PCR-RFLP detection method for complex vertebral malformation of milk cow and crossbred cattle

A syndrome and spine technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long breeding cycle and low accuracy

Inactive Publication Date: 2009-07-08
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The traditional method is often to identify and eliminate harmful genes based on the phenotypic charact

Method used

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  • PCR-RFLP detection method for complex vertebral malformation of milk cow and crossbred cattle
  • PCR-RFLP detection method for complex vertebral malformation of milk cow and crossbred cattle
  • PCR-RFLP detection method for complex vertebral malformation of milk cow and crossbred cattle

Examples

Experimental program
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Embodiment 1

[0025] 1. Collection and processing of dairy cow blood samples

[0026] Take about 10 mL of cow blood sample, add 500 μL of 0.5 mol / L EDTA (to make the final concentration 25 mmol / L) for anticoagulation, centrifuge (8000 rpm) at 4 °C for 10 min, discard the supernatant, separate white blood cells, add distilled water, shake well, and rupture the red blood cells. Centrifuge for 10 min and discard the supernatant. After repeating twice, add 1×SET 1mL and 10% sodium dodecyl sulfate to the precipitate (to make the final concentration 0.5%), then slowly add proteinase K (final concentration is 50-100 μg / mL), and place at 55 ℃ for 8-10 hours in a water bath, and stored in a 4℃ refrigerator for later use.

[0027] 2. Extraction of NDA from blood samples

[0028] (1) Cancel the digested sample, add an equal volume of equilibrated phenol (pH 8.0), slowly invert the centrifuge tube for 10 min, and centrifuge at 8000 rpm for 10 min at 4°C.

[0029] (2) Carefully draw the supernatant c...

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Abstract

The present invention belongs to the field of animal disease detection technology, specifically to the field of cattle recessive genetic disease spondyle malformation syndrome (CVM) detection, includes steps as follows: extracting cattle genome DNA from the cattle sample to be tested, designing the PCR amplification prime pair intruding the restriction enzyme site and performing the PCR reaction based on the cattle SLC35A3 gene sequence, using 2.5% agarose gel electrophoresis to test the PCR amplification products, dyeing, observing bands in the gel imaging system, recovering the PCR products to perform restriction enzyme, performing electrophoresis detection in 10% polyacrylamide gel after the reaction is completed, determining the genetype of the tested sample according to the band distribution of the amplification products.

Description

technical field [0001] The invention belongs to the technical field of animal disease detection, in particular to the detection field of a bovine recessive genetic disease vertebral malformation syndrome (CVM). Background technique [0002] Complex vertebral malformation (CVM) is an autosomal recessive genetic disease of Holstein cattle, which was first reported by Danish scientists Agerholm et al. in 1999. CVM mainly manifests as early death of homozygous fetuses, and homozygous fetuses rarely survive after birth. But CVM heterozygous offspring (carriers) behaved normally and survived. Calves with CVM can be aborted at any point in the gestation of the cow. The most prominent features of CVM-infected calves include: short neck, vertebral deformities, heart deformities, leg deformities, and symmetrical inward rigidity of the calves Wait. [0003] The wide application of artificial insemination technology and embryo transfer has made CVM widely spread all over the world, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张淑君杨利国李翔胡修忠刘文举宋利军陈世林童勤
Owner HUAZHONG AGRI UNIV
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