CN106868194B - A kind of specific primer, kit and the discrimination method of Identification chinese herbs medicine house lizard - Google Patents
A kind of specific primer, kit and the discrimination method of Identification chinese herbs medicine house lizard Download PDFInfo
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- CN106868194B CN106868194B CN201710254140.5A CN201710254140A CN106868194B CN 106868194 B CN106868194 B CN 106868194B CN 201710254140 A CN201710254140 A CN 201710254140A CN 106868194 B CN106868194 B CN 106868194B
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Abstract
The present invention provides specific primer, kit and the discrimination methods of a kind of Identification chinese herbs medicine house lizard, CYTB gene based on house lizard devises a large amount of primer, and it is studied by a large amount of primer screenings, the primer with species specificity is filtered out, it is identified using the Chinese medicine house lizard to be measured of above-mentioned primer pair, house lizard medicinal material and other adulterants can accurately be separated, and its pcr amplification product is only 100~250bp, molecular weight is small, it ensure that it carries out a large amount of Chinese medicine house lizard identification in a short time, qualification result is accurate, stablizes, reliable, high sensitivity.
Description
Technical field
The invention belongs to Materia Medica Identification technical fields, and in particular to a kind of specific primer of Identification chinese herbs medicine house lizard, examination
Agent box and discrimination method.
Background technique
House lizard is entirety of the Gekkonidae animal without web gecko (Gekko Swinhoana Gunther), and pharmacological property is cold, taste
It is salty, it is slightly poisonous, enter lung, kidney channel.With powder detoxifcation, phencyclidine, antispastic cramp and relieves dizziness, warp reaches network and other effects thoroughly.Ancient medicine allusion quotation
Many about recording for house lizard in nationality, as recorded in Compendium of Material Medica, house lizard cures mainly paralysis due to windstroke, and brothers do not lift or joint-running
Wind pain and wind frightened epilepsy, infantile emaciation, blood product scrofula are treated scorpion and are stung；Qing Dynasty Zhao Qiguang mentioned in " book on Chinese herbal medicine ask former " house lizard enter blood system,
Blood disease is controlled, there is the effect of phlegm drops in enriching yin；" Traditional Chinese Medicine in Sichuan will " claims its energy " wind dispelling, blood-breaking product mass, curing oncoma ".
Since the source of house lizard is more complicated, commercially available house lizard often occurs mixing or adulterant, as newt, Chinese scrofula mud eel,
Discoloration Bloodsucker, gekko, red spiral shell wart mud eel, happiness mountain rock lizard, Phrynocephalus ulangalii (strauch), skink and the husky lizard etc. that changes colour.To some of which kind,
Morphological feature difference is subtle therewith for house lizard certified products, is difficult to identify house lizard medicinal material using traditional discrimination method, in view of
House lizard is changed in the links form such as processing, processing, storage and transport, so that the properties and characteristics of house lizard and its mixed adulterant
It disappears, to increase identification difficulty.And criminal, under the driving of economic interests, house lizard " passing a fake product off as a genuine one " " is filled with secondary
Well ", the market phenomenon of " substandard product " is generally existing, is difficult having the personnel of abundant identification of experience according to form spy
Sign is accurately identified.For the quality and clinical efficacy for ensuring Chinese medicine, existing research is expanded using modern molecular biology PCR
Increasing technology can be completed in a short time the identification to Part of Chinese Medicinal, however not propose a kind of utilization PCR skill also in the prior art
Art quickly to identify the specific primer and method of Chinese medicine house lizard kind.
Summary of the invention
The invention solves first technical problem be to provide a kind of specific primer of Identification chinese herbs medicine house lizard, use
The primer Identification chinese herbs medicine house lizard, qualification result is accurate, stablizes, reliable, high sensitivity, can be in a short time to a large amount of to be measured
Chinese medicine house lizard carries out Rapid identification, high-efficient.
