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Chicken B subgroup avian leukosis resistantmolecular marker tvb<3731-3732insA> and molecular diagnosis method thereof

An avian leukemia, molecular marker technology, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problem of uncontrollable occurrence and prevalence of AVL-B

Active Publication Date: 2016-03-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing measures cannot control the occurrence and prevalence of AVL-B in China

Method used

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  • Chicken B subgroup avian leukosis resistantmolecular marker tvb&lt;3731-3732insA&gt; and molecular diagnosis method thereof
  • Chicken B subgroup avian leukosis resistantmolecular marker tvb&lt;3731-3732insA&gt; and molecular diagnosis method thereof
  • Chicken B subgroup avian leukosis resistantmolecular marker tvb&lt;3731-3732insA&gt; and molecular diagnosis method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The genetic variation analysis of embodiment 1tvb receptor gene

[0024] According to the known DNA sequence of tvb receptor gene (GenBank accession number: NC_006109.3), 5 pairs of primers were designed to amplify the full-length 5425 bp sequence of tvb gene. The primer sequences, positions and PCR amplification fragment sizes are shown in Table 1.

[0025] Table 1 shows the PCR amplification information of the full-length sequence of tvb receptor gene

[0026]

[0027] Genomic DNA was extracted from blood samples of Chinese chicken breeds, and the full-length sequence of tvb receptor gene was amplified with the five pairs of primers. Composition of PCR reaction system: template 1 μL, 10×buffer 2.5 μL, dNTPs 2 μL, upstream and downstream primers 1 μL, KOD-FX 0.25 μL, sterile water to 25 μL. PCR reaction program: 94°C pre-denaturation for 3min, 1 cycle; 94°C for 45s, 58-65°C (different primer annealing temperature) for 90s, 72°C for 60s, 35 cycles; 72°C extension fo...

Embodiment 2

[0028] Example 2tvb 3667-3668insAG 、tvb 3731-3672insA Functional validation of mutation-induced host resistance to ALV-B infection.

[0029] 1. Functional verification of in vitro cells: Construct RCASBP(B)EGFP expression plasmid, 7 days after transfection into DF-I cells, collect cell supernatant (supernatant contains RCASBP(B) virus carrying EGFP fluorescent protein, namely ALV- B virus, which can subsequently infect DF-I and CEF cells), after measuring the virus infection unit (IU), aliquot and store at -80°C. RCASBP(B) virus infects tvb respectively 3667 -3668insAG 、tvb 3731-3732insA Different genotype CEF at the mutation site, including wild-type tvb S / S CEF (susceptible to ALV-B), heterozygous mutant tvb S / insAG and tvb S / insA CEF and homozygous mutant tvb insAG / insAG and tvb insA / insA CEF, 1, 2, 4, 7 days after infection, tvb at different time points detected by flow cytometry 3667-3668insAG 、tvb 3731-3732insA Positive cell rate after mutation of different ge...

Embodiment 3

[0035] Embodiment 3 establishes the identification standard of ALV-B genetic resistance chicken

[0036] Utilize the P4 primers in Table 1 to PCR amplify containing tvb 3667-3668insAG and tvb 3731-3732insA For the tvb receptor gene region of the two mutation sites, according to the sequencing results of the P4 primer PCR amplification products, the method standard for determining the genetic resistance of subgroup B avian leukemia chickens was established (see Table 3).

[0037] The identification standard of table 3B subgroup avian leukemia genetic resistance chicken

[0038]

[0039] Remarks: If the tested chicken has A or B or any of A and B at the same time, the chicken is judged to be genetically resistant to ALV-B infection.

[0040] The identification standard of the established B subgroup avian leukemia genetic resistance chicken is: 1, if a certain chicken is in tvb 3667-3668insAG The genotype of the mutation site is tvb insAG / insAG , the chicken will be geneti...

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Abstract

The invention belongs to the technical field of resistant variety breeding, and in particular discloses a chicken B subgroup avian leukosis resistantmolecular marker tvb<3731-3732insA> and a molecular diagnosis method thereof. The invention analyzes genetic variation of a Chinese chicken breed tvb receptor gene for the first time and discovers that the 3731st and the 3732nd sites of the Chinese chicken breed tvb receptor gene have A insertion mutation, and the inventor proves, from in vivo and in vitro experiments two aspects, that the natural mutation causes a host to generate genetic resistance to infection of ALV-B; moreover, the inventor establishes a molecular diagnosis method for a B subgroup avian leukosis resistant genetic marker, and the method can be applied to screening breeding materials of ALV-B genetic resistant chicken varieties (strains) so as to carry out breeding of the ALV-B genetic resistant chicken varieties (strains).

Description

technical field [0001] The present invention relates to the technical field of resistant variety breeding, more specifically, relates to chicken B subgroup avian leukemia resistance molecular marker tvb 3731-3732insA and its molecular diagnostic methods. Background technique [0002] Avian leukemia is an immunosuppressive neoplastic infectious disease caused by avian leukosis virus (Avian Leukosis Virus, ALV), which can cause a variety of infectious benign and malignant tumors. There are 6 subgroups of avian leukosis virus infecting chickens, including ALVA-E and J. Subgroup B avian leukemia virus (ALV-B) is one of the main pathogens of avian leukemia in my country. It can cause immunosuppression, decline in production performance and characteristic tumors in infected chickens, causing huge economic losses to the world's poultry industry. . At present, there is no commercial vaccine and effective treatment for the disease, and it is mainly prevented by eliminating positive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 谢青梅陈伟国蔺文成舒鼎铭李昕键
Owner SOUTH CHINA AGRI UNIV
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