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Method for diagnosing avian leukosis virus subgroup J of sicken chicken flocks

A technology of avian leukaemia, diagnostic method, applied in the biological field, capable of solving problems such as false positives

Inactive Publication Date: 2017-05-24
王干
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA diagnostic method for ALV-J was used to detect group-specific antigens, but false positives occurred due to detection of endogenous virus group-specific antigens

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: The construction of the gp85 gene prokaryotic expression vector PGEX-6P-1 / gp85 lays the foundation.

[0047] 1) Virus culture and virus cDNA extraction, culture chicken embryo fibroblasts, when the cells grow to 80% monolayer, inoculate the virus, amplify the virus, and extract the cell DNA as a template.

[0048] 2) Amplification of the target fragment. In this experiment, the S1 and S2 primers were used to carry out PCR amplification of the virus sequence integrated into the host genome, in order to amplify 960 bp including the complete gp85 gene of the virus, and the reaction system was 100 μl. The reaction conditions were pre-denaturation at 94°C for 4 minutes, followed by 30 cycles. One cycle is: denaturation at 94°C for 45 seconds, annealing at 57°C for 45 seconds, extension at 72°C for 1.5 minutes, and finally extension at 72°C for 10 minutes, and the reaction product is stored at 4°C.

[0049] 3) Prepare 1% low-melting point agarose, mix the PCR reac...

Embodiment 2

[0056] Example 2: The identification of the PGEX-6P-1 / gp85 recombinant plasmid is achieved by linking the gp85 gene to the vector.

[0057] The prepared double-digested purified product was ligated, and a total of 10 μl reaction system was ligated overnight at 16°C. The ligated product will be used to transform Escherichia coli competent cells DH5α. The connection volume is as follows:

[0058] 3 μl of purified gp85 gene with sticky ends

[0059] 1.5 μl of purified sticky-end PGEX-6P-1

[0060] 10×ligase buffer 1μl

[0061] T4DNAligase 1μl

[0062] Water 3.5μl

[0063] Competent cells were prepared by the calcium chloride method. Pick out the frozen strain DH5α with a sterile inoculation loop and streak on the LB plate, and at the same time, streak on the LB plate containing ampicillin as a control, and cultivate overnight in a 37°C incubator. On the next day, a single colony that grew well was picked and inoculated in 5ml LB liquid medium with shaking at 37°C overnight....

Embodiment 3

[0064] Embodiment 3: The method for diagnosing J subtype avian leukemia for diseased flocks, not only includes the detection process of the gp85 fragment of the virus strain cloned by specific primers, but also includes the detection process of NRAMP1 gene;

[0065] The gp85 fragment recognizes the specific viral receptor on the target cell membrane, called the outer membrane protein

[0066] NRAMP1 gene is an immunity-related candidate gene for disease resistance, which is mainly expressed specifically in phagocytes, neutrophils and peripheral blood cells, and affects the innate immunity of animals;

[0067] Include the following steps:

[0068] 1) Virus cultivation and propagation;

[0069] 2) extraction of proviral DNA;

[0070] 3) PCR reaction to amplify the target gene fragment;

[0071] 4) PCR reaction product electrophoresis detection;

[0072] 5) Gel recovery of PCR reaction product;

[0073] 6) Enzyme digestion identification of PCR reaction products;

[0074] 7...

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Abstract

The invention relates to a method for diagnosing avian leukosis virus subgroup J of sicken chicken flocks and belongs to the technical field of molecular biology. According to the method, C type retrovirus characteristics of the avian leukosis virus subgroup J are taken as a breakthrough point, on the theoretical basis of identifying a JL-2 strain of the avian leukosis virus subgroup J, specific primers are utilized to clone gp85 fragments of the virus strain, an expression vector PGEX-6P-1 / gp85 category is constructed, an RFLP method is used to detect the genotype of NRAMP1 genes, the enzyme digestion reaction of the fragments where PCR amplification sites are located is observed, according to the research, it not only is indicated that that cocks carrying T allele have higher resistance to avian leukosis virus subgroup J, but also is found that the specificity diagnosis method established by gp85 is a powerful technical means for detecting specificity of ALV-J and timely purifying populations.

Description

technical field [0001] The patent of the invention belongs to the field of biotechnology, especially in the field of detection of a J subtype avian leukosis resistance-related gene NRAMP1 and specific virus receptor gp85. Background technique [0002] Because ALV-J causes chicken growth and immunosuppression, the host spectrum of the pathogen is wide, and the age range of onset is large, which has caused a huge impact on the poultry industry in the world. Since the discovery of ALV-J, it has been reported many times around the world. However, due to the difficulty in diagnosis of J subgroup leukemia and the rapid mutation of the virus, in the past ten years, ALV-J has evolved from a highly contagious chronic tumorigenic virus to a highly contagious acute tumorigenic virus, bringing new challenges to treatment and prevention. It must be difficult. The control and elimination of ALV-J lies in diagnosing diseased chickens as early as possible. Therefore, it is necessary to st...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6858C12Q1/702C12Q2531/113C12Q2521/301
Inventor 王乾
Owner 王干
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