CN104593358A - Primer for identifying Shaobai chicken as well as application and method for quickly identifying Shaobai chicken - Google Patents
Primer for identifying Shaobai chicken as well as application and method for quickly identifying Shaobai chicken Download PDFInfo
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- CN104593358A CN104593358A CN201410796811.7A CN201410796811A CN104593358A CN 104593358 A CN104593358 A CN 104593358A CN 201410796811 A CN201410796811 A CN 201410796811A CN 104593358 A CN104593358 A CN 104593358A
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Abstract
A primer comprises a primer F1 and a primer F2; nucleotide sequences of the primer F1 and the primer F2 are shown in a sequence table. The invention also provides application as a molecular identification marker of the primer for identifying Shaobai chicken genetic resources. In addition, the invention also provides a method for quickly identifying Shaobai chicken, which comprises the following steps that PCR amplification is carried out by utilizing the primer and using a DNA gene group extracted from blood of tested chicken breeds as a template; a PCR amplification product is detected by means of agarose gel electrophoresis; whether the product includes DW and/or dw genes or not is judged according to strips, and the DW-containing gene chicken only amplifies a single strip with 417 dp; the dw-containing gene chicken only amplifies a single strip with 217 bp; the DW-and-dw-containing gene chickens amplify two strips with 417 bp and 217 bp. According to the primer, the authenticity of different Shaobai chickens are identified by utilizing difference of sex-linked dwarf genes (dw) and the size of amplification strips; the method is not affected by environment, and the method has the advantages of simpleness in operation, quickness in detection, accuracy in results, high generality, low cost and the like.
Description
Technical field
The present invention relates to the qualification of Shao's uncle's father and mother's generation and commercial generation, be specifically related to a kind of method utilizing uncle round pcr Rapid identification Shao chicken, belong to technical field of molecular biology.
Background technology
Uncle Shao chicken breed system is presided over by Jiangsu Inst. of Fowls Science and is tackled key problems, and by importing dw gene in China's high-quality indigenous chicken kind, being bred as blue or green leg fiber crops plumage high quality meat chicken strain (S2 system) of dwarf-type at home and abroad first and participating in supporting.This breed system by national kind (breed system) authorization (agriculture 09 new variety card No. 12nd, word), has been the first national high quality meat chicken breed system with independent intellectual property right.Through qualification, this technological achievement reaches international most advanced level.Sex linkage dwarf gene (dw) is growth hormone receptor (GHR) gene defection type, Dw gene is the sex linked inheritance gene be positioned on Z chromosome, be in hepatic necrosis gene in site and without between wing gene we site, near silver color (S), golden (s) feather genes and slow plumage (K), fast plumage (k) gene.Normal gene DW is dominant to dw, therefore only has the homozygous cock of dwarf-type to show dwarfism with the hen of carrying low and small gene.Dw and DW is incomplete allelotrope, dw gene has the characteristic of qualitative trait gene and Quantitative Trait Genes simultaneously, dwarf chicken is the phenotype of the homozygous sex linkage dwarf gene (dw) of chicken, has the advantages such as individuality is little, feed consumption is few, basal metabolism is low, laying rate is high, stocking density is large, the price of deed is high, cost-saving.
Shao primary chicken breed system is compared with similar chicken kind, and uncle Shao chicken kind chicken Age at first laying shifts to an earlier date 10%, egg laying performance improves 20%, often only kind chicken saves feed more than 25%, and uncle Shao chicken breed system commodity are attractive in appearance on behalf of the numb plumage of blue or green leg, figure and features; Sexual prematurity, meat is good, strong adaptability, can put in a suitable place to breed in knob, is also adapted to mass-producing and raises in cages; Market acceptance level is high, and price is high by about 15 ~ 20%, and raising comprehensive benefit can improve more than 10% ~ 15%.Market application foreground is very wide, how to occur the phenomenon of palming off the sale of uncle Shao chicken, needs the method for simple and fast to be identified.
Round pcr is a kind of Protocols in Molecular Biology, is widely deployed in medical science and biological laboratory, such as, for judging whether to show in a corpse or other object for laboratory examination and chemical testing collection of illustrative plates of certain genetic diseases, the diagnosis of transmissible disease, gene replication and paternity test.
Summary of the invention
The object of this invention is to provide the method for a kind of Shao uncle's chicken qualification primer and application and uncle Rapid identification Shao chicken, the different amplified band size of the chain dwarf gene of usability of the present invention (dw), differentiate uncle different true and false Shao chicken, the method is not affected by environment, have simple to operate, detect fast, result accurately, highly versatile, the advantage such as with low cost.
The invention provides a kind of primer, comprise primers F 1 and primers F 2,
Described primers F 1 upstream primer F:5 '-tcccagactacacttctattca-3 ';
Described primers F 1 downstream primer R:5 '-cgggacagatcaaagacaatac-3 ';
Described primers F 2 upstream primer F:5 '-acctccaaagaaatctgtcgag-3 ';
Described primers F 2 upstream primer R:5 '-tggccaaatcctgaagtcct-3 '.
Present invention also offers the application of above-mentioned primer as the Molecular Identification mark of uncle Shao chicken germ plasm resource.
In addition, invention further provides the method for uncle a kind of Rapid identification Shao chicken, the DNA genome extracted with the blood of tested chicken kind is for template, PCR amplification is carried out with primer described in claim 1, agarose gel electrophoresis detects PCR amplified production, judge whether containing DW and/or dw gene according to band, DW gene chicken only amplifies the single band of 417 bp; Only only amplify the single band of 217 bp containing dw gene chicken; 417 bp and 217 bp, two bands are amplified containing DW and dw gene chicken; Only there is 417bp band in father and mother godfather system, and only there is 217bp band in father and mother godmother system, and commercial generation has 417 bp and 217 bp, two bands.Only occurring the single band of 417 bp or the single band of 217 bp in described PCR amplified production, is uncle false Shao commercial chicken.
The reaction system of described PCR amplification is 20 μ L, and described reaction system consists of the following composition: 10 × PCR Buffer 2.0 μ L, Mg
2+2.2 μ L, dNTP Mixture 0.8 μ L,
taqarchaeal dna polymerase (5 U/ μ L) 0.2 μ L, each 1 μ L of upstream and downstream primer of described primers F 1 and primers F 2, cDNA template 1 μ L, ddH
2o 9.8 μ L.
The reaction conditions of described PCR amplification is: 95 DEG C of denaturation 5 min; 95 DEG C of sex change 30 s, 61.4 DEG C of annealing 30 s, 72 DEG C extend 30 s, 30 circulations; 72 DEG C extend 8 min; 4 DEG C of preservations.
Described agarose gel electrophoresis detecting step is: get 5 μ L pcr amplification products and mix rear 20 min under 1.5% agarose gel electrophoresis 120 V voltage with 1 μ L 6 × Loading Buffer, gel imaging system is taken pictures.
Compared with prior art, the present invention has following beneficial effect:
The first, the present invention extracts the chicken genomic dna of sample to be tested, and only there is 417bp band in pcr amplification father and mother godfather system, and only there is 217bp band in father and mother godmother system, and commercial generation has 417 bp and 217 bp, two bands, and discern the false from the genuine Shao Baiji.The present invention be the DNA genome that directly extracts with the blood of tested chicken kind for template, utilize round pcr to detect the clip size of dw gene.Advantageous of the present invention exists: simple to operate, detect fast, result accurately, highly versatile, the advantage such as with low cost;
The second, the invention discloses the application of dwarf gene (dw) as the molecular identificalion mark of uncle Shao chicken father and mother's generation, commercial generation.
