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Method for determining avian influenza virus subtype by pyrosequencing technology

A technology of avian influenza virus and pyrosequencing, applied in biochemical equipment and methods, measurement/inspection of microorganisms, analysis through chemical reaction of materials, etc. Quantitative detection and typing, unable to realize simultaneous detection of different subtypes, etc., to achieve the effect of fast method and reliable subtype identification results

Inactive Publication Date: 2012-06-27
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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AI Technical Summary

Problems solved by technology

The traditional pathogen isolation and serological diagnosis methods are cumbersome and cannot realize the simultaneous detection of different subtypes, and the RT-PCR method cannot achieve the purpose of rapid, accurate, and high-throughput detection and typing of the virus. Integrated diagnostic technology for high-throughput rapid screening of influenza viruses and accurate identification of subtypes has important practical significance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Identify whether exported chicken products contain avian influenza virus subtypes

[0035] 1. Design specific primer sequences

[0036] 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 each subtype of avian influenza virus, which does not contain sequences of unknown base components. 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.

[0037] PCR amplification primer design and sequen...

Embodiment 2

[0071] Example 2: Identify whether the chickens in a breeding factory in Shandong are infected with avian influenza virus subtypes

[0072] 1. Design specific primer sequences

[0073] With embodiment 1.

[0074] 2. Extract the RNA of the sample to be tested:

[0075]Take the nasal cavity swab samples of the chickens in the farm and mix them fully on the mixer, squeeze out the liquid in the swabs with autoclaved tweezers, let them stand at room temperature for 30 minutes, take the supernatant, and use the RNA extraction kit (TaKaRa MiniBEST Viral RNA / DNAExtraction Kit Ver 3.0), and RNA was extracted according to the instructions.

[0076] 3. Perform RT-PCR amplification with primers for each subtype of avian influenza virus

[0077] With embodiment 1.

[0078] 4. Perform agarose gel electrophoresis

[0079] Perform agarose gel electrophoresis on the PCR product, take 2g of agarose, heat it in 100mL electrophoresis buffer, fully dissolve, add ethidium bromide stock solutio...

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Abstract

The invention belongs to the animal pathogeny detection technical field, relating to a method for determining avian influenza virus subtype by pyrosequencing technology. Firstly avian influenza virus H5, H7, H9, N1 and N2 specific primers and pyrosequencing primer are firstly designed; RNA of sample to be detected is extracted, then reverse transcription-polymerase chain reaction is carried out for amplification by utilizing each subtype primer; agarose gel electrophoresis is utilized to detect amplification product, if the lengths of H5, H7, H9, N1 and N2 primer amplified segments are 127bp,222bp, 174bp, 269bp and 101bp respectively, a pyrosequencing single chain template is prepared, then pyrosequencing reaction is carried out, and finally the subtype of the avian influenza virus in sample is determined according to the amplification result and pyrosequecing result. The invention combines the advantages of RT-PCR and DNA sequencing technology, and determination of specific sequenceis applied to meet the requirement of molecule diagnosis, so that subtype determination result is reliable and the method is rapid.

Description

Technical field: [0001] The invention belongs to the technical field of animal pathogen detection, and relates to a method for identifying avian influenza virus subtypes by adopting pyrosequencing (Pyrosequencing) technology. Background technique [0002] Avian influenza (avian influenza, AI) is a severe infectious disease that seriously endangers animal husbandry and human health. The spread of the disease can cause respiratory diseases and systemic sepsis in poultry. Almost all wild and domestic poultry can be infected. . Now, highly pathogenic avian influenza has been listed as a class A infectious disease by the International Office of Epizootics (OIE), and the Ministry of Agriculture of my country has also designated them as a class I animal disease. Avian influenza virus belongs to the Orthomyxoviridae family Influenzavirus genus, the diameter of the virus particle is 80nm-120nm, and it is spherical rod or long filament. The viral genome consists of eight negative-st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/76
Inventor 徐彪张太翔岳志芹孙涛赵玉然凌宗帅梁成珠
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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