CN108018363A - The specific primer and its identification method of one group of identification tortoise plastron - Google Patents
The specific primer and its identification method of one group of identification tortoise plastron Download PDFInfo
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Abstract
The present invention relates to the specific primer and its identification method of one group of identification tortoise plastron, belong to Chinese traditional medicine identification technical field；Extraction and purification genomic DNA after the present invention first crushes tortoise plastron product, and using this sample as template, the specific nucleotide sequences through designing, screening are selected to carry out PCR amplification for primer, then product is detected into row agarose gel electrophoresis analysis with gel imaging system, it is final according to testing result to the true and false of tortoise plastron product, mix pseudo- situation and judge；Pass through the identification method in the present invention, it can be determined that whether be mixed with adulterant in sample；This method is easy to operate, without sequencing, specificity is strong, strong antijamming capability, reproducible, it is and applied widely, available for the precise Identification of tortoise plastron base, medicinal material and medicine materical crude slice, practicable method is provided to differentiate the true and false of tortoise plastron product and mixing pseudo- situation.
Description
Technical field
The present invention relates to the specific primer and its identification method of one group of identification tortoise plastron, belong to Chinese traditional medicine identification technical field.
Background technology
Tortoise plastron (Testudinis Carapax ET Plastrum), is Testudinidae animal tortoise (Chinemys
Reevesii carapace and plastron), are a kind of traditional animal tcms, its beginning is recorded in《Sheng Nong's herbal classic》, it is listed in
Product.History tree is on the books, is now classified as medicine-food two-purpose class Chinese medicine by National Health Service.Tortoise plastron cold nature, taste is salty sweet, Return liver,
Kidney, the heart channel of Hang-Shaoyin, energy nourishing and suppressing Yang, the strong bone of kidney-nourishing, blood-nourishing bushing, solid warp only collapse, and cure mainly deficiency of Yin hectic fever, hectic fever due to yin night sweat, dizzy mesh
Dizzy, endogenous deficient wind, muscles and bones impotence, forgetful, uterine bleeding of having a guilty conscience are through more etc..
Tortoise plastron is clinical conventional Chinese medicine, its base tortoise breeding is difficult and growth period is longer, generally minimum 5~6 years can
Hyoscine, causes domestic herb resource deficiency.In addition, homonym, medication difference among the people, the lines of affinity species, color etc.
Morphological feature is similar and the factor such as the shortage of professional experiences, often has tortoise plastron adulterant and mixes adulterant and is mixed into market, as Trachemys scripta,
Flower tortoise, harmful effect is brought to the security and validity of medication.At present, the discriminating of tortoise plastron still relies primarily on traditional property
Shape authentication technique, i.e., evaluate from appearance, color and luster, quality, smell etc., its experience dependence is strong, accuracy is relatively low.This
Outside, portioned product is after a series of working process, it is difficult to which the conventional method such as applied morphology investigation is effectively differentiated.
Since DNA is present in most of biological tissues and highly stable, detection dosage is few, therefore tortoise plastron can be selected special
Some DNA fragmentations are as the identification thing for specificity identification.In the research with molecular marking technique identification tortoise plastron, grind
Study carefully personnel and design the diagnostic primers that a pair is exclusively used in tortoise, the identification for tortoise plastron medicinal material and former animal.Also there is researcher's pin
To Cyt B and COI sequences, 2 pairs of specific primers are designed, multiplex PCR is established and true and false discriminating is carried out to tortoise plastron medicinal material；So
And the technology is applicable only to tortoise plastron medicinal material and base sample, it there is no method to differentiate medicine materical crude slice and mix adulterant, application range more office
Limit.In addition, carrying out Molecular Identification to tortoise and common mixed adulterant using based on the DNA bar code technology of COI sequences, this method is also
Medicinal material and base are only used for, and the factor such as fluorescence interference, base mispairing, biological evolution speed causes intraspecific variablity threshold value to be difficult to really
Fixed, the accuracy of inspection result leaves a question open.In addition, the also inherent shortcoming such as complex steps, time and cost height.
PCR has good specificity, high sensitivity (index increase), reproducible, easy to operate, high throughput (every instrument
Amplifiable 96 samples of single) etc. advantage；However, in Standard PCR technology carries out the qualification process of species using DNA sequence dna,
As a result of the universal primer in the regions such as COI, ITS, thus complete expand after, it is necessary to carry out product sequencing and with gene number
Compared according to storehouse, then could judge kind, step is more, time-consuming, needs special equipment；In addition, it can not also realize for mixing
The inspection of adulterant is known, or influences qualification result due to mixing pseudo- phenomenon.
The content of the invention
The purpose of the invention is to overcome problems of the prior art, more perfect tortoise plastron identification body is established
System, i.e. base, medicinal material, medicine materical crude slice.
