Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology

A pyrosequencing, influenza virus technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, analysis by chemical reaction of materials, etc., can solve the problems of cumbersome methods, insufficient accuracy, cross-contamination, etc., Achieving the effect of low cost, small sample volume and easy operation

Inactive Publication Date: 2010-09-29
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The isolation and identification of viruses is time-consuming and laborious, requiring high test conditions, which cannot meet the needs of emergency epidemic prevention and control of acute infectious diseases; serological diagnostic methods mainly include hemagglutination and hemagglutination inhibition tests, neuraminidase inhibition tests, and And test, agar gel diffusion test, immunofluorescence, enzyme-linked immunosorbent assay, the method has the advantages of strong specificity and high sensitivity, but the method is quite cumbersome and not suitable for testing a large number of samples; molecular biology diagnostic methods Mainly including polymerase chain reaction (PCR), real-time fluorescence quantitative polymerase chain reaction, nucleic acid sequence amplification assay (NASBA) method, these methods are relatively fast and sensitive, but the accuracy is not enough, and the problem of cross contamination is relatively serious

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
  • Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology
  • Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology
  • Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Detect whether the exported pork products contain porcine influenza A H1N1 virus

[0029] 1. Design specific primer sequences

[0030] Log in to the National Center for Bioinformatics (NCBI) of the United States through the Internet to query and retrieve the published nucleotide sequences of swine influenza H1N1 virus HA and NA, which do not contain unknown base sequences, and search for the same year and the same place with closer homology Only one strain was selected, and the nucleotide sequences of the HA and NA genes of the selected samples were edited with Editseq and MegAlign software in the DNAStar 7.1 software package, compared one by one, and the default parameters of the Clustal W Method (software) were used to align all The selected HA and NA nucleotide sequences are homologously compared to find the conserved sequence region representing each subtype and the specific nucleotide sequence (target detection sequence) that characterizes this region. ...

Embodiment 2

[0054] Embodiment 2: Detect whether porcine A H1N1 influenza virus is infected in a certain breeding pig herd, sampling is pig nasal cavity swab

[0055] 1. Design specific primer sequences

[0056] With embodiment 1.

[0057] 2. Extract the RNA of the sample to be tested:

[0058] After the pig nasal cavity swab samples were fully mixed on the mixer, the liquid in the swab was squeezed out with autoclaved tweezers, and left at room temperature for 30 minutes, and the supernatant was taken, and the RNA extraction kit (TaKaRa MiniBEST ViralRNA / DNA Extraction Kit Ver 3.0), RNA was extracted according to the instructions.

[0059] 3. Carry out RT-PCR amplification with swine H1N1 influenza virus H1 and N1 primers

[0060] With embodiment 1.

[0061] 4. Perform agarose gel electrophoresis:

[0062]Perform agarose gel electrophoresis on the PCR product, take 2g of agarose, heat it in 100mL of electrophoresis buffer, fully dissolve, add ethidium bromide stock solution to a fina...

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 method for the detection of swine influenza A H1N1 virus based on pyrosequencing technology, belonging to the technical field of animal pathogen detection. The method comprises the steps: firstly designing specific primers and pyrosequencing primers of the swine influenza A H1N1 virus; extracting RNA of a sample to be detected and then amplifying respectively by H1 and N1 primers of the swine influenza A H1N1 virus; detecting the amplified product by agarose gel electrophoresis preparing a pyrosequencing single-chain template in case that the length of H1 primer-amplified fragment is 102bp and the length of N1 primer-amplified fragment is 249bp, implementing pyrosequencing reaction, and finally judging whether the swine influenza A H1N1 virus is contained in the sample according to the amplification result and the pyrosequencing result. The method can realize short DNA sequence analysis accurately and fast by using the pyrosequencing technology and has the characteristics of high flux, low cost, direct use of the product for sequencing, no need of secondary treatment, extremely simple and convenient operation and small number of required sample.

Description

Technical field: [0001] The invention belongs to the technical field of detection of animal pathogens, and relates to a method for detecting swine type A H1N1 influenza virus by adopting pyrosequencing (Pyrosequencing) technology. Background technique: [0002] Porcine influenza A (H1N1) virus is an acute respiratory disease in pigs caused by a series of influenza A viruses with variable antigenic structures. In the nearly 90 years since the influenza virus invaded pig herds, swine influenza has brought catastrophic losses to the swine industry all over the world, and its continuous antigenic variation makes it difficult to completely eliminate swine influenza. And because pigs can serve as intermediate hosts between avian influenza and human influenza, swine influenza virus can spread to humans, thus posing a potential threat to human health. The swine influenza A (H1N1) epidemic that broke out in 2009 not only posed a severe test to the world's public health, but also had...

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
IPC IPC(8): C12Q1/70C12Q1/68G01N21/76
Inventor 徐彪凌宗帅岳志芹张太翔孙涛辛学谦梁成珠
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU