CN109735647A - Identify specific primer, kit and the method for aweto - Google Patents
Identify specific primer, kit and the method for aweto Download PDFInfo
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Abstract
The present invention relates to Materia Medica Identification technical fields, and in particular to identifies the specific primer, kit and method of aweto.Identify the method for aweto the present invention provides the specific primer that can expand aweto ITS conservative fragments specifical and efficiently and using the specific primer.Discrimination method provided by the invention, specific, sensitivity with higher, only need that whether there is or not the accurate identifications that can realize aweto and other easily mixed species according to the electrophoretic band of PCR product, with it is easy to operate quickly, identify that accuracy is high, the advantages such as low in cost, quickly detect aweto suitable for the identification of the true and false of the aweto of great amount of samples or Chinese herbal medicine material.
Description
Technical field
The present invention relates to Materia Medica Identification technical fields, and in particular to identifies specific primer, the reagent of aweto
Box and method.
Background technique
Cordyceps sinensis (CORDYCEPS) is section ergot fungus cordyceps sinensis bacterium (Cordyceps sinensis (Berk.)
Sacc, different name Ophiocordyceps sinensis) it parasitizes on Lepidoptera Hepialidae (Hepialidae) insect larvae
Drying complex (Sung G H, Hywel-Jones N L, Sung the J M, et of stroma and larva corpse
al.Phylogenetic classification of Cordyceps and the clavicipitaceous fungi
[J].Studies in Mycology,2007,57:5.).Cordyceps sinensis is first recorded in " new compilation of materia medica " (Chen Lu, Wan Deguang, state's brocade
The book on Chinese herbal medicine of beautiful jade cordyceps sinensis newly examines the Jilin [J] traditional Chinese medicine, 2014,34 (10): 1022.), for Chinese rare traditional Chinese medicine, having
The effect of kidney tonifying benefit lung, hemostasis and phlegm.Studies have shown that cordyceps sinensis contains, there are many amino acid, peptides, polysaccharide, fatty acid and dimensions
The ingredients such as raw element have certain protective effect to myocardial damage and hepatic injury, and have the function of the (king such as anti-oxidant, antitumor
Sign, Liu Jianli cordyceps sinensis Recent Advances of Chemical Constituents [J] Chinese herbal medicine, 2009,7 (7): 1157；Liu X,Zhong F,
Tang X L,et al.Cordyceps sinensis protects against liver and heart injuries
In a rat model of chronic kidney disease:a metabolomic analysis [J] Chinese Pharmacological
Report, 2014,35 (5): 697；Yamaguchi Y,Kagota S,Nakamura K,et al.Antioxidant activity
of the extracts from fruiting bodies of cultured Cordyceps sinensis[J]
.Phytotherapy Research,2000,14(8):647；Nakamura K,Shinozuka K,Yoshikawa
N.Anticancer and antimetastatic effects of cordycepin,an active component of
Cordyceps sinensis[J].Journal of Pharmacological Sciences,2015,127(1):53.)。
Cordyceps sinensis is distributed mainly in the Alpine meadow of Qinghai-Tibet 3000~5000m of height above sea level, wherein Chinese winter worm
Summer grass accounts for 90% of total output or more, and there is a small amount of distribution on the ground such as Bhutan, Nepal.Wild cordyceps are distributed mainly on China
(weekly society, Zhang Jianchun, Huang Xiaoqing wait Tibet plateau Cordyceps Resources suitability area on the ground such as Tibet, Qinghai, Sichuan, Yunnan
Divide analysis [J] Acta Ecologica Sinica, 2018,38 (8): 2768；Li Y,Wang X L,Jiao L,et al.A survey of the
geographic distribution of Ophiocordyceps sinensis[J].Journal of
Microbiology,2011,49(6):913.).With the development of economy, the market demand of cordyceps sinensis increases year by year, but by
In excessive excavation, growing environment is seriously damaged, and producing region area constantly reduces, and supply falls short of demand for wild product, and price still occupies height not
Under, leading to market often has mixed adulterant to occur.Have when the case where Cordyceps gunnii (Berk.) Berk, liangshan cordyceps herb, Xinjiang cordyceps sinensis etc. pretend to be cordyceps sinensis
Occur.In addition relatively universal (Yao Yisang, the Zhu Jiashi of phenomena such as name of relevant bacteria species fermentation mycelium is chaotic, falsely uses, uses with, misapplying
The Chinese medicine cordyceps sinensis history mixed with contained a variety of aweto latin names and status [J] CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2016,
41(7):1361.).Moreover, being connected to such as genuine cordyceps sinensis stroma on false polypide, or there is also part fraud situation true
Polypide on phenomena such as connecing false stroma (Xiang Li cordyceps sinensis conservation biology research [D]；Beijing Union Medical College,
2013.).Especially discerns the false from the genuine under pulverulence in patent medicine or health product in the correlation and further increase the difficulty of identification.
