CN113604580B - Primer, kit and application for identifying genotype of chicken rose crowns by whole blood method - Google Patents
Primer, kit and application for identifying genotype of chicken rose crowns by whole blood method Download PDFInfo
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- 240000003815 Rhodiola rhodantha Species 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 31
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 24
- 239000008280 blood Substances 0.000 title claims abstract description 24
- 210000004369 blood Anatomy 0.000 title claims abstract description 24
- 230000003321 amplification Effects 0.000 claims abstract description 24
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 24
- 235000012736 patent blue V Nutrition 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 235000012745 brilliant blue FCF Nutrition 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- AUIINJJXRXMPGT-UHFFFAOYSA-K trisodium 3-hydroxy-4-[(2-hydroxy-4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].[Na+].Oc1cc(c2ccccc2c1N=Nc1c(O)c(cc2cc(ccc12)S([O-])(=O)=O)S([O-])(=O)=O)S([O-])(=O)=O AUIINJJXRXMPGT-UHFFFAOYSA-K 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 claims 2
- 238000011901 isothermal amplification Methods 0.000 abstract description 5
- 235000013330 chicken meat Nutrition 0.000 description 18
- 238000012408 PCR amplification Methods 0.000 description 9
- 239000003146 anticoagulant agent Substances 0.000 description 6
- 229940127219 anticoagulant drug Drugs 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000010100 anticoagulation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
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- 238000002360 preparation method Methods 0.000 description 3
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- 238000010257 thawing Methods 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 241000220317 Rosa Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 241000561734 Celosia cristata Species 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000001520 comb Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a primer, a kit and application for identifying a chicken rose crown genotype by a whole blood method, and belongs to the molecular biotechnology. The primer comprises: rose-F3: CCAGCATTCCCTGTGACC; rose-B3: TACAAACCCGACCCCAAAGA; rose-FIP: ACATTCCCAGGCCACGCAGCTGCTCTGGGACGCAAAT; rose-BIP: ATTGCTTCAGGAAGGGGCAGGCACTCAGCAAACAGGAACAGA. The PCR isothermal amplification reaction is carried out by using the primer, and the genotype of the rose crown of the chicken can be identified by observing the color of an amplification system. The method is simple to operate, high in accuracy and easy to popularize.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer and a kit for identifying a chicken rose crown genotype by a whole blood method and application thereof.
Background
The most common crown type in chickens is single crown, the character is mainly controlled by heredity, and the single crown is generally more abundant in size variation among chicken varieties and more consistent in varieties. Mutated cockscomb traits include rose, bean, walnut, rose, pea, multiple and no crowns, etc., wherein the different mutants are divided into several phenotypes.
The rose crown trait is inherited by autosomal dominant on the single crown and the semen quality of homozygous individuals for the rose crown trait is poor (semen of homozygous individuals cannot compete with heterozygous individuals substantially during mixed insemination), resulting in the inability to completely purify the trait during production by just panning of the appearance.
At present, two methods for purifying rose crown traits in a population exist: firstly, by the traditional means of test cross, and secondly, by the existing molecular means. The traditional testing and delivery means involves complex working steps, long period and easy error in the process. The existing molecular means all involve the steps of DNA extraction, PCR amplification, gel electrophoresis and the like, require specialized instruments (centrifuges, PCR instruments, electrophoreses, gel imaging systems and the like) to assist in completion, and also require technicians with a certain level of expertise to perform better operation, so that the method is generally not easy to implement in breeding enterprises. Therefore, a detection means which is simple and easy to operate and has high accuracy is needed for identifying the genotype of the rose crown.
Disclosure of Invention
The invention aims to provide a primer, a kit and application for identifying the genotype of a rose crown by a whole blood method, so as to solve the problems in the prior art, the primer is used for carrying out isothermal amplification on a whole blood template, the genotype of the rose crown individual can be accurately judged by observing the color of an amplification system, and the method is simple to operate, high in accuracy and easy to popularize.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a primer for identifying a chicken rose crown genotype by a whole blood method, which comprises the following steps:
Rose-F3：CCAGCATTCCCTGTGACC；
Rose-B3：TACAAACCCGACCCCAAAGA；
Rose-FIP：ACATTCCCAGGCCACGCAGCTGCTCTGGGACGCAAAT；
Rose-BIP：ATTGCTTCAGGAAGGGGCAGGCACTCAGCAAACAGGAACAGA。
the invention also provides a kit for identifying the genotype of the rose crowns by using the whole blood method, which comprises the primer.
The invention also provides a method for identifying the genotype of the rose crowns by using the whole blood method, which comprises the following steps:
using chicken whole blood as a template, performing PCR amplification reaction by using the primer of claim 1, and then observing the color of the amplified product to distinguish, wherein if the amplified product is sky blue, the amplified product is a single crown or rose crown heterozygote, and if the amplified product is light purple, the amplified product is a rose crown homozygote.
