration centrifugation, to remove in centrifuge tube lid along liquid；200 μ are added
L Buffer AL vibrates 15s, 70 DEG C of water-bath 10min, of short duration centrifugation；Be added 200 μ L dehydrated alcohols, vibrate 15s, it is of short duration from
The heart；Mixed liquor obtained as above is carefully added into centrifugal column, centrifugal column is put into 2mL collecting pipe, with 6000 × g centrifugation
1min takes out centrifugal column, is put into a clean 2mL collecting pipe；500 μ L Buffer AW1 are added into centrifugal column, with
6000 × g is centrifuged 1min, and centrifuge tube is taken out, is put into a clean 2mL collecting pipe；500 μ L Buffer AW2 are added to arrive
In centrifugal column, 3min is centrifuged with 20000 × g, centrifuge tube is taken out, is put into a clean 2mL collecting pipe；50 μ L are added
Buffer AE, stands 1min at room temperature, and 6000 × g is centrifuged 1min, collecting pipe is covered, -20 DEG C spare.
6. discrimination method according to claim 2, it is characterised in that: the PCR reaction, reaction system includes: 25 μ
L includes 1 μ L of template, each 1 μ L of forward primer, reverse primer, 12.5 μ L of archaeal dna polymerase, 9.5 μ L of aqua sterilisa.
7. discrimination method according to claim 2, it is characterised in that: the PCR reaction, response procedures are as follows: 95 DEG C
Initial denaturation 4min, 30 circulations, then 72 DEG C of extension 5min；
30 circulations, specially 95 DEG C denaturation 30s, 58.8 DEG C of renaturation 10s, 72 DEG C of extension 1min.
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Citations (5)
| 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 |
| CN106636397A (en) * | 2016-12-20 | 2017-05-10 | 中国中医科学院中药研究所 | Primer combination for identifying three medicinal snakes and application thereof |
| CN106834535A (en) * | 2017-04-18 | 2017-06-13 | 北京建生药业有限公司 | Specific primer, kit and discrimination method for identifying Bungarus Parvus |
| CN107012230A (en) * | 2017-04-24 | 2017-08-04 | 北京康仁堂药业有限公司 | Differentiate the method for zaocys dhumnade, scorpio and centipede using specific primer |
| CN107663542A (en) * | 2016-07-29 | 2018-02-06 | 周亚伟 | A kind of PCR method for identifying zaocys dhumnade |
-
2018
- 2018-12-13 CN CN201811523650.9A patent/CN109439660A/en active Pending
Patent Citations (5)
| 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 |
| CN107663542A (en) * | 2016-07-29 | 2018-02-06 | 周亚伟 | A kind of PCR method for identifying zaocys dhumnade |
| CN106636397A (en) * | 2016-12-20 | 2017-05-10 | 中国中医科学院中药研究所 | Primer combination for identifying three medicinal snakes and application thereof |
| CN106834535A (en) * | 2017-04-18 | 2017-06-13 | 北京建生药业有限公司 | Specific primer, kit and discrimination method for identifying Bungarus Parvus |
| CN107012230A (en) * | 2017-04-24 | 2017-08-04 | 北京康仁堂药业有限公司 | Differentiate the method for zaocys dhumnade, scorpio and centipede using specific primer |
Non-Patent Citations (3)
| Title |
|---|
| XIAOMEI ZHANG ET AL.: "Development and evaluation of a PCR-based assay kit for authentication of Zaocys dhumnades in traditional Chinese medicine", 《MITOCHONDRIAL DNA PART A》 * |
| 宋礼华: "《生物工程制药研究与实践》", 28 February 2009, 安徽科学技术出版社 * |
| 黄璐琦 等: "《中国药用动物DNA条形码研究》", 30 November 2016, 福建科学技术出版社 * |
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