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

A technology for detecting reagents and disease resistance is applied in the field of animal disease detection. The effect of closing the technology gap

Inactive Publication Date: 2012-05-09
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
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Extraction of Goat Genomic DNA

[0104] The goat genomic DNA template of each genotype used in the invention is a gift from the Italian Institut zooprofilatticsperimen DELLA SARDEGNA, and the virus DNA is extracted according to the instructions of the Roche spinning column DNA extraction kit. Example 2 Design and synthesis of primers and probes for detecting codons 136, 154 and 171 of PRNP alleles

Embodiment 2

[0104] The goat genomic DNA template of each genotype used in the invention is a gift from the Italian Institut zooprofilatticsperimen DELLA SARDEGNA, and the virus DNA is extracted according to the instructions of the Roche spinning column DNA extraction kit. Example 2 Design and synthesis of primers and probes for detecting codons 136, 154 and 171 of PRNP alleles

[0105] According to (GenBank accession number: AY907689) PRNP gene sequence design the PRNP coding region 136 (A) codon MGB probe and primer; according to (GenBank accession number: AY907685) PRNP coding region 136 (V) ) MGB probes and primers for codons; according to (GenBank accession number: AY907685) PRNP gene sequence design MGB probes and primers for the 154th (R) codon of the PRNP coding region; according to (GenBank accession number: AY822666.1) PRNP gene sequence design PRNP coding region 154 (H) codon MGB probe and primer; according to (GenBank accession number: AB373795.1) PRNP gene sequence design PRNP co...

Embodiment 3

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

[0124] The gene amplification master mix 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 tube cap (sealing membrane) were purchased from Roche Company.

[0125] 1 Preparation of PCR reaction mixture

[0126] Take the DNA extracted in Example 1 as a template, and use the various fluorescent primers and probes designed in Example 2 to perform a fluorescent quantitative PCR reaction, and select 20 μL of the following PCR reaction system:

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

[0128] 136 upstream and downstream primers and 136A and 136V probes are a detection system used to identify 136 a...

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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 strong specificity 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 present invention belongs to the field of animal disease detection, and in particular, is a biological agent designed to target the codons 136, 154 and 171 of the PRNP encoding gene PRNP which determines the resistance / sensitivity of scrapie. The real-time fluorescence quantitative PCR (real-time fluorescence quantitative PCR) detection system and its subsequent end-point reading mode detect codons 136, 154, and 171 of PRNP to determine whether the detected sheep has infectious scrapie The resistance gene is a rapid typing detection reagent for the resistance gene of infectious scrapie and its preparation method and application. Background technique [0002] Scrapie, commonly known as "scrapie", is a type of Transmissible spongiform encephalopathies (TSEs), which is a chronic, progressive and lethal cause of adult sheep and goats caused by the scrapie prion. Nervous system diseases (Stanley B Prusiner. The Prion Diseases. Scientific American, 1995, 48-...

Claims

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Application Information

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Patent Type & Authority Patents(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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