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

Multiplex-PCR primer group and kit for simultaneously identifying H5 subtype avian influenza viruses and NA subtypes thereof, and detection method

An avian influenza virus and detection method technology, applied in the field of molecular detection, can solve problems such as no reports yet, and achieve the effects of good repeatability, simple operation and short duration

Inactive Publication Date: 2017-11-03
GUANGZHOU SOUTH CHINA BIOLOGICAL MEDICINE
View PDF6 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the four NA subtypes of N1, N2, N6 and N8 are more common among the popular H5 subtype avian influenza viruses in my country, and the outbreaks of different NA subtypes of H5 avian influenza have the characteristics of acute onset, rapid spread, and similar symptoms. Therefore, the specific NA subtype cannot be judged only by the clinical observation of the disease, and the current detection methods focus on the detection of HA. For HA that can detect H5 and its four common NA subtypes Multiplex PCR method has not been reported yet

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
  • Multiplex-PCR primer group and kit for simultaneously identifying H5 subtype avian influenza viruses and NA subtypes thereof, and detection method
  • Multiplex-PCR primer group and kit for simultaneously identifying H5 subtype avian influenza viruses and NA subtypes thereof, and detection method
  • Multiplex-PCR primer group and kit for simultaneously identifying H5 subtype avian influenza viruses and NA subtypes thereof, and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the design of the multiplex PCR primer composition of simultaneously distinguishing H5 subtype avian influenza virus and NA subtype thereof

[0073] According to the M, H5, N1, N2, N6 and N8 genes of avian influenza virus, download a large number of corresponding sequences from GenBank, analyze and compare the sequences of each gene fragment, find out the relatively conserved region of each gene fragment, and design specific primers, Then, the designed primers were analyzed and screened to obtain the multiplex PCR primer composition of the present invention (see Table 1), which was synthesized by Shanghai Invitrogen and purified by PAGE.

[0074] Table 1. Primer information

[0075]

[0076]

[0077] The primer information is as shown in Table 1, the combined base code R=A / G, Y=C / T, according to the different strains of the pathogens actually detected, the length of the actual amplified product obtained by detection can be within the length of the ex...

Embodiment 2

[0078] Embodiment 2, the kit for simultaneously identifying H5 subtype avian influenza virus and its NA subtype

[0079] A kit for simultaneously identifying H5 subtype avian influenza virus and its NA subtype, comprising multiple PCR primer compositions for simultaneously identifying H5 subtype avian influenza virus and its NA subtype as shown in SEQ ID NO: 1-12, the reaction solution, Ex Taq polymerase.

[0080] The molar concentration ratio of primer pair MU and ML, primer pair H5U and H5L, primer pair N1U and N1L, primer pair N2U and N2L, primer pair N6U and N6L, primer pair N8U and N8L in the multiplex PCR primer composition is 2:2: 1:1:2:1; the reaction solution contains 10×PCR buffer, 2.5mM MgCl 2 , 2.5mM dNTP, the volume ratio of the three is: 2:2:2.5.

Embodiment 3

[0081] Embodiment 3, the detection method of simultaneously distinguishing H5 subtype avian influenza virus and NA subtype thereof

[0082] (1) Nucleic acid extraction: TRIzol virus nucleic acid extraction method was used to extract viral nucleic acid. The specific steps were: 750 μL of TRIzol was fully mixed with 250 μL of virus sample allantoic fluid, and allowed to stand for 5 minutes; 200 μL of chloroform was added to be fully mixed and left for 5 minutes; 4 Centrifuge at 12000rpm for 10min, inhale about 450μL of the supernatant into a new clean EP tube, add 500μL of isopropanol, let stand for 20min, centrifuge at 12000rpm at 4℃ for 10min, discard the supernatant, add 1ML of 70% ice ethanol to the wall, and centrifuge at 12000rpm After 5 minutes, the supernatant was discarded, and 70% ice ethanol was repeatedly added to wash again. After the EP tube was inverted to dry, 25 μL of RNA-free water was added to elute to obtain the nucleic acid of the virus sample.

[0083] (2) ...

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 multiplex-PCR primer group and a kit for simultaneously identifying H5 subtype avian influenza viruses and NA subtypes thereof, and a detection method. Typing and detection primer compositions established aiming at genes M, H5, N1, N2, N6 and N8 of the avian influenza viruses are shown as SEQ ID NO:1-12. The primer group and the kit disclosed by the invention can simultaneously identify the H5 subtype avian influenza viruses and the NA subtypes thereof, are strong in specificity, high in sensitivity, small in cross reaction and good in repeatability and can distinguish the H5 avian influenza viruses with the different NA subtypes through multiplex-PCR amplification detection and intuitive characteristic stripe judgment. The multiplex-PCR primer group, the kit and the detection method disclosed by the invention have the benefits that the detection cost can be greatly reduced, the operation is simple, the used time is short, epidemic viruses of the H5 subtype avian influenza viruses are investigated, prevented and controlled, and a great significance in guaranteeing the healthy and sustainable development of the poultry industry is realized.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and more specifically relates to a multiplex PCR primer set, a kit and a detection method for simultaneously identifying H5 subtype avian influenza virus and its NA subtype. Background technique [0002] Avian Influenza (AI) is an acute infectious disease of poultry caused by avian influenza virus (AIV). At the same time, some subtypes of avian influenza virus can infect humans. Avian influenza virus is a negative-sense single-stranded RNA virus order Orthomyxoviridae Influenza virus belongs to type A influenza virus, its genome consists of 8 gene segments, namely PB2, PB1, PA, HA, NP, NA, M and NS, According to the antigenicity of its glycoprotein hemagglutinin (HA) and neuraminidase (NA), AIV can be divided into 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9). Avian influenza can be divided into highly pathogenic avian influenza and low pathogenic avian influenza according to its...

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): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 彭志仇微红叶贺佳魏榕钟文婷
Owner GUANGZHOU SOUTH CHINA BIOLOGICAL MEDICINE
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