The common discrimination method of cordyceps sinensis include form identify, microscopical characters and chemistry identify (Chen little Qiu, Liu Baoling,
The character and microscopical characters of the such as Zhao Zhongzhen cordyceps sinensis and its adulterant study [J] CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2011,36 (9):
1141；The .HS-SPME/GC-MS combination Chemical Measurement such as Song Yuling, Hu Ping, Zhao Shiyi identifies different strain ferment cordyceps sinensis product
And analysis of volatile components [J] Pharmaceutical Analysis magazine, 2018 (1): 67；The near infrared spectrum such as Wang Gangli, Shi Yan, Wei Yuhai mirror
Other cordyceps sinensis genunie medicinal materials [J] Chinese herbal medicine, 2006,37 (10): 1569；Zhang J,Wang P,Wei X,et al.A
metabolomics approach for authentication of Ophiocordyceps sinensis by liquid
chromatography coupled with quadrupole time-of-flight mass spectrometry[J]
.Food Research International,2015,76(Pt 3):489.].In recent years, DNA barcoding, specificity
The molecules such as PCR, multiplex PCR, Bar-HRM, LAMP authentication technique quickly grow in Chinese traditional medicine identification field (Chen S, Pang X,
Song J,et al.A renaissance in herbal medicine identification:from morphology
to DNA[J].Biotechnology Advances,2014,32(7):1237；Liu Y,Wang X Y,Gao Z T,et
al.Detection of Ophiocordyceps sinensis and its common adulterates using
species-specific primers.[J].Frontiers in Microbiology,2017,8；Luo Yuqin, Jiang Chao, Yuan
The multiple Allele-specific diagnostic PCR identification Dendrobidium huoshanness of the such as beautiful woman, dendrobium candidum and dendrobium devonianum medicinal material [J] Acta Pharmaceutica Sinica, 2017
(6):998；Wei S,Li J J,Chao X,et al.The potential power of Bar-HRM technology
in herbal medicine identification:[J].Frontiers in Plant Science,2016,7
(e8613):367；Zhao M,Shi Y,Lan W,et al.Rapid authentication of the precious
herb saffron by loop-mediated isothermal amplification(LAMP)based on internal
Transcribed spacer 2 (ITS2) sequence [J] .Scientific Reports, 2016,6:25370.), about
There are also reports for the molecular identificalion research of cordyceps sinensis, however, still lacking special, accurate, sensitive molecular identificalion side at present
Therefore method develops efficient cordyceps sinensis discrimination method and is of great significance.
Summary of the invention
In order to solve the technical problems existing in the prior art, the object of the present invention is to provide the spies for identifying aweto
Specific primer, kit and method realize the quick and precisely identification of aweto.
In order to achieve the above object, technical scheme is as follows: the present invention passes through to hundreds of parts of different regions source
It cordyceps sinensis material and is measured and analyzes with the DNA sequence dna of the higher easily mixed material of cordyceps sinensis similitude, discovery is each
From different places the ITS sequence of the aweto in source there are apparent conservative region, and aweto and it is easily mixed
There is also multiple difference sites for the ITS sequence of species of confusing, and on the basis of the sequencing results, target two region of DNA domain conducts
It distinguishes and identifies aweto and its easily obscure the DNA marker of species, and devise two pairs for the two specific fragments
Specific primer provides scientific basis for the identification of aweto and its powder, and for Related product identification.
Specifically, technical scheme is as follows:
Firstly, the present invention provides the specific primer for identifying aweto, including one in following (1), (2) primer pair
Pair or two pairs:
(1) forward primer: ITSSF1:5 '-CCCTACGAACACCACAGCA-3 '；
Reverse primer: ITSSR1:5 '-TGAGATGACACTCGGACA-3；
(2) forward primer: ITSSF2:5 '-CCCTACAAACACCACAGC-3；
Reverse primer: ITSSR2:5 '-GATTCATTTACTTACTTCTT-3.