The invention solves second technical problem be to provide a kind of kit of Identification chinese herbs medicine house lizard, using described
Kit Identification chinese herbs medicine house lizard, qualification result is accurate, stablizes, reliable, high sensitivity, can in a short time to it is a large amount of it is to be measured in
Medicine house lizard carries out Rapid identification, high-efficient.
The invention solves third technical problem be to provide a kind of method of Identification chinese herbs medicine house lizard, using the side
Method Identification chinese herbs medicine house lizard, qualification result is accurate, stablizes, reliable, high sensitivity, can keep in a short time a large amount of Chinese medicines to be measured
Palace carries out Rapid identification, high-efficient.
For this purpose, the primer is as follows the present invention provides a kind of primer for Identification chinese herbs medicine house lizard:
Upstream primer: 5 '-CAACGGGGCCCTATATTCCG-3 '；
Downstream primer: 5 '-AGGGTTGCGTATGGGGTTTC-3 '.
The present invention provides a kind of kits of Identification chinese herbs medicine house lizard, contain above-mentioned primer.
The kit, every kit include the primer liquid part and PCR reaction solution portion of respective independent packaging
Point；The PCR reaction solution part contain for carry out PCR amplification containing Mg2+Amplification buffer, dNTPs, Taq archaeal dna polymerase
And ddH2O。
The present invention also provides described in one kind primer or the kit application in medicine house lizard in authentication.
The present invention provides a kind of methods of Identification chinese herbs medicine house lizard, include the following steps:
(1) total DNA of Chinese medicine house lizard to be measured is extracted, it is spare；
(2) using the DNA extracted in step (1) as template, PCR amplification is carried out using following primer:
Upstream primer: 5 '-CAACGGGGCCCTATATTCCG-3 '；
Downstream primer: 5 '-AGGGTTGCGTATGGGGTTTC-3 '.
(3) size of pcr amplification product obtained in determination step (2), if in the pcr amplification product containing 100~
The DNA fragmentation of 250bp, then the Chinese medicine house lizard to be measured is true；Conversely, then the Chinese medicine house lizard to be measured is false.
The method, in the step (2), the PCR reaction system includes:
DNA profiling, 100ng, 2 μ L；
Upstream primer, 10 μM, 1 μ L；
Downstream primer, 10 μM, 1 μ L；
PCR SuperMix, 10 μ L；
ddH2O, 8 μ L；
Reaction total volume is 22 μ L.
The method, in the step (2), the PCR amplification program is as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
30s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 30 circulations, then proceed to 72 DEG C of extension 5min, be cooled to 4 DEG C, obtain
4 DEG C of amplified production preservation.
The method, in the step (3), the DNA fragmentation of the 100~250bp is the DNA piece of 110~150bp
Section.
The method, the DNA fragmentation of the 100~250bp are the DNA fragmentation of 116bp.
The method, the sequence dna fragment of the 116bp is as shown in SEQ ID NO:3.
Technical solution of the present invention has the advantages that
(1) specific primer of the present invention for Identification chinese herbs medicine house lizard, the CYT 1 B gene based on house lizard devise
A large amount of primer, and studied by a large amount of primer screenings, the primer with species specificity has been filtered out, above-mentioned primer pair is used
Chinese medicine house lizard to be measured is identified, can accurately be separated house lizard medicinal material and other adulterants, and its pcr amplification product is only
100~250bp, molecular weight is small, ensure that it carries out a large amount of Chinese medicine house lizard identification in a short time, qualification result is accurate, steady
Fixed, reliable, high sensitivity.
(2) kit of the present invention for Identification chinese herbs medicine house lizard, containing described for reflecting in the kit
Determine the specific primer of Chinese medicine house lizard, the primer has species specificity, carries out using the Chinese medicine house lizard to be measured of above-mentioned primer pair
Identification, can accurately separate house lizard medicinal material and other adulterants, carry out a large amount of Chinese medicine house lizard identification, identification in a short time
As a result accurate, stable, reliable, high sensitivity.
