CN101712985A - Pelodiscus sinensis DNA fingerprint identification method - Google Patents
Pelodiscus sinensis DNA fingerprint identification method Download PDFInfo
- Publication number
- CN101712985A CN101712985A CN200910155177A CN200910155177A CN101712985A CN 101712985 A CN101712985 A CN 101712985A CN 200910155177 A CN200910155177 A CN 200910155177A CN 200910155177 A CN200910155177 A CN 200910155177A CN 101712985 A CN101712985 A CN 101712985A
- Authority
- CN
- China
- Prior art keywords
- wiegmann
- muscle
- dna
- trionyx sinensis
- time
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a Pelodiscus sinensis DNA fingerprint identification method adopting the technical scheme that high-purity genome DNA is quickly extracted from Pelodiscus sinensis muscle, and then a random amplified primer and an amplified reaction procedure are screened and built. The method comprises the following specific steps: (1) muscle pretreatment comprising drying temperature, moisture content and the like; (2) genome DNA extraction condition optimization, such as water bath time and temperature, centrifugal speed, time and the like; (3) a PCR amplified reaction system and a program establishment comprising a template, a primer and enzyme addition and the like, annealing temperature, time and cycle time design and the like; (4) under the optimal amplified condition, screening different random primers and building the molecular markers of Pelodiscus sinensis in different areas. Compared with the common shape identification technology, the technology is quick and accurate, is not affected by external feeding environmental condition, can quickly identify the Pelodiscus sinensis parent with different regional characteristics and lays the foundation for successively carrying out hybridization breeding.
Description
Technical field
The present invention relates to biological technical field, specifically refer to a kind of Trionyx sinensis (Wiegmann) dna fingerprint authentication method
Background technology
It is very swift and violent that China's Trionyx sinensis (Wiegmann) is cultured development, its breed scale and technology degree of integration have become the leading industry of freshwater aquiculture, according to incompletely statistics, annual production is above 150,000 tons, more than 60 hundred million yuan of the output values, hundred million soft-shelled turtle seedlings of Zhejiang Province's year demand 5-10 only, and soft-shelled turtle is spent in the south of the River that comes from local Taihu Lake strain, the year deliverability of seed has only hundred million of 1-2, a large amount of soft-shelled turtle seedling or ovum are imported by Taiwan, Thailand and other places, cause high-quality Trionyx sinensis (Wiegmann) seed resource to suffer seriously to mix and fail threat, the sign that germplasm is degenerated occurred, the provisions soft-shelled turtle has already brought very big harm.On the one hand, owing to ignore source work such as germplasm protection, purification seed selection, domestic Trionyx sinensis (Wiegmann) population and lineage promiscuity, inbreeding is serious, the descendant inheritting diversity constantly reduces, and deleterious gene constantly is purified, Trionyx sinensis (Wiegmann) germplasm serious degradation, natural best possible merchandise proterties generation serious degradation, disease resistance and immunizing power reduce, poor growth, and commodity soft-shelled turtle quality descends, sexual maturity in advance, the miniaturization that becomes of the individual bodily form of sexual maturity; Commodity soft-shelled turtle quality descends, and the meat delicate flavour goes down, and common people's acceptance level descends, and the phenomenon of " high yield is not had a good harvest " occurs; On the other hand, very different by the seed quality of ground such as Taiwan, Thailand input, bring the threat that mixes degeneration for this area Trionyx sinensis (Wiegmann) germ plasm resource.High-quality Trionyx sinensis (Wiegmann) seed in short supply become restriction and supported one of bottleneck of soft-shelled turtle industry development.