The present invention has separately designed specific primer for the base of tortoise plastron certified products and common adulterant, and provides utilization
The method that primer amplified technology differentiates tortoise plastron.For the method without sequencing, easy to operate, specificity is strong, time-consuming short, can be accurate
Really, the true and false (including mixing puppet) of tortoise plastron is intuitively differentiated.The present invention provides a new approach for the specificity identification of tortoise plastron, has
Help monitor the quality of tortoise plastron product, so as to be conducive to ensure security, the validity of clinical application, valency is applied with important
Value.
The present invention achievees the purpose that quickly to detect tortoise plastron series of products by following steps：
Be used to identifying the specific primer of tortoise plastron present invention firstly provides one group, the primer for PCR-1 or PCR-2 or
The upstream and downstream primer of PCR-3, PCR-1 are as shown in SEQ.ID.NO.1 and 2；The upstream and downstream primer of PCR-2 such as SEQ.ID.NO.3 and 4
It is shown；The upstream and downstream primer of PCR-3 is as shown in SEQ.ID.NO.5 and 6.
Further, in above-mentioned specific primer qualification process, if given the test agent is complete tortoise plastron and the results show
For the positive, be then judged as certified products tortoise plastron, if given the test agent is sliced or crushes, for incomplete tortoise plastron (such as fragment, powder) and
The results show is the positive, then whether need further to identify is to mix adulterant (tortoise plastron containing Trachemys scripta or flower tortoise tortoise plastron or both have),
The above-mentioned specific primer group for being used to identify tortoise plastron further includes the upper and lower of primer PTS-1 or PTS-2, PMS-1 or PMS-2, PTS-1
Primer is swum as shown in SEQ.ID.NO.7 and 8；The upstream and downstream primer of PTS-2 is as shown in SEQ.ID.NO.9 and 10；Above and below PMS-1
Primer is swum as shown in SEQ.ID.NO.11 and 12；The upstream and downstream primer of PMS-2 is as shown in SEQ.ID.NO.13 and 14.
Primer PCR -1, PCR-2 and the PCR-3 is both from tortoise (Chinemys reevesii), same energy
For detecting and achieving the purpose that identification tortoise plastron；Described primer PTS-1, the PTS-2 is both from Trachemys scripta (Trachemys
Scripta), it is same whether to be used to detect and achieve the purpose that in identification tortoise plastron doped with Brazilian tortoise plastron；Primer PMS-1 or
PMS-2 both from flower tortoise (Mauremys sinensis), it is same can be used to detect and reach whether adulterated in identification tortoise plastron
There is the purpose of colored tortoise plastron.
It is specific as shown in the table：
The present invention also provides a kind of kit of specificity identification tortoise plastron, the kit includes above-mentioned specific primer
PCR-1 or PCR-2 or PCR-3 primer pairs, DNA positive controls, PCR reaction systems, electrophoresis test system.
Further, the kit also wraps the specific primer PTS-1 or PTS-2 that states and and/or including primer
To PMS-1 or PMS-2 primer pairs.
The present invention also provides a kind of method that tortoise plastron is identified by specific primer, test solution is prepared first, then
Target fragment is quickly detected using PCR and agarose gel electrophoresis technology.
Wherein the preparation method of test solution carries out as steps described below：
(1) tortoise plastron product is crushed, crosses 40 mesh sieves, collected fine powder, add suitable SDS lysates and Proteinase K (feed liquid
Than for 10-200:1mg/mL), fully mix, bath extraction 1h~6h is incubated in 55 DEG C~65 DEG C, supernatant is taken after centrifugation；
(2) addition and the isometric Tris balance phenols of supernatant, are sufficiently mixed, and supernatant, addition and supernatant are taken after centrifugation
Phenol-chloroform-isoamyl alcohol (25 of liquid equivalent:24:1), it is sufficiently mixed, supernatant is taken after centrifugation；
(3) chloroform-isoamyl alcohol (24 with supernatant equivalent is added:1), it is sufficiently mixed, supernatant is taken after centrifugation, then
96% ethanol of the precooling of 2 times of amounts of 5M potassium acetate solutions and supernatant volume of 1/10 times of amount of supernatant volume is added, it is fully mixed
Close, -20 DEG C of freeze overnights, abandoning supernatant after centrifugation；
(4) 70% ethanol, the 500 μ L washing precipitations of precooling are added, abandoning supernatant after centrifugation, sediment dries in the air at room temperature
Dry or lyophilized, with appropriate TE buffer solutions, quality examination, preserves at -20 DEG C or 4 DEG C, spare.
