Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken

A technology for avian leukemia virus and typing method, which is applied in the field of high-quality chicken resistance typing against avian leukemia virus of subgroup A, and can solve the problem that there is no high-quality chicken anti-avian leukemia A resistance typing breeding method, resistance gene report, etc. To eliminate the threat of large-scale outbreaks, with strong purpose and strong operability

Active Publication Date: 2013-01-02
SOUTH CHINA AGRI UNIV
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AI Technical Summary

Problems solved by technology

[0008] At present, there is no good vaccine for ALV that can be used for the prevention and control of the disease, and there is no report on the resistance gene of high-quality chickens against avian leukosis A, and there is no mature resistance gene that can be applied to the inheritance of high-quality chickens against ALV-A Breeding for disease resistance, not to mention the establishment of a method for genotype breeding of high-quality chickens against avian leukosis A resistance, which poses a huge challenge to the breeding of high-quality chickens for ALV disease resistance

Method used

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  • Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken
  • Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken
  • Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken

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Embodiment 1

[0043] The establishment of embodiment 1 PCR-RFLP

[0044] 1. PCR amplification of TVA619 fragment

[0045] According to the SNP site (C / G) at position 619 on the mRNA sequence of the known TVA gene (GenBank accession number: AY531262.1), design PCR-RFLP amplification primer TVA6195 (5'-AAA CCG ACT GCT ACC CGC TGG AGT GGC TCT T-3', SEQ ID NO:1), TVA6193 (5'-ACT CGT CCC GTC CAT CGT-3', SEQ ID NO:2), with ear I restriction site CTCTT. Chicken genomic DNA was extracted and PCR amplified with primers TVA6195 and TVA6193. The composition of the PCR reaction system is as follows: Genomic DNA template 1 μL, 10× buffer 10 μL, dNTPs 8 μL, primer TVA6195 1 μL, primer TVA6193 1 μL, Bland Taq 1 μL, ddH 2 O to make up to 100 μL. PCR reaction program: pre-denaturation at 94°C for 3min, 1 cycle; 94°C for 40s, 67°C for 40s, 72°C for 1min, 35 cycles; extension at 72°C for 10min. When the PCR product is detected by 3.5% agarose gel electrophoresis, a specific band of about 69bp can be o...

Embodiment 2

[0050] Example 2 Establishment of 4-base Pyrosequence method

[0051] 1. PCR amplification of TVA223 fragment

[0052] According to the 4-base insertion or deletion site (GCTC / ----) at position 305-308 on the mRNA sequence of the known TVA gene (GenBank accession number: AY531262.1), the PCR amplification primer TVA2235 (5 '-CATGGTGCGGTTGTTGGAGC-3', SEQ ID NO:4), TVA2233 (5'-GGGGAACGCGGGGTGC-3', SEQ ID NO:5)), TVA2233 primers were labeled with biotin. Chicken genomic DNA was extracted and PCR amplified with primers TVA2235 and TVA2233. The composition of the PCR reaction system is as follows: Genomic DNA template 1 μL, 10× buffer 10 μL, dNTPs 8 μL, primer TVA2235 1 μL, TVA2233 1 μL, Bland Taq 1 μL, ddH 2 O to make up to 100 μL. PCR reaction program: pre-denaturation at 94°C for 3min, 1 cycle; 94°C for 40s, 62°C for 40s, 72°C for 1min, 35 cycles; extension at 72°C for 10min. The PCR product fragment was 223bp in length and was directly used for pyrosequencing.

[0053] 2...

Embodiment 3

[0056] Example 3 Establishment of Judgment Criteria for TVA Genotyping

[0057] According to the results of PCR-RFLP and 4-base pyrosequence sequencing, the present invention formulates the judgment standard of chicken resistance genotyping against subgroup A avian leukemia virus (ALV-A). The TVA resistance gene defined in the present invention has three alleles, namely TVA*S, TVA*R1 and TVA*R2. There are six genotypes of TVA genes in different chicken strains, TVA*S / S, TVA*S / R1, TVA*S / R2, TVA*R1 / R1, TVA*R1 / R2, TVA*R2 / R2. The standard of genetic resistance typing is: if a chicken carries one or two TVA*S alleles, the chicken will be susceptible to ALV-A, i.e. genetic susceptibility; if a chicken carries There is one TVA*R1 and one TVA*R2, or two TVA*R1 or two TVA*R2 alleles, then this chicken is the genotype of resistance to ALV-A, that is, the genetic resistance type. The genotyping determination is shown in Table 2.

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Abstract

The invention discloses a typing method of resistance for resisting a subgroup A avian leukosis virus by quality chickens, which adopts an SNP locus at the 619th site of a TVA gene sequence as a research object; PCR-RLFP analysis is performed to detect whether mutation occurs at the SNP locus, and also to detect whether 4 bases are inserted into SNP loci at the 305th-308th sites of the TVA gene sequence; the two detection results are combined together to determine whether the detected chicken is a genetic resistance type or a genetic susceptibility type. The resistance typing method of the invention performs detection from the source, and omits troubles of later-stage detection; the detection method is rapid, accurate, strong in purposiveness, and simple in operation, has practice significance, and can realize rapid selection of chicken genetic characters and improvement of disease resistance of strains.

Description

technical field [0001] The invention relates to a method for typing chicken anti-virus genes, in particular to a method for typing resistance of high-quality chickens against subgroup A avian leukemia virus. Background technique [0002] my country is a big producer and consumer of broiler chickens, and the output of broiler chickens ranks second in the world. In 2010, the annual slaughter rate of high-quality chickens in my country reached 5 billion, accounting for about 50% of China's broiler production. China has become the world's largest high-quality chicken production base and consumer market. The dominant position in the international market is obvious. The epidemiological investigation results of our research group for many years show that the incidence of avian leukemia in high-quality chickens is higher than that in fast and large broilers, and it has become one of the main diseases threatening the high-quality chicken industry. [0003] Avian leukemia is a type o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 谢青梅张焕民常爽李鸿鑫毕英佐
Owner SOUTH CHINA AGRI UNIV
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