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Method for appraising gender of chicken blastoderm by using multiple PCR

An application method and gender technology, applied in the field of animal reproduction and poultry sex identification, can solve the problem of difficulty in accurately identifying sex and other problems

Inactive Publication Date: 2011-06-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the two sex identification methods reported above are only effective for relatively abundant templates. For blastoderms with very little DNA content, it is difficult for the above two methods to accurately identify their sex.

Method used

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  • Method for appraising gender of chicken blastoderm by using multiple PCR
  • Method for appraising gender of chicken blastoderm by using multiple PCR
  • Method for appraising gender of chicken blastoderm by using multiple PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Gender identification through DNA samples of known gender, and the specificity of primers was tested.

[0028] 1. DNA extraction

[0029] The blood sample DNA of roosters and hens was first extracted, and then the extracted blood sample DNA was dissolved in 400 μL sterilized ultrapure water, and then 0.5 μL DNA sample was taken for nested PCR amplification.

[0030] 2. Primer design

[0031] According to the chicken W chromosome-specific EE0.6 sequence (GenBank accession number: D85614), the general biological primer design software Primer3 and Primer Premier 5 were used to design nested PCR amplification primers (two pairs of nested primers). According to the chicken wisp1 gene series (GenBank accession number: NC_006089), two pairs of nested primers were designed as internal controls to determine the effectiveness of PCR and prevent false negative results. The above primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0032]...

Embodiment 2

[0052] Example 2: Sex identification of chicken embryonic disc cells

[0053] 1 Removal of non-fertilized eggs

[0054] The total process from egg laying to excretion takes about 23-28 hours. The fertilized eggs continue to develop during this period, and when they are excreted, they have developed into a blastocyst containing about 60,000 cells. There are clear blastodiscs and bright and dark areas, and they are in the xth stage of chicken embryo development. Fertilized eggs can thus be distinguished from fertilized eggs. Fertilized eggs have a blastodisc (with bright and dark areas), while fertilized eggs have no blastodisc but ovules without light and dark areas. Eliminate sperm-free eggs based on this feature.

[0055] 2 Isolation of the blastodisc of the fertilized egg

[0056] First cut the filter paper into a square with a side length of about 1.5 cm, and open a hole in the middle of the square (diameter or side length is about 0.5 cm); separate the egg white and egg...

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Abstract

The present invention belongs to the field of animal propagation technology, in the concrete, said invention belongs to the field of poultry sex identification technology, in particular which relates to a method for identifying chick embryonic desc sex by utilizing multiple PCR. Said method includes the following steps: (1), according to chick EE0.6 sequence whose GenBank accession number is D85614 designing two pairs of nested primers and according to chick wispl gene whose GenBak accession number is NC_006089 designing two pairs of nested primers which can be used as internal contrast; and (2), extracting chick blood sample genome DNA whose sex is defined, making primer specificity check, then extracting chick embryonic disc cell DNA to be detected, making PCR amplification; utilizing the amplified DNA fragment size to identify the chick embryonic disc sex; determining the embryo as male when the amplified DNA fragment is one band of 500 bp; and determining the embryo as female when the amplified DNA fragment has one band of 500 bp and one band of 246 bp. The present invention also discloses a sequence of primers which is used for amplification.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, in particular to the technical field of poultry sex identification, and relates to a method for chicken germinal disc sex identification using multiplex PCR. Background technique [0002] Sex identification plays an important role in the poultry breeding industry. By identifying the sex of early embryos, non-target sex can be eliminated, feeding costs can be reduced, and economic benefits can be improved. At the same time, gender identification is more important in the field of basic research. The use of sex chromosome-specific genetic markers for sex identification of early poultry embryos is beneficial to the study of the synthesis and expression of sex hormones during sex differentiation. Sex determination at the molecular level of poultry is a prerequisite for the study of chicken chimeras of the opposite sex. [0003] The gonads of chicken embryos have no testis and ovary chara...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 张淑君李文明俸艳萍王成吴俊静赵瑞霞刘辉武防杰
Owner HUAZHONG AGRI UNIV
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