A kind of method that tortoise plastron is identified by specific primer, detection method carry out as steps described below：
(1) using the test solution DNA of preparation as template, PCR reaction systems are prepared；
(2) PCR amplification program is set, carries out PCR amplification；
(3) Ago-Gel is prepared, amplified production is subjected to electrophoretic analysis；
(4) it is detected after electrophoresis by Labworks image acquisition and analysis software.
Wherein, the PCR reaction systems described in step (1), by taking 25 μ L of cumulative volume as an example, including：1 × PCR buffer solutions,
2.0mmol/L MgCl2, 0.2mmol/L dNTPs, 0.1 μm of ol/L~0.4 μm ol/L primer pairs, 0.625U~1.0U Taq
Archaeal dna polymerase, template DNA 1ng~150ng, add sterilizing distilled water to complement to 25 μ L.
PCR amplification program described in step (2) is：95℃3min；95 DEG C of 30s, 60 DEG C~68 DEG C 10s~30s, 72 DEG C
1min, period 30~40；72 DEG C of 5min~7min.
Electrophoresis detection described in step (3) refers to that amplified production is coagulated using 2%~3% agarose of the EB containing fluorescent dye
Glue is detected after being analyzed with gel imaging system, and the buffer solution of glue and electrophoresis is TBE, and applied sample amount is the μ L of 3 μ L~10.
Beneficial effects of the present invention are as follows：
(1) preparation method of the invention incubates bath using SDS lysates and Proteinase K as Extraction solvent with constant temperature blending instrument, from
DNA is extracted in tortoise plastron product, is then purified with phenol and chloroform method, not only reduces the interference that impurity identifies follow-up PCR,
And this method operating time is short, cost is low, compared with column purification, the dosage and test solution concentration flexibility and changeability of sample,
Impurity is not easy to plug purification column and avoids adsorption loss caused by purification column so that the method scope of application is more extensive.
(2) primer of the present invention is set compared with specific primer known to disclosure for tortoise plastron and its common adulterant
Multipair primer has been counted, and can have been distinguished on species level, it is possible to achieve has mixed the identification of adulterant；In addition, the purpose production of amplification
Thing is very short (≤120bp), is very useful to the amplification of purpose fragment, reduces the probability that template degradation causes to expand false negative.Should
The method of specific primer identification tortoise plastron is fitted compared with the common DNA bar code technology based on COI genes carries out identification technology
It is wider with scope, it is applicable to tortoise plastron base, medicinal material, medicine materical crude slice and its mixes adulterant.In addition, this method strong antijamming capability, need not
It is sequenced, convenient economy, high throughput, intuitively tortoise plastron can be identified exactly.
Detection method in the present invention can directly, it is easy, precise Identification is carried out to tortoise plastron product with high throughput, without surveying
Sequence, it is more more directly perceived than the DNA bar code investigative technique based on COI genes, economical, quick, and with applied widely, anti-interference
Ability is strong, can recognize that the features such as mixing adulterant, and a kind of new scheme is provided for the true and false Rapid identification of tortoise plastron product.
Brief description of the drawings
Fig. 1 is identification method flow chart.
Fig. 2 is that specific primer screens electrophoresis verification result figure, and a is the verification knot to PCR-1, PCR-2, PCR-3 in figure
Fruit, b are the verification result to PTS-1, PTS-2, PMS-1, PMS-2.
Fig. 3 is specific primer optimization electrophoresis result figure；A is the optimum results to temperature in figure；B is to the excellent of period
Change result.
Fig. 4 is the specificity verification electrophoresis result figure of the specific primer group of the present invention.
Fig. 5 is the sequencing result and purpose fragment comparison chart of primer amplified product of the present invention.
Fig. 6 is that specific primer of the present invention detects electrophoresis result figure to mixing adulterant from CARAPAX ET PIASTRUM TESTUDINIS PREPARATA, and a is primer PCR -2 in figure
With detections of the PTS-2 to tortoise and Trachemys scripta melange；B is primer PCR -2 and PMS-1 to tortoise and the inspection of flower tortoise melange
Survey；C is primer PCR -1 and PMS-2 to tortoise and the detection of flower tortoise melange；D for primer PCR -3 and PTS-1 to tortoise with bar
The detection of western tortoise melange.
Fig. 7 is specific primer PCR-2, PTS-2, PMS-1 of the present invention to the commercially available tortoise plastron base sample detection of different batches
Electrophoresis result.
Fig. 8 is specific primer PCR-2 (a) of the present invention, PTS-2 (b), PMS-1 (c) to the commercially available tortoise plastron medicinal material of different batches
Detect electrophoresis result.
Fig. 9 is specific primer PCR-2 (a) of the present invention, PTS-2 (b), PMS-1 (c) to the commercially available tortoise plastron medicine materical crude slice of different batches
Detect electrophoresis result.
