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Avian leukemia resistance molecular marker tva304-305insGCCC in chicken subgroup A and application of marker

A molecular marker, avian leukemia technology, applied in recombinant DNA technology, DNA/RNA fragment, determination/inspection of microorganisms, etc., to achieve the effect of good application and promotion value

Pending Publication Date: 2019-05-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiency that the prior art cannot fully control the occurrence and prevalence of A-subgroup avian leukemia in China, and provide a molecular marker tva for chicken A-subgroup avian leukemia resistance 304-305insGCCC and its application

Method used

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  • Avian leukemia resistance molecular marker tva304-305insGCCC in chicken subgroup A and application of marker
  • Avian leukemia resistance molecular marker tva304-305insGCCC in chicken subgroup A and application of marker
  • Avian leukemia resistance molecular marker tva304-305insGCCC in chicken subgroup A and application of marker

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Experimental program
Comparison scheme
Effect test

Embodiment

[0053] Genetic Variation Analysis of 1tva Receptor Gene

[0054] 1. Experimental method

[0055] 1. Design of primers for tva receptor gene

[0056] Taking the DNA sequence of the chicken tva gene in the NCBI database (GenBank accession number: AY531262.1) as a reference, 3 pairs of primers were designed and divided into 3 fragments to PCR amplify the full-length sequence of tva gene 3607bp (section A, section B and section C), The primer sequences, positions and PCR amplified fragment sizes are shown in Table 1.

[0057] Table 1 PCR amplification information of the full-length sequence of tva receptor gene

[0058]

[0059] 2. PCR reaction

[0060] Genomic DNA from blood samples of different Chinese chicken breeds (including local chicken breeds and commercial strains) was extracted, and the full-length sequence of the tva gene was amplified by PCR with the three pairs of primers.

[0061] PCR reaction system composition: DNA template 1 μL, 10× buffer 2.5 μL, dNTPs 2 μ...

Embodiment 2

[0069] tva 304-305insGCCC Mutation causes host resistance to ALV-A infection

[0070] 1. Experimental method

[0071] The RCASBP(A)-EGFP expression plasmid (ALV-A reporter vector) was successfully constructed, see image 3 . The RCASBP(A)-EGFP expression plasmid was transfected into DF-1 cells, and 7 days after the transfection, the RCASBP(A)-GFP virus in the cell supernatant (that is, the ALV-A reporter virus, which can infect chickens) was collected. Fibroblast CEF). tva infected with RCASBP(A)-GFP virus 304 -305insGCCC Mutation site wild type tva s / s , heterozygous mutant tva s / insGCCC and homozygous mutant tva insGCCC / insGCCC CEF (the chicken that utilizes detection among the embodiment 1 is prepared), 1,2,4,7 days after infection, utilize flow cytometry to detect RCASBP (A)-GFP virus infection tva 304-305insGCCC The situation of CEF with different genotypes at the mutation site.

[0072] 2. Experimental results

[0073] The result is as Figure 4 As shown, wil...

Embodiment 3

[0075] A detection kit for screening chicken resistance to ALV-A infection

[0076] 1. Composition

[0077] Detection primers and PCR amplification reagents,

[0078] Wherein, the nucleotide sequence of the detection primer is shown in SEQ ID NO: 1,

[0079] PCR amplification reagents include: 10×buffer, dNTPs, KOD-FX, ddH 2 O.

[0080] 2. How to use

[0081] 1. Extract the genomic DNA of the sample to be tested;

[0082] 2. PCR detection

[0083] PCR reaction system composition: DNA template 1 μL, 10×buffer 2.5 μL, dNTPs 2 μL, upstream and downstream detection primers (the nucleotide sequence of which is shown in SEQ ID NO: 1) 1 μL, KOD-FX 0.5 μL, ddH 2 O to 25 μL.

[0084] PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 90 s, a total of 35 cycles; extension at 72°C for 10 min, and finally storage at 4°C.

[0085] 3. After detection by 2% agarose gel electrophoresis, the PCR amp...

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Abstract

The invention discloses an avian leukemia resistance molecular marker tva304-305insGCCC in a chicken subgroup A and an application of the marker. The invention analyzes the genetic variation of the Chinese chicken species tva gene, and finds that GCCC insertion mutation exists between 304th and 305th bases of a Chinese chicken species tva gene DNA sequence (GenBank login number: AY531262.1), and the inventor further studies and proves that the natural mutation of the tva gene can cause host-resistant ALV-A infection. A detection kit for screening the avian leukemia resistant chickens in the subgroup A is prepared by using the molecular marker, can accurately and quickly determine whether a detection sample is the avian leukemia resistant chickens in the subgroup A or susceptible chickens,and can be applied to screening breeding materials of the avian leukemia genetic resistant chicken varieties (strains) in the subgroup A, and thus breeding of the avian leukemia genetic resistant chicken varieties (strains) in the subgroup A is carried out, so that the marker has good application and popularization value.

Description

technical field [0001] The invention belongs to the technical field of disease-resistant breed selection and breeding, and more specifically relates to a chicken A subgroup avian leukemia resistance molecular marker tva 304-305insGCCC and its application. Background technique [0002] A subgroup avian leukemia is an immunosuppressive neoplastic infectious disease of chickens caused by Avian Leukosis Virus subgroup A (ALV-A). ALV-A mainly invades lymphocytes of chickens, and transfers to other organs such as liver, kidney, spleen, etc., leading to lymphocytoma and other malignant tumors. ALV-A can cause immunosuppression in infected chickens, decline in production performance, and even death due to characteristic tumors, causing huge economic losses to the poultry industry. So far, there is no commercial vaccine and effective treatment for the disease, and it is mainly prevented by eliminating positive chickens to purify breeder flocks and biosafety measures. However, in r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
Inventor 谢青梅陈伟国张翔宇张新珩廖立钦
Owner SOUTH CHINA AGRI UNIV
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