Summary of the invention
Technical problem to be solved by this invention is that the present situation at prior art provides a kind of and can carry out the method that the Trionyx sinensis (Wiegmann) dna fingerprint is identified fast, to reach the purpose of discriminating, seed selection and protection Trionyx sinensis (Wiegmann) kind.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: this Trionyx sinensis (Wiegmann) dna fingerprint authentication method is characterized in that comprising the steps:
1. muscle samples pre-treatment: get Trionyx sinensis (Wiegmann) sample muscle to be measured, this muscle is dried 40-45 ℃ of temperature, the muscle moisture content after the oven dry is 3-5%;
2. extract genomic dna: adopt the genomic dna in the sample muscle after test kit trace extraction method is extracted above-mentioned oven dry, extraction conditions is that bath temperature is that 50-55 ℃, water-bath time are 40-45min, centrifugal rotational speed is that 10000-12000r/min, centrifugation time are 5-10min, obtain high purity DNA genome, this genomic A
260/ A
280Be 1.7～1.9;
3. set up the pcr amplification reaction system: the reaction cumulative volume is 20-25 μ L, wherein 10 * Buffer 2-2.5 μ L, 25mMMgCl
21.6-2.0 μ L, 2mM dNTP0.4-0.5 μ L, Taq enzyme 0.25-0.4 μ L, 5 μ M random primers, 2 μ L (synthetic), dna profiling 1 μ L by Shanghai biotechnology company limited; The annealing temperature of PCR reaction is 36-38 ℃, and cycle index is 40-45 time;
4. establish Trionyx sinensis (Wiegmann) characteristic molecule marker: adopt RAPD random primer S105, S327 and the characteristic molecule marker of S474 as the different regions of differentiation Trionyx sinensis (Wiegmann).
Compared with prior art, the present invention has characteristics fast and accurately, is not raised the influence of external environmental condition, can identify rapidly to have the differently Trionyx sinensis (Wiegmann) parent of characteristic of field, lays the foundation for successfully carrying out cross-breeding.
Description of drawings
Fig. 1 is the DNA electrophorogram, from left to right is followed successively by: south of the River flower soft-shelled turtle 2., south of the River flower soft-shelled turtle 1., the Yellow River soft-shelled turtle 2., the Yellow River soft-shelled turtle 1., Japanese soft-shelled turtle 2., Japanese soft-shelled turtle 1.
Fig. 2 is the pcr amplification figure of RAPD random primer S105 and S327 in the embodiment of the invention;
Fig. 3 is the pcr amplification figure of RAPD random primer S474 in the embodiment of the invention.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
Sample pretreatment: the used material of this experiment is Japanese soft-shelled turtle, the Yellow River soft-shelled turtle and south of the River flower soft-shelled turtle.Pick up from the national Trionyx sinensis (Wiegmann) seed farm of ShaoXing,ZheJiang.Head, neck and limb muscle tissue are got in each sample vivisection, mix, and dry in 40-45 ℃ thermostat container.Wear into the muscle powder with pulverizer afterwards, tinfoil subpackage sealing is stored under the room temperature.
The research of Trionyx sinensis (Wiegmann) extracting genome DNA condition: because classical extraction method had both needed more sample, sample generally needs 50-100g, and spended time is longer.Expensive in view of the price comparison of Trionyx sinensis (Wiegmann) original seed sample, consider problems such as extraction time again, the test kit that this patent adopts Shanghai biotechnology company limited to produce carries out the extraction of genomic dna, and sample only needs 0.05-0.1g.Analyze the concentration and the purity of the Trionyx sinensis (Wiegmann) genomic dna that extracts under the different condition.The concrete operations step is as follows:
(1) take by weighing three kinds of soft-shelled turtle samples of 50mg biased sample, put into the 1.5ml centrifuge tube, the TE buffering suspension of producing with 200 μ l Shanghai biotechnology company limiteds suspends;
(2) add cell pyrolysis liquid (the Cell Lysis Solution) mixing that 400 μ l are provided by test kit, and then add the Proteinase K mixing that 6 μ l Shanghai biotechnology company limiteds produce, place 50-60 ℃ of water-bath 30-50min, during mixing 5-6 time up and down;
(3) add 600 μ l chloroforms, mixing, it is too violent to vibrate;
(4) 4 ℃ of centrifugal 3min of following 10000r/min, be divided into 3 layers this moment, gets 400 μ l supernatant liquors, places the 1.5ml centrifuge tube;
(5) add DNA precipitated liquid (Precipication Solution) mixing that 400 μ l are provided by test kit, room temperature is placed 2min, 8000-12000r/min, centrifugal 3-8min;
(6) supernatant liquor is removed in suction, adds 100 μ l 1.2mol/L NaCl immediately, vibrates gently and dissolves fully until the DNA sample, adds 3 μ l RNase A, places 37 ℃ of 5-10min;
(7) add 300 μ l-20 ℃ following precooled ethanol, place 30min, the centrifugal 5-8min of 10000r/min for-20 ℃, ethanol is removed or outwells in suction, washes twice with 70% ethanol again, is inverted drying at room temperature 10min, DNA fully dissolves with 100 μ l sterilized waters, and it is standby to place 4 ℃ of refrigerators to preserve.