Figure 10 is specific primer PCR-1 (a), PCR-3 (b), PTS-1 (c) and PMS-2 (d) of the present invention to different batches
Commercially available tortoise plastron medicine materical crude slice detects electrophoresis result.
In figure, CR is tortoise base sample, TS is Trachemys scripta base sample, MS is colored tortoise base sample, PS is soft-shelled turtle base
Sample, AF are Florida soft-shelled turtle base sample；M：Low ladder SN127；P：Positive control；N：Negative control；CRM：Tortoise plastron
Reference substance 121494-201403.
CR:TS is that adulterant is mixed in the self-control that tortoise mixes with Trachemys scripta；CR:MS is that tortoise mixes puppet with the self-control that flower tortoise mixes
Product；7:1、3:1、1:1、1:3、1:7 for five kinds of tortoises and Trachemys scripta or colored tortoise it is different mix pseudo- ratio.
CR1~10 are 10 batches of tortoise base samples, TS1~2 are 2 batches of Trachemys scripta base samples, MS1~2 are 2 batches of colored tortoise bases
Raw sample, YG1~10 are 10 batches of tortoise plastron medicinal materials, SG1~10 are 10 batches of tortoise plastron medicine materical crude slice.
Embodiment
To make those skilled in the art be better understood from technical scheme, with reference to specific embodiments and the drawings
Further illustrate.
Involved in the present invention to specimen material be all from commercially available, respectively write a Chinese character in simplified form and title be as follows：
CR4：Tortoise base sample, from jiangsu wuxi；TS1：Trachemys scripta base sample, from Zhenjiang, Jiangsu；MS1：Flower
Tortoise base sample, from Zhejiang Hangzhou；PS1：Soft-shelled turtle base sample, from jiangsu wuxi；AF1：Florida soft-shelled turtle base sample, comes
From Law Firm Suzhou Jiangsu；CRM：Tortoise plastron reference substance 121494-201403.
CR is tortoise base sample, and 1 comes from Zhejiang Huzhou, and 4 come from jiangsu wuxi；TS is Trachemys scripta base sample, and 1 comes from
Zhenjiang, Jiangsu, 2 come from jiangsu wuxi；MS is flower tortoise base sample, and 1 comes from Zhejiang Hangzhou, and 2 come from jiangsu wuxi；PS is soft-shelled turtle base
Raw sample, 1 comes from jiangsu wuxi, and 4 come from Jiangsu Taizhou；AF is Florida soft-shelled turtle base sample, and 1,2 are all from Law Firm Suzhou Jiangsu.
CR1~10 are 10 batches of tortoise base samples, and 1,2 come from Zhejiang Huzhou difference market, and 3 come from Shanghai, and 4,5 come from river
Su Wuxi differences market, 6 come from Yancheng, Jiangsu Province, and 7 come from Zhuji, zhejiang, and 8 come from Zhenjiang, Jiangsu, and 9 come from Zhejiang Hangzhou, and 10 come from
Nanyang, henan；
TS1~2 are 2 batches of Trachemys scripta base samples, and 1 comes from Zhenjiang, Jiangsu, and 2 come from jiangsu wuxi；
MS1~2 are 2 batches of colored tortoise base samples, and 1 comes from Zhejiang Hangzhou, and 2 come from jiangsu wuxi；
YG1~10 are 10 batches of tortoise plastron medicinal materials, and 1~6 engraves a medicine company long purchased from Hui nationality, and 7~8 are purchased from the peaceful cloud in Hui nationality
Hall medicine company, 9~10 purchased from Baoding Qi state hall Chinese medicine；
SG1~10 are 10 batches of tortoise plastron medicine materical crude slice, are purchased from one hundred Shi Xin prepared slices of Chinese crude drugs Co., Ltd of Bozhou City.
Embodiment 1：Design, screening, optimization and the verification of tortoise plastron kit specific primer
(1) reagent
Lauryl sodium sulfate, sodium chloride, trishydroxymethylaminomethane, hydrochloric acid, disodium ethylene diamine tetraacetate, Proteinase K
(20mg/mL, Sigma-Aldrich, P6556), Tris balance phenols (LIFE SCIENCES, T0250), three chloromethanes
Alkane, isoamyl alcohol, absolute ethyl alcohol, Ezup pillar Animal genome DNA extraction agents box (raw work, B518251), DreamTaq
Green DNA Polymerase (Thermo Scientific, #EP0712), dNTP Mix (Thermo Scientific, #
R0192), Agarose, Ethidium Bromide (SunShineBio, SN314-1), boric acid, reagent are that analysis is pure.
