Pyrosequencing detection method for dairy cow leukocyte adhesion deficiency

A technology of pyrosequencing and detection method, applied in the field of detection of dairy cow leukocyte adhesion deficiency (BLAD)

Inactive Publication Date: 2014-10-08
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report about detecting BLAD by pyrosequencing at present, and the present invention intends to establish a convenient and specific BLAD detection method on the basis of pyrosequencing

Method used

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  • Pyrosequencing detection method for dairy cow leukocyte adhesion deficiency
  • Pyrosequencing detection method for dairy cow leukocyte adhesion deficiency
  • Pyrosequencing detection method for dairy cow leukocyte adhesion deficiency

Examples

Experimental program
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Effect test

Embodiment 1

[0101] The bovine leukocyte adhesion deficiency pyrosequencing detection method of the present invention is used to detect Holstein cattle blood samples, and the specific steps are as follows:

[0102] (1) Design specific PCR primers and sequencing primers for SNP sites:

[0103] Upstream primer CD18F1: 5'-GTTGCGTTCAATGTGACCTTC-3',

[0104] Downstream primer CD 18R2: 5'-GGTCATCCACCATGGAGTAGG-3',

[0105] The 5' end of downstream primer R2 was labeled with biotin.

[0106] Sequencing primer S2: 5'-TCCGGAGGGCCAAGG-3'.

[0107] (2) Genomic DNA was extracted from blood samples of Holstein cows using the method of the kit. For the method, please refer to the technical scheme in the instruction manual, and details will not be repeated here.

[0108] (3) Using the specific PCR primers designed in step (1) as primers, configure a 50 μL PCR Mix reaction system: 25 μL 2×PCR TaqMix, 1 μL upstream primer CD18F1, 1 μL downstream primer CD18R2, 2 μL bovine blood genomic DNA, 21 μL ddH 2...

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Abstract

The invention relates to a BLAD detection method, and especially relates to a BLAD pyrosequencing detection method. A specific PCR primer and a sequencing primer are designed based on BLAD CD18 sequences published in Genbank, wherein the upstream primer CD18F1 is represented by 5'-GTTGCGTTCAATGTGACCTTC-3', the downstream CD18R2 is represented by 5'-GGTCATCCACCATGGAGTAGG-3', the 5' terminal of the downstream CD18R2 is labeled with a biotin, and the sequencing primer S2 is represented by 5'-TCCGGAGGGCCAAGG-3'. The BLAD pyrosequencing detection method provided by the invention has the characteristics of convenience, rapidness, sensitivity, stability, specificity and reliability, and is suitable for routine detection and large scale detection of the BLAD CD18 gene.

Description

technical field [0001] The invention relates to a detection method for dairy cow leukocyte adhesion deficiency (BLAD), in particular to a detection method for dairy cow leukocyte adhesion deficiency pyrosequencing (Pyrosequcing). Background technique [0002] Bovine leukocyte adhesion deficiency (BLAD) is a recessive hereditary immunodeficiency disease that occurs in Holstein cows. It is caused by a single base mutation (A→G) in the CD18 gene Caused by CD18 gene expression defects, homozygous recessive gene causes clinical disease, mainly severe repeated infection, reduced pus formation, slow wound healing and leukocytosis are characterized. In the past 30 years, my country has imported a large number of live cattle from the United States, Canada and Australia, most of which are Holstein cattle. Since most of the harmful BLAD genes exist in a heterozygous state, the carriers do not develop the disease, and there is not enough disease for this disease. Therefore, it is inevit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 贾广乐周莉廖娟红林祥梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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