(3) method of Identification chinese herbs medicine house lizard of the present invention includes the following steps: that (1) extracts Chinese medicine house lizard to be measured
Total DNA, it is spare；(2) using the DNA extracted in step (1) as template, primer designed by the invention carries out PCR amplification；(3)
The size of pcr amplification product obtained in determination step (2), if the DNA piece in the pcr amplification product containing 100~250bp
Section, then the Chinese medicine house lizard to be measured is true；Conversely, then the Chinese medicine house lizard to be measured is false；The method can by house lizard medicinal material with
Other adulterants accurately separate, and pcr amplification product is only 100~250bp, and molecular weight is small, ensure that it in a short time
A large amount of Chinese medicine house lizard identification is carried out, qualification result is accurate, stablizes, reliable, high sensitivity.
(4) method of Identification chinese herbs medicine house lizard of the present invention, in the step (3), the DNA piece of the 100~250bp
Section is the DNA fragmentation of 116bp, and pcr amplification product molecular weight is small, ensure that it carries out a large amount of Chinese medicine house lizard mirror in a short time
It is fixed.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it is clear that the accompanying drawings in the following description is
Some embodiments of the present invention, for those of ordinary skill in the art, without creative efforts, also
Other drawings may be obtained according to these drawings without any creative labor.
Fig. 1 is the agarose gel electrophoresis figure in the embodiment of the present invention 3.
Specific embodiment
Main agents used in the present invention are as follows:
Genomic DNA Kit (OMEGA company),PCR SuperMix(+dye)
With Trans2K Plus DNA Marker (Beijing Quan Shijin biotech company).
Capital equipment used in the present invention is as follows:
ST16R high speed freezing centrifuge is purchased from Wei Taike, Dolphin View purchased from match silent winged generation you, V-GES electrophoresis apparatus
Gel imaging system is purchased from Wei Taike.
Embodiment 1
A kind of specific primer for Identification chinese herbs medicine house lizard is present embodiments provided, is set for the CYT 1 B gene of house lizard
The specific primer of meter is as follows, referring in sequence table shown in SEQ ID NO:1-2:
Upstream primer: 5 '-CAACGGGGCCCTATATTCCG-3 '；
Downstream primer: 5 '-AGGGTTGCGTATGGGGTTTC-3 '.
Above-mentioned primer is synthesized by Shanghai Invitrogen Corp. (Beijing combining unit).
Embodiment 2
A kind of kit for Identification chinese herbs medicine house lizard is present embodiments provided, every kit includes respectively independent
The primer liquid part and PCR reaction solution part of packaging；The primer liquid is containing the specific primer in the embodiment 1；
The PCR reaction solution part contain for carry out PCR amplification containing Mg2+Amplification buffer, dNTPs, Taq archaeal dna polymerase and
ddH2O, the formula of every kit are as follows:
Upstream primer, 10 μM, 1 μ L；
Downstream primer, 10 μM, 1 μ L；
PCR SuperMix, 10 μ L；
ddH2O, 8 μ L.
Embodiment 3
The kit Identification chinese herbs medicine for present embodiments providing a kind of specific primer using embodiment 1 or embodiment 2 is kept
The method in palace, includes the following steps:
(1) medicinal material is collected in the provinces such as Shanxi, Anhui, Guangxi and Hebei (table 1), is tested all samples in table 1 and is
Commercial product can acquire or buy by being commercially available, or from source place.