The present invention includes: the upstream and downstream primer mixture of dw gene PCR amplification and PCR reaction reagent.PCR reaction reagent is 10 × buffer, dNTP, rTaq enzyme.The present invention includes the upstream and downstream primer mixture of DW gene PCR amplification, reagent needed for PCR.The inventive method Be very effective, detection method simple and fast, and not by the impact of external environment.The present invention is applied to the qualification of uncle Shao chicken breed, be mainly used in Shao Baiji for generations, the qualification of father and mother's generation and commercial generation, the present invention is reasonable in design, easy to use, reliable results and with low cost.
Accompanying drawing explanation
Fig. 1 is the amplification figure of father and mother godfather system cock;
Fig. 2 is the amplification figure of father and mother godmother system hen;
Fig. 3 is the amplification figure of commercial generation.
Embodiment
A kind of primer, comprises primers F 1 and primers F 2,
Described primers F 1 upstream primer F:5 '-tcccagactacacttctattca-3 ';
Described primers F 1 downstream primer R:5 '-cgggacagatcaaagacaatac-3 ';
Described primers F 2 upstream primer F:5 '-acctccaaagaaatctgtcgag-3 ';
Described primers F 2 upstream primer R:5 '-tggccaaatcctgaagtcct-3 '.
Above-mentioned primer is as the application of the Molecular Identification mark of uncle Shao chicken germ plasm resource.
A kind of method of uncle Rapid identification Shao chicken, it is characterized in that, the DNA genome extracted with the blood of tested chicken kind is for template, PCR amplification is carried out with primer described in claim 1, agarose gel electrophoresis detects PCR amplified production, judge whether containing DW and/or dw gene according to band, DW gene chicken only amplifies the single band of 417 bp; Only only amplify the single band of 217 bp containing dw gene chicken; 417 bp and 217 bp, two bands are amplified containing DW and dw gene chicken; Only there is 417bp band in father and mother godfather system, and only there is 217bp band in father and mother godmother system, and commercial generation has 417 bp and 217 bp, two bands.Only occurring the single band of 417 bp or the single band of 217 bp in PCR amplified production, is uncle false Shao commercial chicken.
The reaction system of PCR amplification is 20 μ L, and described reaction system consists of the following composition: 10 × PCR Buffer 2.0 μ L, Mg
2+2.2 μ L, dNTP Mixture 0.8 μ L,
taqarchaeal dna polymerase (5 U/ μ L) 0.2 μ L, each 1 μ L of upstream and downstream primer of described primers F 1 and primers F 2, cDNA template 1 μ L, ddH
2o 9.8 μ L.
The reaction conditions of PCR amplification is: 95 DEG C of denaturation 5 min; 95 DEG C of sex change 30 s, 61.4 DEG C of annealing 30 s, 72 DEG C extend 30 s, 30 circulations; 72 DEG C extend 8 min; 4 DEG C of preservations.
Agarose gel electrophoresis detecting step is: get 5 μ L pcr amplification products and mix rear 20 min under 1.5% agarose gel electrophoresis 120 V voltage with 1 μ L 6 × Loading Buffer, gel imaging system is taken pictures.
Embodiment 1
1, test materials
Test uncle Shao used chicken from Jiangsu Inst. of Fowls Science, 10 dwarf-type father and mother godmother system cock (Z
dwz
dw), 10 heterozygote commercial generation cock (Z
dwz
dw), 10 normal type father and mother godfather system cock (Z
dwz
dw), 10 maternal hen (Z of dwarf-type
dww), 10 the paternal chicken hen of normal type (Z
dww), all chicken wings venous blood collections, heparin sodium anti-freezing.
2, operation steps
Pcr amplification: PCR amplification system is 20 μ L:10 × PCR Buffer 2.0 μ L, Mg
2+2.2 μ L, dNTP Mixture 0.8 μ L,
taqarchaeal dna polymerase (5 U/ μ L) 0.2 μ L, each 1 μ L of F1 and F2 upstream and downstream primer, cDNA template 1 μ L, ddH
2o 9.8 μ L.Reaction conditions: 95 DEG C of denaturation 5 min; 95 DEG C of sex change 30 s, 61.4 DEG C of annealing 30 s, 72 DEG C extend 30 s, 30 circulations; 72 DEG C extend 8 min; 4 DEG C of preservations.