Preferably, the amplification reaction system is: 2 Xreaction buffer 12.5. Mu. L, rose-F3 and Rose-B3 primers 1. Mu. L, rose-FIP each and Rose-BIP primers 1. Mu. L, bstDNA polymerase 1. Mu.L, hydroxynaphthol blue 1. Mu.L, template 2. Mu.L, deionized water was supplemented to 25. Mu.L.
Preferably, the amplification conditions are: the prepared amplification reaction system is placed in a constant temperature plate and reacted for 60 minutes at 64 ℃.
The invention also provides application of the primer or the kit in identifying the genotype of the rose crowns of the chickens.
The invention discloses the following technical effects:
the primer disclosed by the invention can identify the gene difference of a specific genome position by using the anticoagulation sample which is simply treated as a template and performing isothermal amplification by using the primer disclosed by the invention, so that the genotype of a rose crown locus can be accurately identified. The method can judge the genotype of the rose crown individuals by using a water bath kettle or a constant temperature plate, and can carry out experimental operation without professional technicians, and finally observing the color of an amplification system by naked eyes, which has important significance for selecting homozygous individuals with the rose crown characteristics by using the genotype of the rose crown. Therefore, the identification method disclosed by the invention does not need a DNA extraction process, special equipment, special personnel and the like, is simple to operate and easy to popularize, and provides theoretical basis and data support for breeding and breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the amplification results of example 2 of the present invention; the first 3 single crown white-ear yellow chickens from the left, the last 6 rose crown homozygotic silk feather black-bone chickens and the last negative control.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 method for identifying genotype of rose crowns by Whole blood method
The embodiment aims at providing a molecular detection method, which uses anticoagulated blood after simple treatment as a template, and identifies the gene difference of a specific genome position by a normal temperature PCR method, so as to identify the genotype of a rose crown locus. The method specifically comprises the following steps:
step 1: template preparation
(1) And (5) blood sample collection. About 0.5mL of blood from the individual chicken to be tested was collected by the subpterygoid vein and placed in a centrifuge tube containing 70. Mu.L of 2% EDTA anticoagulant.
(2) Blood sample treatment (100-fold dilution). 1. Mu.L of anticoagulation was withdrawn and placed in a centrifuge tube containing 99. Mu.L TE and freeze-thawing was repeated.
Step 2: PCR amplification and target site typing
(1) PCR amplification was performed using the anticoagulants treated in step 1 as templates and the primers shown in Table 1.
TABLE 1 amplification primers
The amplification reaction system is shown in Table 2.
TABLE 2 amplification reaction System
(2) And (5) amplifying at constant temperature. The prepared amplification reaction system is placed in a constant temperature plate and reacted for 60 minutes at 64 ℃.
(3) And (5) judging results. Under natural light, if the amplification system is sky blue, it is an individual carrying a wild-type allele (heterozygote); if the amplification system is light purple, the amplification system is rose crown homozygote.
Example 2 verification of amplification efficiency and specificity of primers
1. Experimental materials
3 white-ear yellow chickens of single crown and 6 silk feather black-bone chickens known as rose crown homozygotes are selected.
2. Experimental method
Step 1: template preparation
(1) And (5) blood sample collection. About 0.5mL of blood of the white-ear yellow chicken and silky fowl were collected by the subpterygoid vein, and placed in a centrifuge tube containing 70. Mu.L of 2% EDTA anticoagulant.
(2) Blood sample treatment (100-fold dilution). 1. Mu.L of anticoagulation was withdrawn and placed in a centrifuge tube containing 99. Mu.L TE and freeze-thawing was repeated.
Step 2: PCR amplification
(1) PCR amplification was performed using the anticoagulants treated in step 1 as templates, and the primers shown in Table 1 and the amplification reaction system shown in Table 3 in example 1.
TABLE 3 amplification reaction System
(2) And (5) amplifying at constant temperature. Isothermal amplification conditions were as in example 1.
(3) And (5) judging results. Under natural light, sky blue is single crown white ear yellow chicken, and light purple is rose crown homozygotic silk feather black-bone chicken (figure 1).
The result shows that the method can accurately distinguish the individual with single crown character from the rose crown homozygote.
Example 3 reliability of Single Blind detection decision method
In this example, a single blind approach was used to genotype samples of known genotypes. The test materials are selected from 12 rose crown heterozygote silky fowl individuals (Rr) and 12 rose crown homozygous silky fowl individuals (RR) determined by test crossing. The specific judging method is as follows:
step 1: template preparation
(1) And (5) blood sample collection. The subpter vein collects about 0.5mL of blood from the subject and places it in a centrifuge tube containing 70 μl of 2% edta anticoagulant.