Get 50 μ l dna solution to be measured in the 1.5ml centrifuge tube, with 10 times of sterilized water dilutions, mixing; Surveying wavelength on ultraviolet spectrophotometer respectively is the absorbance A value at 260nm and 280nm place; DNA concentration (μ g/ml)=A wherein
260* 50 * extension rate, DNA purity=A
260/ A
280According to A
260/ A
280The purity of total DNA of being extracted of value comparison, work as A
260/ A
280Value illustrates that the DNA that extracts is purer when 1.6-1.9; Work as A
260/ A
280Value has RNA to exist greater than 2.0 in the interpret sample; Work as A
260/ A
280Value has small amount of albumen matter to exist less than 1.6 in the interpret sample.
Shown in the table 1 is the influence to Trionyx sinensis (Wiegmann) extracting genome DNA effect of different bath temperatures and time, and table 2 is depicted as the influence to Trionyx sinensis (Wiegmann) extracting genome DNA effect of different centrifugal speeds and time.A from table 1 and table 2
260/ A
280Can find out, under 55 ℃, 40min and 10000rpm, the 5min extraction conditions near 1.8.Think that thus the test kit method extraction conditions of Trionyx sinensis (Wiegmann) genomic dna is that 40-45min, centrifugal condition are that 10000-12000r/min, 5-10min are advisable with bath temperature 50-55 ℃, water-bath time.
Different bath temperatures of table 1 and time are to the influence of Trionyx sinensis (Wiegmann) extracting genome DNA effect
Different centrifugal speeds of table 2 and time are to the influence of Trionyx sinensis (Wiegmann) extracting genome DNA effect
The foundation of PCR reaction system and program: whether successful the PCR reaction system be one of the key factor that increases, and comprises substrate dNTP, Taq enzyme, random primer, MgCl
2, the concentration of dna profiling and reaction buffer and ratio etc.Through repeatedly groping, PCR reaction conditions reference standard method i.e. 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 45s, 36 ℃ of annealing 45s, 72 ℃ extend 90s, 45 circulations, 72 ℃ are extended 10min again and carry out.According to the demonstration situation of pcr amplification product electrophorogram specific band, get 5 μ L pcr amplification products and 1 μ L tetrabromophenol sulfonphthalein sample solution and be mixed and carry out electrophoretic analysis.Gel strength is 1.2%, and voltage is 60V, and electrophoresis time is 1.5-2h.Electrophoretic buffer should be identical with electrophoretic buffer with glue in the electrophoresis chamber, and electrophoretic buffer was not just to have gel 1mm for well.Electrophoresis finishes and is placed on the 30min that dyes in ethidium bromide (EB) staining fluid, and sepharose is placed under the UV-light of Gel Doc 2000 gel imaging systems and observes, take pictures and preserve image, so just set up the pcr amplification system of Trionyx sinensis (Wiegmann) genomic dna random primer, the reaction cumulative volume is 20 μ L, and each condition of PCR reaction system sees table 3 for details.