Agarose gel electrophoresis detects: get 5 μ L pcr amplification products and mix rear 20 min under 1.5% agarose gel electrophoresis 120 V voltage with 1 μ L 6 × Loading Buffer, gel imaging system is taken pictures.
3, result
Dwarf-type father and mother godmother system cock amplification figure as shown in Figure 2, heterozygote commercial generation cock amplification figure as shown in Figure 3, father and mother godfather system cock amplification figure as shown in Figure 1, dwarf-type maternal hen amplification figure as shown in Figure 2,1 normal type paternal chicken hen amplification figure as shown in Figure 1.As can be seen here, pcr amplified fragment size can be used as the method for uncle Shao chicken breed qualification.
<110> Jiangsu Inst. of Fowls Science
The method of <120> Shao's uncle's chicken qualification primer and application and uncle Rapid identification Shao chicken
<160> 4
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<220>
<221>F1 upstream primer
<223>
<400> 1
tcccagacta cacttctatt ca 22
<210>2
<211> 22
<212> DNA
<213> artificial sequence
<220>
<221> F1 downstream primer
<223>
<400> 2
cgggacagat caaagacaat ac 22
<210>3
<211> 22
<212> DNA
<213> artificial sequence
<220>
<221>F2 upstream primer
<223>
<400> 3
acctccaaag aaatctgtcg ag 22
<210>4
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221>F2 downstream primer
<223>
<400> 4
tggccaaatc ctgaagtcct 20
Claims (6)
1. a primer, is characterized in that, described primer comprises primers F 1 and primers F 2,
Described primers F 1 upstream primer F:5 '-tcccagactacacttctattca-3 ';
Described primers F 1 downstream primer R:5 '-cgggacagatcaaagacaatac-3 ';
Described primers F 2 upstream primer F:5 '-acctccaaagaaatctgtcgag-3 ';
Described primers F 2 upstream primer R:5 '-tggccaaatcctgaagtcct-3 '.
2. primer as claimed in claim 1 is as the application of the Molecular Identification mark of uncle Shao chicken germ plasm resource.
3. the method for uncle Rapid identification Shao chicken, it is characterized in that, the DNA genome extracted with the blood of tested chicken kind is for template, PCR amplification is carried out with primer described in claim 1, agarose gel electrophoresis detects PCR amplified production, judge whether containing DW and/or dw gene according to band, DW gene chicken only amplifies the single band of 417 bp; Only only amplify the single band of 217 bp containing dw gene chicken; 417 bp and 217 bp, two bands are amplified containing DW and dw gene chicken; Only there is 417bp band for father and mother godfather system, only have 217bp band for father and mother godmother system, have 417 bp and 217 bp, two bands to be commercial generation; Only occurring the single band of 417 bp or the single band of 217 bp in described PCR amplified production, is uncle false Shao commercial chicken.
4. the method for uncle Rapid identification Shao chicken according to claim 3, is characterized in that, the reaction system of described PCR amplification is 20 μ L, and described reaction system consists of the following composition: 10 × PCR Buffer 2.0 μ L, Mg
2+2.2 μ L, dNTP Mixture 0.8 μ L, 5 U/ μ L
taqarchaeal dna polymerase 0.2 μ L, each 1 μ L of upstream and downstream primer of described primers F 1 and primers F 2, cDNA template 1 μ L, ddH
2o 9.8 μ L.
5. the method for uncle Rapid identification Shao chicken according to claim 4, is characterized in that, the reaction conditions of described PCR amplification is: 95 DEG C of denaturation 5 min; 95 DEG C of sex change 30 s, 61.4 DEG C of annealing 30 s, 72 DEG C extend 30 s, 30 circulations; 72 DEG C extend 8 min; 4 DEG C of preservations.