(2) method
Design specific primer：
The tortoise compared according to DNAMAN softwares and its gene difference Chinemys reevesii of common adulterant
(Accession No.：NC_006082.1, AY676201.1, FJ469674.1, KJ700438.1), Trachemys
scripta(Accession No.：NC_011573.1, FJ392294.1, KM216749.1), Mauremys sinensis
(Accession No.：NC_016685.1, FJ871126.1, KC333650.1), drawn using 7 Software for Design specificity of Oligo
Thing, primer sequence is as shown in the table, and by Shanghai, Sheng Gong Co., Ltds synthesize.
Screen specific primer：
Tortoise plastron base sample (including tortoise and its common adulterant) is crushed, weighs the following powder 50mg of 40 mesh, adds 995
The SDS lysates of μ L and 5 μ L Proteinase Ks, fully mix, in constant temperature blending instrument (Tuo Pusen scientific instrument Co., Ltd) at 56 DEG C
600rpm incubates bath extraction 6h, and supernatant is taken after centrifugation；The Tris balance phenols of 1 times of amount of supernatant volume are added, are fully mixed, centrifugation
After take supernatant；Add phenol-chloroform-isoamyl alcohol (25 of 1 times of amount of supernatant volume:24:1), fully mix, taken after centrifugation
Supernatant；Add chloroform-isoamyl alcohol (24 of 1 times of amount of supernatant volume:1), fully mix, supernatant is taken after centrifugation；Add
Enter 96% ethanol of the precooling of 2 times of amounts of 5M potassium acetate solutions and supernatant volume of 1/10 times of amount of supernatant volume, it is fully mixed
Close, -20 DEG C overnight, abandoning supernatant after centrifugation；Add 70% ethanol, the 500 μ L washing precipitations of precooling, supernatant discarding after centrifugation
Liquid；Room temperature is dried or freezed, and with 25 μ L TE buffer solutions, obtains genomic DNA test solution, quality examination, -20 DEG C
Or preserved at 4 DEG C, it is spare.
Prepare PCR reaction systems：1 × PCR buffer solutions, 2.0mmol/LMgCl2, 0.2mmol/L dNTPs, 0.2 μm of ol/L
Upstream and downstream primer, 0.625U Taq archaeal dna polymerases, template DNA 50ng, adds sterilizing distilled water to complement to 25 μ L.After centrifugation, put
Expanded, reaction condition in PCR instrument (5331Thermal Cycler, Eppendorf)：95℃3min；95 DEG C of 30s, 65 DEG C
30s, 72 DEG C of 1min, 35 circulations；72℃7min.8 μ L amplified productions are analyzed using 2% Ago-Gel containing EB.Electricity
After swimming, detected by gel imaging system, and according to the presence or absence of each electrophoretic band and brightness, molecular size range to screen spy
Specific primer.
The results are shown in Figure 2, and each specific primer carries out Amplification Analysis result to its corresponding species bright band,
Its molecular weight is consistent with the purpose fragment length of expected amplification.Wherein, PTS-1, PTS-2 pin are in five tested species specificities
It is very good；Two primer of PMS-1, PMS-2 to Trachemys scripta species in addition to having very faint non-specific amplification, to remaining four
The specificity of species is very good；In three pairs of PCR primers of tortoise the specificity of PCR-2 better than PCR-1 and PCR-3, PCR-1 and
PCR-3 is very good to the specificity of remaining four species in addition to having faint non-specific amplification to flower tortoise.Pass through each amplification
The optimization of parameter is expected to be used for the authenticity of tortoise plastron sample.
Optimize specific primer：
According to the selection result of specific primer, by taking primer PCR -2, PTS-2, PMS-1 as an example.By annealing temperature, draw
The parameters such as thing concentration, period optimize the specific primer of screening.
Show as shown in Figure 3, under 35 cyclic amplification reactions, for PCR-2 when annealing temperature is 66 DEG C, expanding effect is most
It is good；For PTS-2 when annealing temperature is 64 DEG C, expanding effect is optimal；And PMS-1 annealing temperature be 68 DEG C when, Trachemys scripta species
Still there is extremely faint non-specific amplification, and the amplification of flower tortoise species in itself has also been suppressed.Thus, pass through period pair
PMS-1 further optimizes, the results showed that, the lower PMS-1 expanding effects of 31 circulations are best；Since medicine materical crude slice is the mixing of multiple samples
Thing, comprehensive detection efficiency consider that the more excellent amplification condition of PCR-2/PTS-2 primers is 95 DEG C of 3min；95 DEG C of 30s, 66 DEG C of 30s,
72 DEG C of 1min, 35 circulations；72℃7min.The more excellent amplification condition of PMS-1 primers is 95 DEG C of 3min；95 DEG C of 30s, 66 DEG C of 30s,
72 DEG C of 1min, 31 circulations；72℃7min.Same method, optimizes other primer pairs.
Verify tortoise plastron kit primer：
Each 2 batches of crust for randomly selecting the base sample of the tortoise identified and common adulterant carries out under optimal conditions
PCR amplification, agarose gel electrophoresis analysis and gel imaging system detection, according to the presence or absence of electrophoretic band and molecular size range
To be verified to specific primer.It is confirmed whether in addition, carrying out further bidirectional sequencing by amplified production after purification
For purpose fragment.