(2) Blood sample treatment (100-fold dilution). 1. Mu.L of anticoagulation was withdrawn and placed in a centrifuge tube containing 99. Mu.L TE and freeze-thawing was repeated.
Step 2: PCR amplification
(1) PCR amplification was performed using the anticoagulants treated in step 1 as templates, and the primers shown in Table 1 of example 1 and the amplification system shown in Table 4.
TABLE 4 amplification reaction System
(2) And (5) amplifying at constant temperature. Isothermal amplification conditions were as in example 1.
Step 3: result determination
And judging the genotype according to the color of the PCR amplification system. Sky blue individuals were judged as rose crown heterozygotes (Rr) and purple as homozygotes (Rr). The determination results are shown in Table 5.
Table 5 molecular detection identification of filoplume black-bone chicken rose crown genotype
Note that: "AB" means that the individual being tested is a rose crown heterozygote;
"AA" means that the subject is homozygous for rose crown.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Sequence listing
<110> Nanchang university Tai and Aoque black-bone chicken development Co., ltd
<120> primer and kit for identifying genotype of chicken rose crowns by whole blood method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ccagcattcc ctgtgacc 18
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tacaaacccg accccaaaga 20
<210> 3
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
acattcccag gccacgcagc tgctctggga cgcaaat 37
<210> 4
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
attgcttcag gaaggggcag gcactcagca aacaggaaca ga 42
Claims (2)
1. The method for identifying the genotype of the chicken rose crowns by using the whole blood method is characterized by comprising the steps of taking the whole chicken blood as a template, carrying out PCR (polymerase chain reaction) amplification by using primers, and identifying the genotypes of the chicken crowns and the rose crowns by using the color of amplified products;
the primers were as follows:
Rose-F3：CCAGCATTCCCTGTGACC；
Rose-B3：TACAAACCCGACCCCAAAGA；
Rose-FIP：ACATTCCCAGGCCACGCAGCTGCTCTGGGACGCAAAT；
Rose-BIP：ATTGCTTCAGGAAGGGGCAGGCACTCAGCAAACAGGAACAGA；
the reaction system of the amplification is as follows: 2 Xreaction buffer 12.5. Mu. L, rose-F3 and Rose-B3 primers 1. Mu. L, rose-FIP each and Rose-BIP primers 1. Mu. L, bstDNA polymerase 1. Mu.L, hydroxynaphthol blue 1. Mu.L, template 2. Mu.L, deionized water was supplemented to 25. Mu.L;
the color of the amplified product is observed under natural light to distinguish, if the amplified product is sky blue, the amplified product is judged to be single crown or rose crown heterozygote, and if the amplified product is light purple, the amplified product is judged to be rose crown homozygote.
2. The method of claim 1, wherein the amplification conditions are: the prepared amplification reaction system is placed in a constant temperature plate, and the amplification is carried out for 60 minutes at the constant temperature of 64 ℃.
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| CN114854880B (en) * | 2022-06-17 | 2023-03-31 | 华南农业大学 | Molecular marker of silky feather black-bone chicken rose corolla based on KASP technology and application thereof |
| CN114959064A (en) * | 2022-06-17 | 2022-08-30 | 华南农业大学 | Molecular detection method for identifying recessive white feather genotype of chicken by anticoagulation whole blood method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102041310A (en) * | 2010-09-13 | 2011-05-04 | 李宁 | Method for detecting rose cockscomb character |
| CN108570506A (en) * | 2017-10-13 | 2018-09-25 | 江苏兴牧农业科技有限公司 | A kind of primer, kit and detection method for detecting rose comb locus gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102041310A (en) * | 2010-09-13 | 2011-05-04 | 李宁 | Method for detecting rose cockscomb character |
| CN108570506A (en) * | 2017-10-13 | 2018-09-25 | 江苏兴牧农业科技有限公司 | A kind of primer, kit and detection method for detecting rose comb locus gene |
Non-Patent Citations (4)
| Title |
|---|
| The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility;Freyja Imsland等;PLoS Genet.;第8卷(第6期);e1002775 * |
| 玫瑰冠鸡冠型分子鉴定方法的研究;张梅等;玫瑰冠鸡冠型分子鉴定方法的研究;第39卷(第17期);6-9 * |
| 环介导等温扩增技术及其在基因突变筛查中的应用研究;王雯雯等;中国病院生物学杂志;第15卷(第4期);492-497 * |
| 鸡冠常见冠型及其遗传基础研究进展;许继国等;广东农业科学;第40卷(第03期);162-166、193 * |
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