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Genetic diagnosis kit and use method thereof

A gene diagnosis and kit technology, which is applied in the field of C125T-SNP typing kits, can solve the problems such as no reports on the correlation between NRAMP1 gene polymorphism sites and J subtype avian leukemia resistance.

Inactive Publication Date: 2016-11-09
王干
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After searching the existing domestic and foreign literature and patents, there has been no report on the correlation between NRAMP1 gene polymorphism and J subtype avian leukemia resistance

Method used

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  • Genetic diagnosis kit and use method thereof
  • Genetic diagnosis kit and use method thereof
  • Genetic diagnosis kit and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: In practice, this kit consists of 1, box body; 2, liner; 3, upstream primer mixture P; 4, restriction endonuclease; 5, DNATag enzyme; 6, downstream primer mixture P, wherein The details of the upstream and downstream primer mix P are as follows:

[0026] The upstream and downstream primer mixture P represents the upstream and downstream primer mixture used for PCR amplification of the NRAMP1 gene, and the primer information is as follows:

[0027] Upstream primer 5'-CCCTTGGATATGTGTTTGCAGA-3'

[0028] Downstream primer 5'-CTGGCTCCAATGATGCCA-3'

[0029] How to use this kit:

[0030] 1PCR amplification of the fragment where the SNP site is located

[0031] 1) PCR amplification reaction system preparation

[0032] The PCR reaction system is 20 μl, including:

[0033] Template DNA (about 50ng) 1μl

[0034] Amplification buffer (10×buffer) 2μl

[0035] 2.5mM dNTP 0.8μl

[0036] Primer P (5uM) 0.8μl

[0037] 5U / ul DNA polymerase (rTaq) 0.1μl

[0038] Ad...

Embodiment 2

[0054] Example 2: This kit was used to type the C125T-SNP site of NRAMP1 gene of 100 negative and 100 Gushi chickens naturally infected with J-ALV.

[0055] 1. Sample

[0056]The blood of 100 negative and 100 Gushi chickens naturally infected with J-ALV was collected, the DNA was extracted by the traditional phenol / chloroform method, and the DNA concentration was diluted to 50ng / μl.

[0057] 2. PCR amplification reaction and results

[0058] The PCR amplification and detection results were carried out according to step 1 of the kit use method, and the size of the NRAMP1 gene PCR product was 268bp, which was consistent with the expected result (see figure 2 )

[0059] 3. Enzyme digestion and identification of NRAMP1 gene PCR amplification products

[0060] Enzyme digestion identification of NRAMP1 gene PCR product is carried out according to step 2 of the kit use method. The basis for the detection results is that two bands of 119bp and 101bp of the enzyme digestion product...

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Abstract

The invention relates to a genetic NRAMP1 diagnosis kit which is closely related to J-subtype avian leukosis resistance, and a use method of the genetic NRAMP1 diagnosis kit, and belongs to the technical field of molecular biology. The kit comprises an upper / lower stream primer mixture of NRAMP1 gene PCR amplification, restrictive incision enzyme TscAI and rTaq enzyme. The use method comprises the following steps: detecting the gene type of an NRAMP1 gene and a segment of a PCR amplification locus by using an RFLP method, and performing enzyme digestion reaction on the incision enzyme TscAI. NRAMP1 exon C125T mutation has influence on the resistance of J-subtype avian leukosis, an individual carrying T-allele belongs to a J-subtype avian leukosis resistant group, and particularly a cock carrying the T-allele has relatively high resistance on J-subtype avian leukosis.

Description

technical field [0001] The patent of the invention belongs to the field of biotechnology, and provides a C125T-SNP typing kit for NRAMP1 (Natural Resistance Associated Macrophage protein), a gene related to resistance to J subtype avian leukaemia, by using PCR-RFLP technology. Background technique [0002] NRAMP1 gene is also called solute transport 11 member 1 (Solute carrier family 11 member 1, SLC11A1). This gene is a relatively conserved gene. Studies by Melder D C and others found that it is related to the disease resistance of humans, mice and livestock, and mainly affects the innate immunity of animals (Melder D C et al., 1995)). Hu et al. cloned and analyzed the chicken NRAMP1 gene, which consists of 554 amino acids, has 15 exons, contains 12 putative transmembrane regions and 2 glycosylation sites, and is mainly used for ion channels and transport. function (Hu et al., 1996). NRAMP1 gene has 68% homology with chicken, mouse and human. The Northern hybridization ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/156
Inventor 王乾
Owner 王干
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