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Detection method for universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses

A general-purpose technology for avian influenza virus, applied in the field of fluorescence quantitative RT-PCR detection, can solve the problem of not being able to cover the latest branches

Inactive Publication Date: 2019-11-22
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the previous influenza virus detection methods were aimed at a specific subtype, and could not cover the latest branch currently circulating in our country.

Method used

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  • Detection method for universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses
  • Detection method for universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses
  • Detection method for universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Design and screening of primers and probes

[0062] The applicant selects the relatively conservative M gene of influenza virus to design primers and probes for a general detection method, and selects the HA gene to design primers and probes for typing and detecting influenza viruses of H5 subtype, H7 subtype and H9 subtype.

[0063] Find and download 1641 publicly available M gene sequences containing currently reported avian influenza subtypes from the NCBI database, analyze the conserved regions based on gene alignment, and identify the bases at 124-275 after homology analysis (Reference sequence GenBank accession number: KU042441) Design upstream primers, downstream primers and probes for the target fragment, and perform combined screening for the best primer and probe set. figure 1 .

[0064] Find and download the HA gene sequence of 4636 published H5 subtype avian influenza viruses from the NCBI database, analyze the conserved region according to the g...

Embodiment 2

[0102] Embodiment 2: detection sensitivity and specificity of primer probe

[0103] 1) Detection sensitivity

[0104] Seven sets of RNA templates corresponding to the universal type, H5 subtype, H7 subtype and H9 subtype avian influenza virus with different concentrations were set up, and the nucleic acid amplification was carried out under the optimal conditions of fluorescent quantitative RT-PCR.

[0105] According to the instructions of the RNA extraction kit, extract the RNA of the general-purpose, H5 subtype, H7 subtype and H9 subtype avian influenza virus respectively, measure the original concentration of the extracted RNA template, dilute to 1ng / μL according to the ratio, and then dilute it in a 10-fold gradient into 10 - 1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL, take 2 μL respectively as the reaction template, and perform real-time fluorescence quantitative RT-PCR nucleic acid amplification according to the aforementioned sample ...

Embodiment 3

[0112] Embodiment 3: detection application to actual sample

[0113] 1. Sample collection:

[0114] A total of 90 throat swab samples of various types of poultry in a live poultry wholesale market were collected. PBS solution (pH7.0-7.4, 0.01mol / L) was used as the preservation solution (containing penicillin 2000IU / mL, streptomycin 2000IU / mL, nystatin 1000IU / mL, BSA5mg / mL). After collection, the samples were sealed in an incubator with ice and sent to the laboratory for processing or stored at -70°C within 24 hours.

[0115] 2. Sample Preparation

[0116] Place the cotton swab in a centrifuge tube filled with 1 mL of sample preservation solution, vortex and mix well, centrifuge at 10,000 r / min at 4°C for 5 min, and take the supernatant for nucleic acid extraction.

[0117] 3. Nucleic acid extraction

[0118] According to the instructions of the RNA extraction kit, 90 clinical samples to be tested, positive control and negative control RNA were extracted. The extracted RNA...

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Abstract

The invention aims to provide a quadruple real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection method for universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses so as to realize safe, specific, sensitive, rapid and convenient detection of the universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses. The invention provides a quadruple fluorescent RT-PCR detection method for rapidly detecting universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses based on molecular biology. Therefore, safe, specific, rapid,sensitive, simple and high-throughput rapid detection of universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses is realized, and the defects of the existing traditional detection technology are overcome. Moreover, the method is very suitable for high-throughput detection; a large number of samples can be detected at the same time and a result can be quickly judged; and pollutionand low specificity caused by a traditional SYBR Green staining method are avoided by using a TaqMan fluorescent probe.

Description

technical field [0001] The invention belongs to the technical field of fluorescent quantitative RT-PCR detection, in particular to a detection method for quadruple real-time fluorescent RT-PCR of general-purpose, H5 subtype, H7 subtype and H9 subtype avian influenza virus, including Primer pairs and probes for universal, H5 subtype, H7 subtype and H9 subtype avian influenza viruses. Background technique [0002] Avian influenza virus (AIV) is a single-stranded negative-sense RNA virus composed of 8 independent RNA segments. One of the remarkable biological characteristics is that it has many subtypes and frequent mutations. Thousands of strains with different virulence have been isolated from poultry so far. According to the antigenic differences of hemagglutinin (HA) and neuraminidase (NA) on the surface of their virions, they are divided into 18 HA subtypes. Types (H1-H18) and 11 NA subtypes (N1-N11). The pathogenicity of different subtype strains to the host is signific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2521/107C12Q2537/143C12Q2563/107C12Q2561/113
Inventor 王楷宬王素春黄保续
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT