Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Specific PCR primer, kit and detection method for detecting bovine HCD carrier

A kit and carrier technology, applied in the field of specific PCR primers and kits, can solve the problems of unclear carrier status of HCD haplotypes

Inactive Publication Date: 2016-11-02
北京奶牛中心
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, my country has not yet carried out relevant research and detection work, and the carrying status of HCD haplotypes in my country's Holstein cattle is still unclear

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Specific PCR primer, kit and detection method for detecting bovine HCD carrier
  • Specific PCR primer, kit and detection method for detecting bovine HCD carrier
  • Specific PCR primer, kit and detection method for detecting bovine HCD carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Primer Design

[0034] According to the bovine APOB gene sequence (ID: 494004) on GenBank, and referring to the primer sequence designed by Schütz et al. primer3-0.4.0 / ) to design specific primers for detecting HCD, and obtain primer pairs: upstream primer APOB-Primer-F: 5'-TGCAAAGCCCACCTAGCCTAT-3'; downstream primer APOB-Primer-R1: 5'-AGATGATGCCCCTCTTGATG-3 ', the primer pair spans the 1299bp insertion sequence of the APOB gene. Theoretically, normal individuals can amplify a 171bp fragment; HCD carrier individuals can amplify a 171 / 1470bp fragment, and HCD homozygotes can amplify A 1470bp length fragment was added. After the first round of primer screening and optimization, when the HCD carrier is amplified, the primer pair can only amplify a 171bp fragment, and the 1470bp length fragment cannot be amplified normally, which may be due to the 171bp short fragment Amplification inhibits the amplification of long fragments, and long fragments are inherently e...

Embodiment 2

[0046] Example 2 Optimization of PCR reaction program

[0047] Based on the designed specific PCR primers, the present invention further optimizes the reaction program of PCR.

[0048] Finally, the PCR reaction procedure and reaction system for identifying HCD carriers were determined.

[0049] Described reaction procedure is:

[0050] Pre-denaturation at 94°C for 5 minutes; 35 cycles of denaturation at 94°C for 30s, annealing at 62°C for 30s, and extension at 72°C for 30s; extension at 72°C for 7 minutes, and then storage at 4°C. Among them, the optimum annealing temperature is 62°C

[0051] The PCR reaction system: a total volume of 25 μL

[0052]

Embodiment 3

[0054] 1. Extraction of total bovine DNA

[0055] Holstein bulls from the Beijing Bull Breeding Station were selected as experimental materials to extract total DNA, which can be obtained from blood (for example, fresh or frozen), semen (for example, fresh or frozen), tissue samples (such as ear tissue, etc.) Or extraction, separation and purification from bovine hair samples containing hair follicles.

[0056] 2. PCR amplification and genotype determination

[0057] According to the specific primer pair screened in Example 1, and the PCR reaction conditions and reaction system optimized in Example 2, multiplex PCR amplification was performed.

[0058] Take 4 μL of PCR products and detect them by 2% agarose gel electrophoresis. The results of PCR amplification are shown in the attached figure 1 shown.

[0059] Genotype determination:

[0060] Healthy dairy cows: the genotype is AA, and only 171bp electrophoresis band appears;

[0061] HCD recessive carrier: the genotype i...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to detection primers and kits, and particularly discloses a specific PCR primer and a kit for detecting a bovine HCD carrier. The primer includes a nucleic acid sequences represented as the SEQ ID No. 1-3. With total DNA of a to-be-tested sample as a template, PCR amplification is carried out with the primer. Through agarose gel electrophoresis detection, when only the electrophoresis band of 171 bp exists in a PCR amplification product, the to-be-tested sample does not carry the HCD, when the electrophoresis bands of both 171 bp and 366 bp exist in the PCR amplification product, the to-be-tested sample carries the heterozygote of the HCD, and when only the electrophoresis band of 366 bp exists in the PCR amplification product, the to-be-tested sample carries homozygote of the HCD.

Description

technical field [0001] The invention relates to detection primers and a kit, in particular to specific PCR primers and a kit for detecting bovine HCD carriers. Background technique [0002] In dairy cattle breeding, due to the application of modern breeding techniques such as progeny determination and genomic selection, the selection accuracy has been greatly improved, and the selection intensity has also been potentially increased. Due to the application of artificial insemination (AI) and other reproductive biotechnologies, individual excellent bulls are widely used, which increases the inbreeding coefficient of the population, reduces the effective population content, increases the probability of homozygous recessive genes, and makes bad genes appear in the population. With the continuous deepening of genomics research and the rapid development of high-throughput sequencing technology, the data mining, identification and application of recessive genetic defects in dairy c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2600/16C12Q2537/143
Inventor 李艳华麻柱刘林赵凤吕小青韩广文朱玉林孙东晓
Owner 北京奶牛中心
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com