1 house lizard of table and its common adulterant sample source
The freeze-dried powder of sample in selection in table 1 usesGenomic DNAKit extracts total DNA, according to
Specification operation, steps are as follows:
(1) total DNA of Chinese medicine house lizard to be measured is extracted
1) freeze-dried powder≤25mg for taking each sample respectively is placed in a sterile 1.5mL centrifuge tube；
2) the Proteinase K (Proteinase K) of the LB2 (lysate) and 20 μ L of 100 μ L is added；
3) 55 DEG C of metal baths are incubated for until cracking completely, about 1 hour, every 15min was mixed by inversion once；
4) the RNase A (RNA enzyme) of 20 μ L is added in sample, 2min is incubated at room temperature, to remove the interference of RNA；
5) 12000rpm is centrifuged 5min, shifts supernatant in a sterile centrifuge tube；
6) BB2 (combination buffer) of 500 μ L is added, be vortexed 5s immediately, is incubated at room temperature 10min；
7) whole solution is added in centrifugal column, 12000rpm is centrifuged 30s, discards efflux；
8) CB2 (cleaning buffer solution) of 500 μ L is added, 12000rpm is centrifuged 30s, discards efflux；
9) it is primary to repeat step 8)；
10) WB2 (washing buffer) of 500 μ L is added, 12000rpm is centrifuged 30s, discards efflux；
11) it is primary to repeat step 10)；
12) 12000rpm is centrifuged 2min, completely removes remaining WB2；
13) centrifugal column is placed in a clean centrifuge tube, 50 μ L, 65 DEG C of preheating EB are added in the center of column, and (elution is slow
Fliud flushing), it is stored at room temperature 1min, 12000rpm is centrifuged 1min, eluted dna, and the DNA eluted is placed in -20 as test solution
DEG C save.
(2) using the DNA extracted in step (1) as template, PCR amplification is carried out using following primer:
Upstream primer: 5 '-CAACGGGGCCCTATATTCCG-3 '；
Downstream primer: 5 '-AGGGTTGCGTATGGGGTTTC-3 '.
The PCR reaction system includes:
DNA profiling, 100ng, 2 μ L；
Upstream primer, 10 μM, 1 μ L；
Downstream primer, 10 μM, 1 μ L；
PCR SuperMix, 10 μ L；
ddH2O, 8 μ L；
Reaction total volume is 22 μ L.
The PCR amplification program is as follows:
95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 30 circulations, then
Continue 72 DEG C of extension 5min, is cooled to 4 DEG C, 4 DEG C of amplified production preservations of acquisition.
(3) it takes the pcr amplification product in step (2) to carry out agarose gel electrophoresis, using V-GES electrophoresis apparatus, measures each
The size of the pcr amplification product of sample, for agarose gel electrophoresis method according to three IV B of annex of version Chinese Pharmacopoeia in 2015, glue is dense
Degree is 1%, and electrophoretic buffer is TAE buffer (Tris-EDTA- acetate buffer, pH 8.3), is obtained in above-mentioned steps (2)
The applied sample amount of PCR reaction solution of each sample be respectively 10 μ L, DNA molecular amount marks (Trans2K Plus DNA
Marker) applied sample amount is 2 μ L, after electrophoresis, gel is taken to dye 20min in ethidium bromide solution (0.5 μ g/mL),
Observation on Dolphin View gel imaging system is as a result, 1 be wherein DNA Marker, 2 be house lizard, and 3 be gekko, and 4 be discoloration
Lizard is set, 5 be Eremias argus, and 6 be he Chinese skink Eumeces chinensis (Fig. 1), and gekko, Discoloration Bloodsucker, Eremias argus and he Chinese skink Eumeces chinensis do not have item
Band, and the PCR reaction solution of the freeze-dried powder of house lizard respectively has a DNA band in 100~250bp and 250~500bp respectively, wherein
250~500bp amplified band is non-specific amplification band, and the DNA band in the position 100~250bp is that the PCR of design of primers is produced
Object size (116bp) shows that the primer that the present invention designs has species specificity, can be accurate using the primer and the method for the present invention
House lizard medicinal material and other adulterants are distinguished to come, and the DNA fragmentation expanded is only 116bp, and molecular weight is small, detection rates
Fastly, the DNA band using the DNA Gel Extraction Kit purification and recovery of OMEGA company in the position 100~250bp is
Then the sequence dna fragment of 116bp is sequenced by Shanghai Invitrogen Corp. (Beijing combining unit), the 116bp's
Sequence dna fragment is as shown in SEQ.ID NO:3, by the sequence SEQ.ID NO:3 it is found that the sequence dna fragment of the 116bp
For specific amplification band, the primer for further proving that the present invention designs has species specificity.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
SEQUENCE LISTING
<110>Beijing JianSheng pharmacy Co., Ltd
<120>a kind of specific primer, kit and the discrimination method of Identification chinese herbs medicine house lizard
<130> HA201602557
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
caacggggcc ctatattccg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
agggttgcgt atggggtttc 20
<210> 3
<211> 116
<212> DNA
<213>house lizard Gekko swinhoana gunther
<400> 3
caacggggcc ctatattccg accactcact cgattaacac ttttaatcct agccaacaac 60
atcatcctac tgacctgatt aggcggagaa ccagttgaaa ccccatacgc aaccct 116
Claims (10)
1. a kind of specific primer of Identification chinese herbs medicine house lizard, which is characterized in that the primer is as follows:
Upstream primer: 5 '-CAACGGGGCCCTATATTCCG-3 '；
Downstream primer: 5 '-AGGGTTGCGTATGGGGTTTC-3 '.
2. a kind of kit of Identification chinese herbs medicine house lizard, which is characterized in that contain primer described in claim 1.
3. kit according to claim 2, which is characterized in that every kit includes drawing for respective independent packaging
Thing liquid part and PCR reaction solution part；The PCR reaction solution part contain for carry out PCR amplification containing Mg2+Amplification buffering
Liquid, dNTPs, Taq archaeal dna polymerase and ddH2O。
4. a kind of primer as described in claim 1 or kit described in claim 2 or 3 answering in medicine house lizard in authentication
With.
5. a kind of method of Identification chinese herbs medicine house lizard, which comprises the steps of:
(1) total DNA of Chinese medicine house lizard to be measured is extracted, it is spare；
(2) using the DNA extracted in step (1) as template, PCR amplification is carried out using following primer:
Upstream primer: 5 '-CAACGGGGCCCTATATTCCG-3 '；
Downstream primer: 5 '-AGGGTTGCGTATGGGGTTTC-3 '；
(3) size of pcr amplification product obtained in determination step (2), if containing 100~250bp in the pcr amplification product
DNA fragmentation, then the Chinese medicine house lizard to be measured is true；Conversely, then the Chinese medicine house lizard to be measured is false.
6. according to the method described in claim 5, it is characterized in that, in the step (2), the PCR reaction system includes:
DNA profiling, 100ng, 2 μ L；
Upstream primer, 10 μM, 1 μ L；
Downstream primer, 10 μM, 1 μ L；
PCR SuperMix, 10 μ L；
ddH2O, 8 μ L；
Reaction total volume is 22 μ L.
7. according to the method described in claim 5, it is characterized in that, the PCR amplification program is as follows: 95 in the step (2)
DEG C initial denaturation 5min, 95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 30 circulations, then proceed to 72 DEG C and prolong
5min is stretched, is cooled to 4 DEG C, 4 DEG C of amplified production preservations of acquisition.
8. according to the described in any item methods of claim 5-7, which is characterized in that in the step (3), the 100~250bp
DNA fragmentation be 110~150bp DNA fragmentation.
9. according to the method described in claim 8, it is characterized in that, the DNA fragmentation of 100~250bp described in the step (3)
For the DNA fragmentation of 116bp.
10. according to the method described in claim 9, it is characterized in that, the sequence dna fragment of the 116bp such as SEQ ID
Shown in NO:3.
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| CN114292921B (en) * | 2021-12-20 | 2023-09-26 | 南方医科大学 | Method for quality control of Jinlong capsules by detecting copy number of Gekko Swinhonis specific fragment based on molecular quantification technology, primer and probe |
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Non-Patent Citations (3)
| Title |
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| 5种壁虎科动物Cyt b基因的序列分析;韦素玲等;《安徽农业科学》;20080630;第36卷(第6期);第2268-2269页 |
| 基于COI条形码序列的蛤蚧及其混伪品的DNA分子鉴定;张红印等;《世界科学技术_中药现代化 专题讨论：中药材研究与开发》;20140228;第16卷(第2期);第269-273页 |
| 快速PCＲ技术鉴别中药材蛤蚧的方法研究;蒋超等;《中国现代中药》;20170131;第19卷(第1期);第21-25页 |
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