The results are shown in Figure 4, and specific primer is by different batches tortoise and its common mixes adulterant under optimal conditions
Carry out specificity verification, the DNA of tortoise expanded by specific primer after 120bp at generation band；Its common mixed puppet
After product are then expanded (from Trachemys scripta, flower tortoise) with respective specific primer, bar is produced at 81bp, 105bp respectively
Band, and no cross reaction between primer, have embodied very strong specificity.In addition, sequencing comparison result such as Fig. 5, sequencing result with
Purpose product sequence is basically identical, and it is really the purpose fragment of expected amplification to show obtained amplified production.
Embodiment 2：Tortoise plastron kit primer is used to make the detection application for mixing adulterant by oneself
With crush in advance tortoise, Trachemys scripta, flower tortoise base crust powder be mixed with tortoise plastron and mix adulterant, two kinds of tortoise plastrons
Mix adulterant (CR:TS、CR:MS) in 5 kinds of different ratios (7:1、3:1、1:1、1:3、1:7) mixed, the end of each sample
Quality is 50mg.Then extraction purification is carried out by SDS methods, and is analyzed under optimal conditions.
Test solution is prepared first, then quickly detects target fragment using PCR and agarose gel electrophoresis technology.
Wherein the preparation method of test solution carries out as steps described below：
(1) tortoise plastron product is crushed, crosses 40 mesh sieves, collected fine powder, add suitable SDS lysates and Proteinase K (feed liquid
Than for 10-200:1mg/mL), fully mix, bath extraction 6h is incubated in 56 DEG C, supernatant is taken after centrifugation；
(2) addition and the isometric Tris balance phenols of supernatant, are sufficiently mixed, and supernatant, addition and supernatant are taken after centrifugation
Phenol-chloroform-isoamyl alcohol (25 of liquid equivalent:24:1), it is sufficiently mixed, supernatant is taken after centrifugation；
(3) chloroform-isoamyl alcohol (24 with supernatant equivalent is added:1), it is sufficiently mixed, supernatant is taken after centrifugation, then
96% ethanol of the precooling of 2 times of amounts of 5M potassium acetate solutions and supernatant volume of 1/10 times of amount of supernatant volume is added, it is fully mixed
Close, -20 DEG C of freeze overnights, abandoning supernatant after centrifugation；
(4) 70% ethanol, the 500 μ L washing precipitations of precooling are added, abandoning supernatant after centrifugation, sediment dries in the air at room temperature
Dry or lyophilized, with appropriate TE buffer solutions, quality examination, preserves at -20 DEG C or 4 DEG C, spare.
Using the test solution DNA of preparation as template, PCR reaction systems are prepared, set PCR amplification program, carry out PCR expansions
Increase.
The PCR reaction systems, 25 μ L of cumulative volume, including：1 × PCR buffer solutions, 2.0mmol/L MgCl2、
0.2mmol/L dNTPs, 0.1 μm of ol/L~0.4 μm ol/L primer pair, 0.625U~1.0U Taq archaeal dna polymerases, template
DNA50ng, adds sterilizing distilled water to complement to 25 μ L.
PCR amplification program is：95℃3min；95 DEG C of 30s, 60 DEG C~68 DEG C 10s~30s, 72 DEG C of 1min, period 30~
40；72 DEG C of 5min~7min.
Amplified production uses 2% Ago-Gel of the EB containing fluorescent dye to be detected after being analyzed with gel imaging system,
The buffer solution of glue and electrophoresis is TBE, and applied sample amount is 8 μ L.
The results are shown in Figure 6, and mix two kinds of 5 kinds of different proportions mix specific primer of the adulterant by corresponding species
PCR-2, PTS-2, PMS-1 Amplification Analysis, its pillar location is consistent with the purpose fragment length of expected amplification, shows the present invention
Specific primer can identify the corresponding species mixed in adulterant.Likewise, PCR-1, PCR-3, PTS-1 and PMS-2 amplification point
Also the corresponding species mixed in adulterant can be identified after analysis.
Embodiment 3：The detection of the commercially available tortoise plastron sample of different batches
Method is the same as embodiment 2.
