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

One-step double-fluorescent quantitative RT-PCR detecting kit for duck tembusu virus and H9-subtype avian influenza virus

A technology for duck Tembusu virus and avian influenza virus is applied in the field of PCR detection of viruses, which can solve the problems of detection and diagnosis that have not yet been seen, and achieve the effect of high detection sensitivity

Inactive Publication Date: 2015-04-08
GUANGXI VETERINARY RES INST
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the detection and diagnosis of duck Tembusu virus and H9 subtype avian influenza virus using one-step real-time dual fluorescent quantitative RT-PCR

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
  • One-step double-fluorescent quantitative RT-PCR detecting kit for duck tembusu virus and H9-subtype avian influenza virus
  • One-step double-fluorescent quantitative RT-PCR detecting kit for duck tembusu virus and H9-subtype avian influenza virus
  • One-step double-fluorescent quantitative RT-PCR detecting kit for duck tembusu virus and H9-subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, design and synthesis of primers and Taqman probes

[0031] According to the conserved sequences of duck Tembusu virus and H9 subtype avian influenza virus in GenBank, multiple sets of probes and primers were designed by using Primer Premier 5.0 software, and two pairs of specific primers were selected by analyzing the dimers between the primers. Primers and two Taqmam probes (Table 1).

[0032] Table 1 Primers and TaqMan probe sequences (5'-3')

[0033]

[0034] Table 2 Standard Primer Sequence

[0035]

Embodiment 2

[0036] Embodiment 2, fluorescent quantitative PCR detection

[0037] 1. Determination of fluorescent quantitative PCR detection method

[0038] 1. Sample preparation

[0039] 1), nucleic acid extraction

[0040] Refer to the instructions of the TIANamp virus genome DNA / RNA extraction kit to extract the RNA of H9 subtype AIV, duck paramyxovirus, duck hepatitis virus and DTMUV, Muscovy duck parvovirus, duck circovirus, duck plague virus, and duck egg drop syndrome The DNA of the virus.

[0041] 2), reverse transcription of RNA

[0042] Duck Tembusu virus and H9 subtype avian influenza virus were reverse-transcribed to synthesize cDNA, specifically as follows: the following reverse transcription system was established with a total reaction volume of 20 μL, and the above 1) duck Tembusu virus RNA and H9 subtype avian influenza Viral RNA is 2 μL (approximately 20 μg), 4 μL 8mM MgCl 2 (Dalian Bioengineering Co., Ltd., catalog number: DRR0019A), 2 μL 10×PCR buffer (Dalianbao Bioen...

Embodiment 3

[0071] Embodiment 3, the assembly of detection kit

[0072] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

[0073] Liquid A: PCR amplification buffer, primer 1, primer 2, probe A, primer 3, primer 4, probe B; the final concentrations of primer 1, primer 2, and probe A in liquid A are all 0.25 μM, and primer 3 , Primer 4, and Probe B were all at a final concentration of 0.2 μM.

[0074] Liquid B: DTMUV+AIV-H9 template, as a positive control;

[0075] Solution C: ddH 2 O, as a negative control.

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

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention discloses a one-step double-fluorescent quantitative RT-PCR detecting kit for a duck tembusu virus and an H9-subtype avian influenza virus. The one-step double-fluorescent quantitative RT-PCR detecting kit comprises aprimer group and a probe group, wherein the primer group comprises primers 1, 2, 3 and 4 which respectively have a base sequence as shown in SEQ.ID.No.1 and 2 as well as SEQ.ID.No.4 and 5 in a sequence table; and the probe group comprises a probe A and a probe B which respectively have a base sequence as shown in SEQ.ID.No.3 and 6 in the sequence table. Experiments prove that the one-step double-fluorescent quantitative RT-PCR detecting kit has the advantages of short detection time, high detection sensitivity and strong specificity and can be used for simultaneously detecting and identifying two pathogens including the duck tembusu virus and the H9-subtype avian influenza virus and quantifying the corresponding pathogen contents of the duck tembusu virus and the H9-subtype avian influenza virus, so that the one-step double-fluorescent quantitative RT-PCR detecting kit can be used for evaluating the curative effects of vaccines and drugs for the duck tembusu virus and the H9-subtype avian influenza virus and researching the pathogenesis of the duck tembusu virus and the H9-subtype avian influenza virus, and furthermore the one-step double-fluorescent quantitative RT-PCR detecting kit has important significance for preventing and controlling the duck tembusu virus and the H9-subtype avian influenza virus.

Description

technical field [0001] The invention belongs to the technical field of virus detection by PCR, and in particular relates to a one-step dual fluorescence quantitative RT-PCR detection kit for duck Tembusu virus and H9 subtype avian influenza virus. Background technique [0002] Duck Tembusu Virus (DTMUV) was first discovered in Fujian, Zhejiang, Jiangsu and other major laying duck breeding areas in the east coast of China. It is a new pathogenic flavivirus. DTMUV infection of ducks can lead to a serious decline in egg production of laying ducks, and the egg production rate can drop from 90% to less than 10%, or even extinction; infection of ducklings can cause ducklings to appear unstable, falling down and other nerves Symptoms, which in turn lead to an increase in the elimination rate of ducklings, even as high as 80% in severe cases. Avian influenza virus (AIV) belongs to the Orthomyxoviridae Influenza A virus genus. According to the degree of virulence, AIV can cause diff...

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): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16
Inventor 谢芝勋赵虹谢丽基刘加波罗思思谢志勤邓显文黄莉黄娇玲曾婷婷
Owner GUANGXI VETERINARY RES INST
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