On the basis of above-mentioned specific primer, the present invention is provided to be used to identify the worm summer in winter comprising the specific primer
The kit of careless bacterium.
To realize PCR detection, the kit further includes PCR reaction buffer, dNTPs, archaeal dna polymerase and positive control
Template.
Further, the present invention provides a kind of method for identifying aweto, to utilize the specific primer
Or the kit carries out PCR detection to sample to be tested, according to the sequence signature of PCR product or type of strip judgement it is described to
Whether whether sample contain or be aweto.
Preferably, the method for identifying aweto includes the following steps:
(1) DNA of sample to be tested is extracted；
(2) using the DNA as template, PCR amplification is carried out using the specific primer or the kit；
(3) judge whether sample to be tested contains or whether be aweto according to the type of strip of PCR product.
The response procedures of PCR amplification are as follows in above-mentioned steps (2): 94~98 DEG C, 5~10min；94~98 DEG C, 10~
30s, 52~58 DEG C, 10~30s, 72 DEG C, 10~30s, 25~40 circulations；72 DEG C, 2~10min.
To realize higher identification specificity and accuracy, preferably, when the primer of the PCR amplification is ITSSF1/
When ITSSR1, the response procedures of PCR amplification are as follows in above-mentioned steps (2): 94~98 DEG C, 5~10min；94~98 DEG C, 10~
30s, 54 DEG C, 10~30s, 72 DEG C, 10~30s, 25~40 circulations；72 DEG C, 2~10min.
The response procedures of PCR amplification are such as when the primer of the PCR amplification is ITSSF2/ITSSR2, in above-mentioned steps (2)
Under: 94~98 DEG C, 5~10min；94~98 DEG C, 10~30s, 52 DEG C, 10~30s, 72 DEG C, 10~30s, 25~40 circulations；
72 DEG C, 2~10min.
Preferably, 25 μ L reaction systems of PCR amplification are as follows in above-mentioned steps (2): 2 × Taq PCR Mix, 12.5 μ
L, 2.5 μM of 2 μ L, 10~50ng/ μ L DNA profilings of primer mixed liquor 1 μ L, ddH2O supply surplus；In the primer mixed liquor just
It is 1:1 to the molar ratio of primer and reverse primer.
Judge whether sample to be tested contains or whether be the worm summer in winter according to the type of strip of PCR product in above-mentioned steps (3)
Careless bacterium, judgment criteria are as follows: the unique DNA of 297bp occur according to the PCR product of primer pair ITSSF1/ITSSR1 amplification
Band, then detected cordyceps sinensis sample contains aweto or is aweto；If without the band, which is not
Or aweto is not contained；There is the unique DNA item of 136bp according to the PCR product of primer pair ITSSF2/ITSSR2 amplification
Band is then detected cordyceps sinensis sample and is containing aweto or is aweto；If without the band, which is not
Or aweto is not contained.
The present invention also provides the specific primer or the kits or the identification cordyceps sinensis method in the detection winter
Application in worm summer grass bacterium or the identification aweto true and false.
The method of specific primer of the present invention or kit or identification aweto containing specific primer
Suitable for can extract the identification of the complete cordyceps sinensis or the powder containing aweto of DNA.
The beneficial effects of the present invention are: it is conservative the present invention provides aweto ITS can be expanded specifical and efficiently
The specific primer of segment and the method for identifying aweto using the specific primer, identification side provided by the invention
Method, specific, sensitivity with higher, without sequencing, it is only necessary to which whether there is or not can realize the winter according to the electrophoretic band of PCR product
Worm summer grass bacterium and other accurate identifications for easily obscuring species have quick, low in cost, identification accuracy height easy to operate etc. excellent
Gesture, the true and false for being suitable for use in large sample cordyceps sinensis identify or quickly detect cordyceps sinensis in Chinese herbal medicine material.
Detailed description of the invention
Fig. 1 is that cordyceps sinensis and its easy PCR for obscuring species identify electrophoretogram in the embodiment of the present invention 2；Wherein, M DNA
Marker, marker band are followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom；Swimming lane 1-11
It is the specific amplified product of primer pair ITSSF1/ITSSR1 with CK1；Swimming lane 12-22 and CK2 are primer pair ITSSF2/
The specific amplified product of ITSSR2；Wherein, swimming lane 1-3 and swimming lane 12-14 is the amplified production of cordyceps sinensis；Swimming lane 4-6 and swimming
Road 15-17 is the amplified production of Cordyceps gunnii (Berk.) Berk；Swimming lane 7 and 18 is the amplified production of Xinjiang cordyceps sinensis；Swimming lane 8 and 19 is Cordyceps nutans
Amplified production；Swimming lane 9-10 and swimming lane 20-21 is the amplified production of liangshan cordyceps herb；Swimming lane 11 and 22 is that the amplification of golden cicada flower produces
Object；Swimming lane CK1 and CK2 are negative control.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The design of 1 aweto of embodiment identification specific primer
1, ITS sequence is analyzed
The present embodiment includes 81 parts of complete cordyceps sinensis and 35 parts of mixed adulterants by extraction, the 116 of totally 6 species part sample
DNA, using ITS sequence universal primer ITS5F/ITS4R (ITS5F:5 '-GGAAGTAAAAGTCGTAACAAGG-3 '；
ITS4R:5 '-TCCTCCGCTTATTGATATGC-3 ') amplification each sample ITS sequence, by sequencing and sequence analyze, determine
The conserved region of ITS sequence and region of variability, and the specific primer according to this design cordyceps sinensis ITS sequence.Above-mentioned 116 parts of samples
As shown in table 1.
Table 1 is used for 116 parts of samples of ITS sequence analysis
2, specific primer design
It is analyzed according to above-mentioned ITS sequence as a result, according to the ITS sequence conserved region of cordyceps sinensis and other easily obscuring species
Sequence variations area devises two couples of awetos specific primer I TSSF1/ITSSR1 and ITSSF2/ITSSR2, and sequence is as follows:
Forward primer: ITSSF1:5 '-CCCTACGAACACCACAGCA-3 '；
Reverse primer: ITSSR1:5 '-TGAGATGACACTCGGACA-3 '；
Forward primer: ITSSF2:5 '-CCCTACAAACACCACAGC-3 '；
Reverse primer: ITSSR2:5 '-GATTCATTTACTTACTTCTT-3 '.
The PCR of 2 aweto of embodiment identifies
Identify aweto using PCR amplification and its 5 kinds of mixed adulterants, the PCR discrimination method of aweto be as follows:
(1) DNA of sample is extracted；
(2) using the DNA extracted in step (1) as template, the primer pair ITSSF1/ITSSR1 that is designed using embodiment 1 into
Row pcr amplification reaction；
Forward primer: ITSSF1:5 '-CCCTACGAACACCACAGCA-3 '；
Reverse primer: ITSSR1:5 '-TGAGATGACACTCGGACA-3 '；
PCR reaction system is 25 μ L:2 × Taq PCR Mix, 12.5 μ L, 1 μ L of forward primer (2.5 μM), 1 μ of reverse primer
L (2.5 μM), 1 μ L of DNA profiling, adds ddH2O is supplemented to 25 μ L；
PCR reaction condition: 94 DEG C of 5min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃10min；
(3) using the DNA extracted in step (1) as template, the primer pair ITSSF2/ITSSR2 that is designed using embodiment 1 into
Row pcr amplification reaction；
Forward primer: ITSSF2:5 '-CCCTACAAACACCACAGC-3 '；
Reverse primer: ITSSR2:5 '-GATTCATTTACTTACTTCTT-3 '.
PCR reaction system is 25 μ L:2 × Taq PCR Mix, 12.5 μ L, 1 μ L of forward primer (2.5 μM), 1 μ of reverse primer
L (2.5 μM), 1 μ L of DNA profiling, adds ddH2O is supplemented to 25 μ L；
PCR reaction condition: 94 DEG C of 5min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃5min；
(4) agarose gel electrophoresis detection is carried out to above-mentioned amplified production, is identified according to electrophoretic band type, judged
Standard is as follows: the unique DNA band of 297bp occurs according to the PCR product of primer pair ITSSF1/ITSSR1 amplification, is then detected
Survey cordyceps sinensis sample is aweto；If the cordyceps sinensis sample is not aweto without the band；According to primer pair
There is the unique DNA band of 136bp in the PCR product of ITSSF2/ITSSR2 amplification, then being detected cordyceps sinensis sample is cordyceps sinensis
Bacterium；If the cordyceps sinensis sample is not aweto without the band.