Table 3PCR reaction system
Annotate: Taq enzyme used in the table 3 is produced by Shanghai biotechnology company limited; Random primer is produced by Shanghai biotechnology company limited
Reaction parameter: it is crucial especially that the pcr amplification reaction parameter directly influences the design of the pcr amplification effect, particularly annealing temperature of Trionyx sinensis (Wiegmann) genomic dna and cycle index.Be respectively 36 ℃, 38 ℃ and 40 ℃ in annealing temperature, cycle index is respectively under 40,42,45 times, other steps are identical with existing method, test of many times by the grads PCR instrument, demonstration situation according to pcr amplification product electrophorogram specific band, we have set up the suitableeest RAPD amplification reaction condition of Trionyx sinensis (Wiegmann) genomic dna, and are specific as follows:
By Fig. 2 and Fig. 3 as can be seen, Kuo Zeng RAPD-PCR product is compared with standard program under these conditions, has advantages such as band is more clear and abundanter.
Establish Trionyx sinensis (Wiegmann) characteristic molecule marker: the RAPD primer such as has fast, makes things convenient at characteristics.Present embodiment adopts successively more than 100 at random that the RAPD primer carries out pcr amplification to the Trionyx sinensis (Wiegmann) genomic dna, found that and has only 15 random primers can amplify specific band.These random primers are further screened and test, filter out random primer S105, S327 and S474 etc. respectively, the electrophorogram of these primer PCR amplified productions is seen Fig. 2 and Fig. 3.By Fig. 2 and Fig. 3 as seen, random primer S105 occurs and the Yellow River soft-shelled turtle, the different specific band of Japanese soft-shelled turtle in the flower soft-shelled turtle of the south of the River, random primer S327 occurs in the soft-shelled turtle of the Yellow River and south of the River flower soft-shelled turtle, the different specific band of Japanese soft-shelled turtle, and random primer S474 occurs in Japanese soft-shelled turtle and the Yellow River soft-shelled turtle, the different specific band of south of the River flower soft-shelled turtle.Therefore random primer S105, S327 and S474 can be respectively as the dna molecular markers of identifying south of the River flower soft-shelled turtle, the Yellow River soft-shelled turtle and Japanese soft-shelled turtle.
Claims (1)
1. a Trionyx sinensis (Wiegmann) dna fingerprint authentication method is characterized in that comprising the steps:
1. muscle samples pre-treatment: get Trionyx sinensis (Wiegmann) sample muscle to be measured, this muscle is dried 40-45 ℃ of temperature, the muscle moisture content after the oven dry is 3-5%;
2. extract genomic dna: adopt the genomic dna in the sample muscle after test kit trace extraction method is extracted above-mentioned oven dry, extraction conditions is that bath temperature is that 50-55 ℃, water-bath time are 40-45min, centrifugal rotational speed is that 10000-12000r/min, centrifugation time are 5-10min, obtain high purity DNA genome, this genomic A
260/
280Be 1.7～1.9;
3. set up the pcr amplification reaction system: the reaction cumulative volume is 20-25 μ L, wherein 10 * Buffer 2-2.5 μ L, 25mMMgCl
21.6-2.0 μ L, 2mM dNTP0.4-0.5 μ L, Taq enzyme 0.25-0.4 μ L, 5 μ M primer 2 μ L, dna profiling 1 μ L; The annealing temperature of PCR reaction is 36-38 ℃, and cycle index is 40-45 time;
4. establish Trionyx sinensis (Wiegmann) characteristic molecule marker: adopt RAPD random primer S104, S105, S327 and S474 as the characteristic molecule marker of distinguishing different regions Trionyx sinensis (Wiegmann).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910155177A CN101712985A (en) | 2009-12-02 | 2009-12-02 | Pelodiscus sinensis DNA fingerprint identification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910155177A CN101712985A (en) | 2009-12-02 | 2009-12-02 | Pelodiscus sinensis DNA fingerprint identification method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101712985A true CN101712985A (en) | 2010-05-26 |
Family
ID=42416983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910155177A Pending CN101712985A (en) | 2009-12-02 | 2009-12-02 | Pelodiscus sinensis DNA fingerprint identification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101712985A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013023442A1 (en) * | 2011-08-18 | 2013-02-21 | 江中药业股份有限公司 | Pharmaceutical application of peptide of soft-shell turtle |