The commercially available tortoise plastron sample of different batches (base, medicinal material and medicine materical crude slice) is crushed, the following powder 50mg of 40 mesh is weighed, passes through
SDS methods carry out extraction purification and quality examination.Hereafter, PCR reaction systems are prepared, are put condition of the PCR instrument by above-mentioned optimization
Expanded.Then, 8 μ L amplified productions are analyzed using 2% Ago-Gel containing EB and tbe buffer liquid, electrophoresis knot
Shu Hou, is detected by gel imaging system, and according to the presence or absence of electrophoretic band and molecular size range to judge the true and false of tortoise plastron.
In addition, the positive control substance of tortoise, Trachemys scripta and flower tortoise is passed through by weighing the following base sample powder 50mg of 40 mesh respectively
After SDS methods carry out extraction purification and quality examination, freeze and prepare DNA positive control substances, and indicate the wherein quality of DNA.
As a result as shown in figs. 7-9, in 34 batches of commercially available tortoise plastron samples, 2 batches of tortoise plastron base samples are expanded by PMS-1
Band is produced at 105bp afterwards, is accredited as colored tortoise plastron.2 batches of tortoise plastron base samples, 2 batches of tortoise plastron medicinal materials are expanded by PTS-2
Band is produced at 81bp afterwards, is accredited as Brazilian tortoise plastron.Remaining 10 batches of tortoise plastron base sample, 8 batches of tortoise plastron medicinal materials, 10 batches of tortoise plastron drinks
Piece produces band after being expanded by PCR-2 at 120bp, and certified products tortoise plastron product is accredited as in addition to medicine materical crude slice；Due to drink
Piece is multiple sample mixtures, needs further to verify whether by PTS-2, PMS-1 to mix adulterant.Detection finds 10 batches of tortoise plastrons
The no positive reaction of medicine materical crude slice, the results showed that be also certified products tortoise plastron product.In addition, as shown in Figure 10, by PCR-1, PCR-3,
PTS-1 and PMS-2 has found that result is consistent with the amplification of PCR-2, PTS-2 and PMS-1 to tortoise plastron medicine materical crude slice Amplification Analysis,
Show to can be equally used for the identification of tortoise plastron product.
Sequence table
<110>Jiangsu University
<120>The specific primer and its identification method of one group of identification tortoise plastron
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Tortoise (Chinemys reevesii)
<400> 1
tatcgttaca gcccatgcct 20
<210> 2
<211> 20
<212> DNA
<213>Tortoise (Chinemys reevesii)
<400> 2
gcgctccgat cattaaaggt 20
<210> 3
<211> 20
<212> DNA
<213>Tortoise (Chinemys reevesii)
<400> 3
aacctggcat attatggtct 20
<210> 4
<211> 20
<212> DNA
<213>Tortoise (Chinemys reevesii)
<400> 4
caatcaactt gaacgagggt 20
<210> 5
<211> 20
<212> DNA
<213>Tortoise (Chinemys reevesii)
<400> 5
caacccaaaa ccttccgaac 20
<210> 6
<211> 21
<212> DNA
<213>Tortoise (Chinemys reevesii)
<400> 6
ggattctatt ggcatgacca c 21
<210> 7
<211> 21
<212> DNA
<213>Trachemys scripta (Trachemys scripta)
<400> 7
agagaaggac tttaaccctc g 21
<210> 8
<211> 21
<212> DNA
<213>Trachemys scripta (Trachemys scripta)
<400> 8
gtttatgccc gatagacctc a 21
<210> 9
<211> 21
<212> DNA
<213>Trachemys scripta (Trachemys scripta)
<400> 9
gcccaaacta acagacaacc g 21
<210> 10
<211> 21
<212> DNA
<213>Trachemys scripta (Trachemys scripta)
<400> 10
cagcgaagta agtagttcac c 21
<210> 11
<211> 21
<212> DNA
<213>Flower tortoise (Mauremys sinensis)
<400> 11
tcctcgggat aatccacgaa c 21
<210> 12
<211> 21
<212> DNA
<213>Flower tortoise (Mauremys sinensis)
<400> 12
ccatggcttt atcgtcttgg t 21
<210> 13
<211> 21
<212> DNA
<213>Flower tortoise (Mauremys sinensis)
<400> 13
tgtcacctat tacgctggca a 21
<210> 14
<211> 21
<212> DNA
<213>Flower tortoise (Mauremys sinensis)
<400> 14
acaataaagc ccaggaaacc g 21
Claims (10)
1. one group of specific primer for being used to identify tortoise plastron, it is characterised in that the primer is PCR-1 or PCR-2 or PCR-3,
The upstream and downstream primer of PCR-1 is as shown in SEQ.ID.NO.1 and 2；The upstream and downstream primer of PCR-2 is as shown in SEQ.ID.NO.3 and 4；
The upstream and downstream primer of PCR-3 is as shown in SEQ.ID.NO.5 and 6.
