Real-time fluorescence quantification RT-PCR kit for detecting border disease virus and application

A real-time fluorescence quantification and sheep border disease virus technology, which is applied in the field of microbial detection, can solve the problems of complicated operation, unfavorable rapid diagnosis of sheep border disease virus, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2017-08-18
GUANGXI VETERINARY RES INST
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The isolation and identification of the virus, the results are accurate and reliable, but there are also disadvantages such as complicated operation, complex equipment, long time required, and many affected factors, which are not conducive to the rapid diagnosis of sheep border disease virus
Fluorescence immunoassay is a relatively fast and sensitive detection method, but its sensitivity is poor

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
  • Real-time fluorescence quantification RT-PCR kit for detecting border disease virus and application
  • Real-time fluorescence quantification RT-PCR kit for detecting border disease virus and application
  • Real-time fluorescence quantification RT-PCR kit for detecting border disease virus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Real-time fluorescent quantitative RT-PCR detection of sheep border disease virus (BDV)

[0048] 1. Preparation of materials

[0049] Ovine border disease virus, Mycoplasma ovis, Brucellosis, Pasteurella and Escherichia coli were either commercially available vaccines or preserved by Guangxi Veterinary Research Institute. 2×UltraSYBR Mixture, Viral Genomic DNA / RNA Extraction Kit (CW0548S), Reverse Transcription Kit HiFiScript cDNA Synthesis Kit (CW2569M) were purchased from Kangwei Century Biotechnology Co., Ltd.

[0050] 2. Design and synthesis of RT-PCR primers

[0051] According to the homology comparative analysis of the 5' non-coding region gene sequence of sheep border disease virus in GenBank, the conserved sequence region was selected as the amplification region, and specific amplification primers were designed by using Oligo 6.0 primer design software and BLAST software program, among which The sequences of the primers are:

[0052] Forward primer:...

Embodiment 2

[0080] Example 2 Real-time fluorescent quantitative RT-PCR reaction conditions optimization results

[0081] The annealing temperature and primer concentration were optimized respectively to obtain the best reaction conditions and system for real-time fluorescent quantitative PCR of sheep border disease virus. The total reaction system was 20 μL, including 11.25 μL of 2× reaction buffer, 0.4 μL of upstream and downstream primers (2 pmol / L), 2 μL of cDNA template and ddH 2 O 5.95 μL. Cycling conditions were: 95°C for 5 min; followed by 40 cycles of 95°C for 20 s, 60°C for 20 s, and 68°C for 20 s; and a final extension at 68°C for 5 min. The fluorescent RT-PCR amplification product was subjected to agarose gel electrophoresis, and the target band 115bp was detected ( figure 2 ).

Embodiment 3

[0082] Example 3 Real-time fluorescent quantitative RT-PCR melting curve analysis

[0083] The melting curve analysis results are shown in figure 1 , the melting temperature is 86.5°C, and there is only one specific peak, which excludes the possibility of non-specific products and the formation of primer-dimers affecting the results. good optimization.

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 discloses a real-time fluorescence quantification RT-PCR kit for detecting the border disease virus and application, and belongs to the technical field of microbiological detection. The kit contains liquid A, liquid B and liquid C. The liquid A is fluorescent quantification RT-PCR reaction liquid, 2 X UltraSYBR Mixture, Forward primer and Reverse Primer. The liquid B is cDNA containing the border disease virus and serves as positive control. The liquid C is RNase-Free water and serves as negative control. It is proved through specificity detection and sensitivity detection that the real-time fluorescence quantification RT-PCR kit has high specificity and sensitivity, and can monitor reaction in real time, detect the copy number of the border disease virus in a quantitative mode, and acquire a detection result quickly and accurately. The real-time fluorescence quantification RT-PCR kit can serve as a quick, accurate and convenient detection tool for quick diagnosis and epidemic surveillance of a border disease laboratory.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a fast, simple and real-time fluorescent quantitative RT-PCR kit capable of real-time quantitative detection of sheep border disease virus (BDV) and its application. Background technique [0002] Border disease (BD), named after it was first discovered in the border area between England and Wales, also known as Hairyshaker disease of lambs, is caused by the border disease virus in newborn lambs with hairy body, A congenital infectious disease characterized by poor growth and neurological abnormalities. [0003] Border disease virus (BDV) belongs to the Pestivirus genus of the Flaviviridae family, and is an enveloped single-stranded positive-sense RNA virus. Borderline disease virus has an immunosuppressive effect. After being infected with borderline disease virus during pregnancy, the virus can persist in various tissues in the body and become a carrier of the virus...

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): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107C12Q2527/107C12Q2531/113C12Q2545/113
Inventor 李军冯世文彭昊潘艳刘丽娅陈泽祥胡帅杨威钟舒红马春霞陶立谢永平
Owner GUANGXI VETERINARY RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap