Molecular marker tvb3667-3668insag for resistance to avian leukemia in subgroup b of chicken and its molecular diagnostic method
A technology of molecular markers and avian leukosis virus, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of uncontrollable occurrence and prevalence of AVL-B
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0018] Example 1 tvb Genetic Variation Analysis of Receptor Genes
[0019] according to tvb The known DNA sequence of the receptor gene (GenBank accession number: NC_006109.3), designed 5 pairs of primers for amplification tvb The full-length sequence of the gene is 5425bp, and the primer sequences, positions and PCR amplification fragment sizes are shown in Table 1.
[0020] Table 1 is tvb Receptor gene full-length sequence PCR amplification information
[0021]
[0022] Genomic DNA extracted from blood samples of Chinese chicken breeds and amplified with the 5 pairs of primers tvb The full-length sequence of the receptor gene. The composition of the 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
[0023] Example 2 tvb 3667-3668insAG Functional validation of mutation-induced host resistance to ALV-B infection.
[0024] 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 carrier EGFP Fluorescent protein RCASBP (B) virus, that is, ALV-B virus, can subsequently infect DF-I and CEF cells), after measuring the virus infection unit (IU), aliquots were stored at -80°C. RCASBP(B) virus infection tvb 3667 -3668insAG Different genotype CEF at the mutation site, including wild type tvb S / S CEF (susceptible to ALV-B), heterozygous mutant tvb S / insAG CEF and homozygous mutant tvb insAG / insAG CEF, 1, 2, 4, 7 days after infection, different time points detected by flow cytometry tvb 3667-3668insAG The positive cell rate after mutant CEFs of different genotypes infected with RCASBP (B) virus was determined tvb 3667-3668insAG The tendency of ...
Embodiment 3
[0033] Example 3 Establishing identification criteria for ALV-B genetically resistant chickens
[0034] Using the P4 primers in Table 1 to PCR-amplify the tvb 3667-3668insAG mutation site tvb For the receptor gene region, according to the sequencing results of the PCR amplification product of the P4 primer, the method standard for determining the genetic resistance of subgroup B avian leukemia chickens was established (see Table 3).
[0035] Table 3 Identification criteria of subgroup B avian leukemia genetically resistant chickens
[0036]
[0037] Remarks: If the tested chicken carries A, the chicken is judged to be genetically resistant to ALV-B infection.
[0038] 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 , then the chicken is genetically resistant to ALV-B infection.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More