Infectivity scrapie resistance gene rapid typing detection reagent, preparation method and application thereof

A detection reagent and genotyping technology, applied in biochemical equipment and methods, material stimulation analysis, microbial measurement/inspection, etc., can solve the problems of long time, high cost, and inability to use daily detection and monitoring, and shorten detection time, shortening the technology gap, and safeguarding human health and the effectiveness of the public health environment

Inactive Publication Date: 2010-03-17
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no rapid screening platform for TSE resistance genes in my country. Currently, only sequencing technology can be used to diagnose PRNP genotypes. However, this method is expensive and takes a long time. It can only be used for laboratory analysis and research, but not for daily detection. monitor

Method used

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  • Infectivity scrapie resistance gene rapid typing detection reagent, preparation method and application thereof
  • Infectivity scrapie resistance gene rapid typing detection reagent, preparation method and application thereof
  • Infectivity scrapie resistance gene rapid typing detection reagent, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0103] The extraction of embodiment 1 sheep genome DNA

[0104] The sheep genomic DNA templates of each genotype used in the invention were donated by Institut zooprofilatticsperimen DELLA SARDEGNA, Italy, and viral DNA was extracted according to the instructions of the Roche spin column DNA extraction kit. Example 2 Design and synthesis of primers and probes for detection of codons 136, 154 and 171 of PRNP alleles

Embodiment 2

[0104] The sheep genomic DNA templates of each genotype used in the invention were donated by Institut zooprofilatticsperimen DELLA SARDEGNA, Italy, and viral DNA was extracted according to the instructions of the Roche spin column DNA extraction kit. Example 2 Design and synthesis of primers and probes for detection of codons 136, 154 and 171 of PRNP alleles

[0105] According to (GenBank accession number: AY907689) PRNP gene sequence design the MGB probe and primer of the 136th (A) codon of PRNP coding region; design the 136th (V ) codon MGB probe and primer; according to (GenBank accession number: AY907685) PRNP gene sequence design the MGB probe and primer of the 154th (R) codon in the PRNP coding region; according to (GenBank accession number: AY822666.1) Design the MGB probe and primer of the 154th (H) codon of the PRNP coding region according to the PRNP gene sequence; design the MGB probe of the 171st (R) codon of the PRNP coding region according to the (GenBank access...

Embodiment 3

[0123] Real-time fluorescent PCR amplification of embodiment 3 target gene

[0124]The gene amplification premix Taqman Universal PCR MasterMix was purchased from Applied Biosystems Reagent Company, the real-time fluorescent quantitative PCR instrument was Roche LightCycler 480, and the fluorescent PCR tube (plate) and cap (parafilm) were purchased from Roche Company.

[0125] 1 Preparation of PCR reaction mixture

[0126] Using the DNA extracted in Example 1 as a template, the fluorescent quantitative PCR reaction was carried out with various fluorescent primers and probes designed in Example 2, and 20 μL of the following PCR reaction system was selected:

[0127] Template DNA: 2 μL; Upstream primer (10 μmol / L): 2 μL; Downstream primer (10 μmol / L): 2 μL; FAM-labeled probe (10 μmol / L): 0.5 μL; VIC-labeled probe (10 μmol / L): 0.5μL; 2×mastermix: 10μL; make up with double distilled water.

[0128] The upstream and downstream primers of 136 and 136A and 136V probes are a detecti...

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Abstract

The invention discloses an infectivity scrapie resistance gene rapid typing detection reagent, and a preparation method and application thereof, which is characterized in that three sets of idiosyncratic primers and Taqman probes are designed and synthesized; and 136-position, 154-position, 171-position codons of sheep prion protein encoding gene PRNP which respectively determine scrapie resistance / sensibility are detected for judging whether sheep to be detected contain infectivity scrapie resistance gene. The invention establishes a rapid, simple and convenient gene typing method with strongspecificity and high sensitivity with the help of a real-time fluorescence quantitative PCR detection system and a subsequent end-point reading plate model; the detection time is only a plurality ofhours; and with the help of the method, a whole set of thorough early warning and monitoring system is established for preventing scrapie, can be applied to a conventional laboratory diagnosis method,is used as a test item for introducing live sheep by entry-exit inspection and quarantine departments so as to put an end to the introduction of the scrapie sensitivity varies.

Description

technical field [0001] The invention belongs to the field of animal disease detection, in particular to a biological agent designed to contain the 136th, 154th and 171st codons of the sheep prion protein coding gene PRNP that determines the resistance / sensitivity of sheep scrapie. Fluorescent quantitative PCR (real-time fluorescence quantitative PCR) detection system and the subsequent end-point plate reading mode are used to detect the 136th, 154th and 171st codons of PRNP to determine whether the detected sheep contains infectious scrapie The resistance gene is a rapid genotyping detection reagent for infectious sheep scrapie resistance gene and its preparation method and application. Background technique [0002] Scrapie, commonly known as "scrapie", is a kind of transmissible spongiform encephalopathy (Transmissible spongiform encephalopathies, TSEs), which is a chronic progressive and fatal disease of adult sheep and goats caused by scrapie prions. Nervous system disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 董志珍李琳肖妍蔡国瑞栾慎顺陈本龙
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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