2. specific primer as claimed in claim 1, it is characterised in that the primer further includes primer pair PTS-1 or PTS-2
And and/or the upstream and downstream primer including primer pair PMS-1 or PMS-2, PTS-1 is as shown in SEQ.ID.NO.7 and 8；PTS-2's
Upstream and downstream primer is as shown in SEQ.ID.NO.9 and 10；The upstream and downstream primer of PMS-1 is as shown in SEQ.ID.NO.11 and 12；PMS-2
Upstream and downstream primer as shown in SEQ.ID.NO.13 and 14.
3. application of the primer in identifying the tortoise plastron sample true and false or mixing puppet described in claim 1 or 2.
4. application according to claim 3, it is characterised in that the application combines power for the primer described in claim 1
Profit requires the primer described in 2 to identify whether be mixed with Brazilian tortoise plastron or flower tortoise plastron in tortoise plastron.
5. application according to claim 3, it is characterised in that the tortoise plastron sample refers to tortoise plastron base, medicinal material and medicine materical crude slice.
6. a kind of kit of specificity identification tortoise plastron, it is characterised in that the kit includes drawing described in claim 1
Thing, DNA positive controls, PCR reaction systems, electrophoresis test system.
7. the kit stated according to claim 6, it is characterised in that the kit further includes the primer described in claim 2.
A kind of 8. method of specificity identification tortoise plastron, it is characterised in that carry out as steps described below：
(1) the genomic DNA test solution of tortoise plastron sample is prepared, quality examination is carried out by nucleic acid-protein analyzer；
(2) PCR amplification is carried out to DNA test samples using the specific primer described in claim 1 or 2；
(3) amplified production is detected with agarose gel electrophoresis analysis and gel imaging system, and according to the presence or absence of electrophoretic band and
Molecular size range the true and false of tortoise plastron and mixes pseudo- situation to identify.
9. the method for a kind of specificity identification tortoise plastron according to claim 8, it is characterised in that described in step (2)
PCR amplification, its reaction system include：1 × PCR buffer solutions, 2.0mmol/L MgCl2, 0.2mmol/L dNTPs, 0.1 μm of ol/L
~0.4 μm of ol/L primer pair, 0.625U~1.0U Taq archaeal dna polymerases, template DNA 1ng~150ng, adds sterilizing distilled water
Complement to 25 μ L.
10. the method for a kind of specificity identification tortoise plastron according to claim 8, it is characterised in that described in step (2)
PCR amplification, its program are：95℃ 3min；95 DEG C of 30s, 60 DEG C~68 DEG C 10s~30s, 72 DEG C of 1min, period 30~
40；72 DEG C of 5min~7min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486227A (en) * | 2018-05-24 | 2018-09-04 | 广东省生物资源应用研究所 | Based on environment DNA technology to the primer and its method of red ear tortoise biomass evaluation |
| CN110317883A (en) * | 2019-07-29 | 2019-10-11 | 湖北省药品监督检验研究院 | One group for identifying the SNP marker of tortoise, flower tortoise and its cenospecies |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255220A (en) * | 2013-05-04 | 2013-08-21 | 吉林市雷博科技有限公司 | Tortoise shell DNA detection kit and identification method |
-
2017
- 2017-12-29 CN CN201711487467.3A patent/CN108018363A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255220A (en) * | 2013-05-04 | 2013-08-21 | 吉林市雷博科技有限公司 | Tortoise shell DNA detection kit and identification method |
Non-Patent Citations (4)
| Title |
|---|
| JIANG Y: "Comparison of complete mitochondrial DNA control regions among five Asian freshwater turtle species and their phylogenetic relationships", 《GENET MOL RES.》 * |
| 刘中权: "中药材龟甲及原动物的高特异性PCR鉴定研究", 《药学学报》 * |
| 刘晓帆: "基于COI基因的龟甲及其混伪品的DNA条形码研究", 《中国中药杂志》 * |
| 王淼: "中药材龟甲基因组DNA提取及PCR鉴定特征", 《北华大学学报（自然科学版）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486227A (en) * | 2018-05-24 | 2018-09-04 | 广东省生物资源应用研究所 | Based on environment DNA technology to the primer and its method of red ear tortoise biomass evaluation |
| CN108486227B (en) * | 2018-05-24 | 2022-01-04 | 广东省科学院动物研究所 | Primer and method for evaluating red-eared turtle biomass based on environmental DNA technology |
| CN110317883A (en) * | 2019-07-29 | 2019-10-11 | 湖北省药品监督检验研究院 | One group for identifying the SNP marker of tortoise, flower tortoise and its cenospecies |
| CN110317883B (en) * | 2019-07-29 | 2022-10-21 | 湖北省药品监督检验研究院 | SNP markers for identifying tortoise, pond turtle and hybrid species thereof |
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