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Bovine degenerative axonopathy carrier screening method and kit thereof

A degenerate, kit-based technology, applied in the field of animal husbandry, can solve the problems of low reliability and inaccuracy, and achieve the effects of simple operation, broad application prospects and strong reliability.

Inactive Publication Date: 2014-04-09
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Bovine degenerative axon disease is a recessive genetic disease controlled by autosomal single gene mutation, so it is inaccurate to determine whether it is a carrier of degenerative axon disease only by pedigree analysis, and it is only an inference on the population. For cattle, its reliability is not strong

Method used

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  • Bovine degenerative axonopathy carrier screening method and kit thereof
  • Bovine degenerative axonopathy carrier screening method and kit thereof
  • Bovine degenerative axonopathy carrier screening method and kit thereof

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Experimental program
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Effect test

Embodiment 1

[0042] 1. Genomic DNA was extracted from cattle blood by phenol-chloroform extraction, including DNA from 80 Holstein cows, 63 Luxi yellow cattle, and 18 Swiss brown cattle. Design specific primers for amplifying bovine MFN2 gene fragments, the primer sequences used are:

[0043] The sequence of the upstream primer MFN2F is: SEQ ID NO.1:

[0044] 5′-AAGCCCGAGTTTATTTCC-3′

[0045] The sequence of the downstream primer MFN2R is: SEQ ID NO.2:

[0046] 5′-GCACTGTCCTCCCCCACA-3′

[0047] 2. According to the above kit for detecting bovine degenerative axon disease carriers, the following steps are used to amplify the bovine MFN2 gene fragment, and the amplified fragment is detected by 1.5% agarose gel electrophoresis with a mass concentration. The results are as follows: figure 1 shown.

[0048] (1) PCR reaction system: DNA (100ng / μl) 1μl, upstream and downstream primers (10μmol / L) 0.5μl each, 2×Taq PCR MasterMix 12.5μl, double distilled water (ddH 2 O) Make up to 25 μl.

[004...

Embodiment 2

[0060] 1. The genomic DNA of 18 gray cattle was extracted from the blood of cattle by phenol-chloroform extraction. Design specific primers for amplifying bovine MFN2 gene fragments, the primer sequences used are:

[0061] The upstream primer sequence is: SEQ ID NO.1:

[0062] 5′-AAGCCCGAGTTTATTTCC-3′

[0063] The downstream primer sequence is: SEQ ID NO.2:

[0064] 5′-GCACTGTCCTCCCCCACA-3′

[0065] 2. According to the above kit for detecting bovine degenerative axon disease carriers, the following steps are used to amplify the bovine MFN2 gene fragment, and the amplified fragment is detected by 1.5% agarose gel electrophoresis with a mass concentration. The results are as follows: figure 1 shown.

[0066] (1) PCR reaction system: DNA (100ng / μl) 1μl, upstream and downstream primers (10μmol / L) 0.5μl each, 2×Taq PCR MasterMix 12.5μl, double distilled water to make up to 25μl.

[0067] (2) PCR reaction conditions: denaturation at 94°C for 4 min; denaturation at 94°C for 30 s...

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Abstract

The invention provides a bovine degenerative axonopathy carrier screening method and a kit thereof, and belongs to the field of animal husbandry technology. According to the method, genomic DNA of bovine to be tested is used as a template; an upstream primer and a downstream primer are designed to carry out a PCR amplification reaction on the template; then a PCR amplification fragment is retrieved and digested by FastDigest restriction enzyme TspRI; and gel testing is performed after the digestion of the reaction system. Bovine to be tested which shows a digested DNA fragment of 307bp by the gel testing after digestion is normal bovine. Bovine to be tested which shows digested DNA fragments of 307bp, 225bp and 82bp after digestion is a degenerative axonopathy carrier. Bovine to be tested which shows the digested DNA fragments of 225bp and 82bp after digestion is degenerative axonopathy bovine. The screening method has advantages of simple and fast operation, low cost and high reliability. The method provided by the invention can be used to eliminate the bovine degenerative axonopathy carrier at an early stage and greatly reduce the loss of cattle industry.

Description

technical field [0001] The invention relates to the technical field of animal husbandry, in particular to a method for screening bovine degenerative axon disease carriers and a kit thereof. Background technique [0002] Degenerative axonopathy (Degenerative axonopathy) is a recessive genetic disease controlled by an autosomal single gene in cattle. Through genome-wide association analysis and haplotype map, it was finally determined that it was caused by a point mutation of the mitochondrial fusion protein 2 (MFN2) gene located on bovine chromosome 16 ( C, Reichart U, Seuberlich T, et al. An unusual splice defect in the mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Gray cattle. PLoS One, 2011, 6(4): e18931). et al. analyzed the DNA sequencing of 44 cows with degenerative axon disease, and found a common synonymous mutation site, SNP c.2229C>T, which was located in the 18th exon of the MFN2 gene. The c.2229C>T mutation affects a CpG din...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 黄金明王秀革王长法鞠志花李秋玲齐超张燕赵秀新李荣岭李建斌杨宏军王玲玲仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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