| CN106811514A (en) * | 2015-12-01 | 2017-06-09 | 中华人民共和国上海出入境检验检疫局 | Soft-shelled turtle subfamily biotic component specificity real-time fluorescence detection method and its kit |
| CN108918725A (en) * | 2018-08-31 | 2018-11-30 | 浙江工商大学 | Effectively identify the analyzing detecting method of variety classes Shelled Turtle Trionyx Sinensis |
-
2009
- 2009-12-02 CN CN200910155177A patent/CN101712985A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013023442A1 (en) * | 2011-08-18 | 2013-02-21 | 江中药业股份有限公司 | Pharmaceutical application of peptide of soft-shell turtle |
| CN106811514A (en) * | 2015-12-01 | 2017-06-09 | 中华人民共和国上海出入境检验检疫局 | Soft-shelled turtle subfamily biotic component specificity real-time fluorescence detection method and its kit |
| CN106811514B (en) * | 2015-12-01 | 2020-10-16 | 中华人民共和国上海出入境检验检疫局 | Specific real-time fluorescence detection method for biological components in Amydae and kit thereof |
| CN108918725A (en) * | 2018-08-31 | 2018-11-30 | 浙江工商大学 | Effectively identify the analyzing detecting method of variety classes Shelled Turtle Trionyx Sinensis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102732973B (en) | Construction method for DNA fingerprint database of high flux cotton variety | |
| CN105154550B (en) | A kind of method using 1217 purity of EST-SSR molecular labeling Rapid identification variety of watermelon treasure sweet tea | |
| CN103993011B (en) | Semen Sesami dominant genic male sterility gene molecule marker and preparation method and application | |
| CN104115747B (en) | Tissue culture method is utilized to produce apple class flavones | |
| CN102851364A (en) | SSR fingerprint of Cr02 strain of Lentinus edodes, and construction method and application thereof | |
| CN103571852B (en) | Sea island cotton chromosome segment and a SSR marker thereof that significantly can increase upland cotton ginning outturn | |
| CN104711361A (en) | Method for quickly identifying purity of new watermelon species namely red peace hybrid seeds as well as primer and kit adopted by method | |
| CN105154529A (en) | Molecular specific marker primer and identification method for fine varieties of camellia oleifera | |
| CN102732621A (en) | Rapid identification method of genetic purity of muskmelon hybrid seeds based on PCR | |
| CN101586102A (en) | Genomic DNA extraction method of peanut leaf blades | |
| CN110894470A (en) | Shiitake mushroom strain nongxiang No. 5 suitable for industrial cultivation and molecular identification method thereof | |
| CN101712985A (en) | Pelodiscus sinensis DNA fingerprint identification method | |
| CN103409420A (en) | Y-chromosome specific molecular marker of Nile tilapia, and genetic sex determination method and supermale producing method both based on molecular marker | |
| CN103866038B (en) | For detecting tobacco to the N gene-specific primer of TMV resistance to, detection method and test kit | |
| CN104087579A (en) | Molecular marker closely linked with corn bacterial wilt resistance genes and primer and application thereof | |
| CN102876777B (en) | The special primer of brown croaker EST microsatellite marker and screening method | |
| CN104878115B (en) | Utilize the fine fiber quality QTLs auxiliary backcrossing polymerization quality breeding methods of positioning | |
| CN105255866A (en) | Method for auxiliary screening of yellow rust resisting wheat and special primers for method | |
| CN106544439A (en) | The SSR authentication methods of new 55 seed purity in cabbage hybrid Henan | |
| CN103233069B (en) | Molecular identification method for purity of seeds of Yunnan hybrid type japonica rice Yunnan hybrid 31 and special primer thereof | |
| CN101984057A (en) | Gender difference molecular marker of Tilapia nilotica and application thereof | |
| CN108707645B (en) | Method for identifying purity of watermelon variety Sumi No. 9 seed | |
| CN106893773A (en) | Identification alfalfa seed carries the kit and detection method of Semen Cuscutae seed | |
| CN103409421A (en) | X-chromosome specific molecular marker of Nile tilapia | |
| CN108359745A (en) | The dual RT-PCR method of synchronous detection wheat dwarf virus and Wheat Yellow strip virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20100526 |