Identification result is as shown in Figure 1, the results showed that, using ITSSF1/ITSSR1 primer pair amplifies, aweto exists
279bp has single band, mixes adulterant then without the specific band, using ITSSF2/ITSSR2 primer pair amplifies, cordyceps sinensis
Bacterium has single band in 136bp, mixes adulterant then without the specific band.Sequence verification through ITS sequence, it was demonstrated that above-mentioned presence
The sample of specific amplification band is aweto, shows 2 couples of primer spies with higher that the embodiment of the present invention 1 provides
The opposite sex can be used for the efficiently and accurately identification of aweto.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Sequence table
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Claims (9)
1. identifying the specific primer of aweto, which is characterized in that including a pair or two in following (1), (2) primer pair
It is right:
(1) forward primer: ITSSF1:5 '-CCCTACGAACACCACAGCA-3 '；
Reverse primer: ITSSR1:5 '-TGAGATGACACTCGGACA-3；
(2) forward primer: ITSSF2:5 '-CCCTACAAACACCACAGC-3；
Reverse primer: ITSSR2:5 '-GATTCATTTACTTACTTCTT-3.
2. identifying the kit of aweto, which is characterized in that including specific primer described in claim 1.
3. kit according to claim 2, which is characterized in that further include PCR reaction buffer, dNTPs, DNA polymerization
Enzyme and positive control template.
4. a kind of method for identifying aweto, which is characterized in that utilize specific primer or right described in claim 1
It is required that kit described in 2 or 3 carries out PCR detection to sample to be tested, judged according to the sequence signature of PCR product or type of strip
Whether whether the sample to be tested contain or be aweto.
5. according to the method described in claim 4, it is characterized by comprising the following steps:
(1) DNA of sample to be tested is extracted；
(2) using the DNA as template, using specific primer described in claim 1 or reagent described in claim 2 or 3
Box carries out PCR amplification；
(3) judge whether sample to be tested contains or whether be aweto according to the type of strip of PCR product.
6. method according to claim 4 or 5, which is characterized in that the response procedures of the PCR amplification are as follows: 94~98
DEG C, 5~10min；94~98 DEG C, 10~30s, 52~58 DEG C, 10~30s, 72 DEG C, 10~30s, 25~40 circulations；72 DEG C,
2~10min.
7. according to the method described in claim 6, it is characterized in that, when the primer of the PCR amplification is ITSSF1/ITSSR1
When, the response procedures of the PCR amplification are as follows: 94~98 DEG C, 5~10min；94~98 DEG C, 10~30s, 54 DEG C, 10~30s,
72 DEG C, 10~30s, 25~40 circulations；72 DEG C, 2~10min；
And/or
When the primer of the PCR amplification is ITSSF2/ITSSR2, the response procedures of the PCR amplification are as follows: 94~98 DEG C, 5
~10min；94~98 DEG C, 10~30s, 52 DEG C, 10~30s, 72 DEG C, 10~30s, 25~40 circulations；72 DEG C, 2~
10min。
8. method according to any one of claim 4 to 6, which is characterized in that 25 μ L reaction systems of the PCR amplification are such as
Under: 2 × Taq PCR Mix 12.5 μ L, 2.5 μM of 2 μ L, 10~50ng/ μ L DNA profilings of primer mixed liquor 1 μ L, ddH2O is supplied
Surplus；
The molar ratio of forward primer and reverse primer is 1:1 in the primer mixed liquor.
9. any one of specific primer described in claim 1 or kit described in claim 2 or 3 or claim 4~8
Application of the method in detection cordyceps sinensis or the identification cordyceps sinensis true and false.
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| CN201910044788.9A CN109735647A (en) | 2019-01-17 | 2019-01-17 | Identify specific primer, kit and the method for aweto |
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| CN201910044788.9A CN109735647A (en) | 2019-01-17 | 2019-01-17 | Identify specific primer, kit and the method for aweto |
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Application publication date: 20190510 |