6. the method for uncle Rapid identification Shao chicken according to claim 3, it is characterized in that, described agarose gel electrophoresis detecting step is: get 5 μ L pcr amplification products and mix rear 20 min under 1.5% agarose gel electrophoresis 120 V voltage with 1 μ L 6 × Loading Buffer, gel imaging system is taken pictures.
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Cited By (2)
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| CN104962636A (en) * | 2015-07-07 | 2015-10-07 | 华南农业大学 | Wenchang chicken white spot phenotype related molecular marker and application of molecular marker |
| CN113430277A (en) * | 2021-07-30 | 2021-09-24 | 中国农业大学 | Primer group for identifying sex-linked dwarf gene and application thereof |
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| CN102618649A (en) * | 2012-04-01 | 2012-08-01 | 广州市权诚生物科技有限公司 | Chicken dwarf foot related molecular detection method |
| CN102747077A (en) * | 2012-06-18 | 2012-10-24 | 中国农业大学 | Method for identifying chicken sex-linked dwarf gene and special primer thereof |
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| CN102002530A (en) * | 2010-11-24 | 2011-04-06 | 广东智威农业科技股份有限公司 | Method for detecting gene mutation |
| CN102618649A (en) * | 2012-04-01 | 2012-08-01 | 广州市权诚生物科技有限公司 | Chicken dwarf foot related molecular detection method |
| CN102747077A (en) * | 2012-06-18 | 2012-10-24 | 中国农业大学 | Method for identifying chicken sex-linked dwarf gene and special primer thereof |
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| S K AGARWAL ET AL.: "Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken: evidence for a mutation in the intracellular domain", 《JOURNAL OF ENDOCRINOLOGY》 * |
| YAN WANG ET AL.: "A Rapid and Cost Effective Method for Genotyping Two Types of GHR Mutations in Sex-linked Dwarf Chicken", 《JOURNAL OF ANIMAL AND VETERINARY ADVANCES》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962636A (en) * | 2015-07-07 | 2015-10-07 | 华南农业大学 | Wenchang chicken white spot phenotype related molecular marker and application of molecular marker |
| CN104962636B (en) * | 2015-07-07 | 2017-12-26 | 华南农业大学 | A kind of Wenchang Chicken hemp phenotype related molecular marker and its application |
| CN113430277A (en) * | 2021-07-30 | 2021-09-24 | 中国农业大学 | Primer group for identifying sex-linked dwarf gene and application thereof |
| WO2022258078A1 (en) * | 2021-07-30 | 2022-12-15 | 中国农业大学 | Primer set for identifying sex-linked dwarf gene and application thereof |
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| TA01 | Transfer of patent application right |
Effective date of registration: 20160323 Address after: 225261 Jiangsu Province, Yangzhou city Jiangdu District Shaobo Town Street No. 2 Applicant after: Yangzhou Xianglong Poultry Development Co., Ltd. Address before: 225125 Jiangsu province Hanjiang District of Yangzhou city CHANGJEI Road No. 58 Applicant before: Jinagsu Poultry lnstitute |
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| CB03 | Change of inventor or designer information |
Inventor after: Zhao Zhenhua Inventor after: Li Shoufeng Inventor after: Zhang Jingxin Inventor after: Huang Huayun Inventor after: Li Chunmiao Inventor after: Wang Qianbao Inventor after: Wu Zhaolin Inventor after: Chen Lianyi Inventor before: Zhao Zhenhua Inventor before: Li Taifeng Inventor before: Zhang Jingxin Inventor before: Huang Huayun Inventor before: Li Chunmiao Inventor before: Wang Qianbao Inventor before: Wu Zhaolin Inventor before: Chen Lianyi |
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Application publication date: 20150506 |