Detection method for H5 and H7N9 subtype high-pathogenicity avian influenza viruses

An avian influenza virus, highly pathogenic technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc. The effect of high sensitivity and high hardware requirements

Active Publication Date: 2019-04-02
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Huge economic losses to the poultry industry after the H7N9 virus mutated into a highly pathogenic strain
The symptoms of its infection are very similar to those of the H5 subtype highly pathogenic avian influenza virus, and it is difficult to distinguish them clinically

Method used

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  • Detection method for H5 and H7N9 subtype high-pathogenicity avian influenza viruses
  • Detection method for H5 and H7N9 subtype high-pathogenicity avian influenza viruses
  • Detection method for H5 and H7N9 subtype high-pathogenicity avian influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Determination of conserved sequence regions and screening of primer probes

[0034] The applicant studied the HA nucleotide sequences of different NA subtypes and different branches of the H5 subtype influenza virus by molecular biology methods (Table 1), and compared them with the HA sequences of H5 strains isolated and preserved in the applicant's laboratory , to determine its conserved region ( figure 1 ). Sequence comparison found that the HA sequence of the H5 subtype influenza virus is in the nt106–nt174 region, and the sequence of this region is as follows:

[0035] ATGGAAAAARAACGTYACTGTWACRCATGCYMAAGACATACTGGARAAGACACAYAAYGGGARGCTYTG;

[0036] In the nt210-nt242 region, the sequence is ATTGYAGTGTRGCWGGATGGCTHCTYGGRAAYCC, which is highly conserved; where, R=A or G, Y=C or T, M=or C, W=A or T.

[0037] Table 1: H5 virus strain information table used in the design of primers and probes in the present invention

[0038]

[0039]

[0040] Accordi...

Embodiment 2

[0056] Embodiment 2: Establish detection method

[0057] After establishing the detection reaction system and reaction conditions, 25 μl of 2×RT-PCR reaction buffer (containing dNTPs, Mg 2+ ), 1.5 μl of water, 2.0 μl of the upstream primer synthesized in the first step (the concentration is 10 μmol / L), 2.0 μl of the downstream primer synthesized in the first step (the concentration is 10 μmol / L), the probe synthesized in the first step 1.5 μl (concentration: 10 μmol / L), enzyme mixture (reverse transcriptase, RNase inhibitor, Taq enzyme with 5'→3' exo-cutting activity) 2.5 μl, viral nucleic acid to be detected 10.0 μl (from clinical samples or other samples extracted with a nucleic acid extraction kit); then the reaction system is sealed and placed on a fluorescent quantitative PCR instrument for reaction. Reaction conditions: first stage, reverse transcription 50°C / 10min; second stage, pre-denaturation 95°C / 2min; third stage, 95°C / 10s, 60°C / 30s, 40 cycles; Fluorescence was c...

Embodiment 3

[0063] Embodiment 3: the effect detection of primer and probe

[0064] 1. Sensitivity testing of H5 and H7N9 subtype highly pathogenic avian influenza viruses was carried out using the primer probes and methods established by the above screening, including the following steps:

[0065] The first step: extract viral RNA, and measure the viral RNA content with a micro-nucleic acid analyzer. Dilute the RNA by 10 times, take 10.0 μl of the diluted RNA template, and add it to 40.0 μl of qRT-PCR master mix;

[0066] The second step: use the established real-time fluorescent quantitative RT-PCR method to detect and determine its sensitivity;

[0067] The result shows that the real-time fluorescent quantitative RT-PCR method that the present invention establishes can detect the H5 virus RNA template of 0.1fg, can detect the virulent RNA template of 0.004fg H7N9 ( image 3 ).

[0068] 2. The specificity test of the real-time fluorescent quantitative RT-PCR detection method for H5 an...

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Abstract

The invention provides a fast detection method for detecting and distinguishing H5 and H7N9 subtype high-pathogenicity avian influenza viruses at the same time. The nucleotide sequences of HA genes ofan H5 subtype high-pathogenicity avian influenza virus and an H7N9 subtype high-pathogenicity avian influenza virus separated in recent years are respectively compared, a primer and a probe are designed at a conserved region, and a fast detection method for nucleic acid in accordance with the H5 and H7N9 subtype high-pathogenicity avian influenza viruses in a real-time fluorescent quantitative RT-PCR method is established. A real-time fluorescent RT-PCR method for the H5 and H7N9 subtype high-pathogenicity avian influenza viruses provided by the invention is high in sensitivity and short in detection time, open loops of electrophoresis and the like are not needed, only one fluorescent PCR apparatus is needed, and detection of two subtype avian influenza viruses can be completed in an airtight reaction tube; and besides, in the detection course, a reaction curve can be examined in a real-time manner, and results can be quickly judged.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a rapid detection method for simultaneously detecting and distinguishing H5 and H7N9 subtype highly pathogenic avian influenza viruses. Background technique [0002] Avian influenza viruses can be divided into highly pathogenic and low pathogenic avian influenza viruses according to their pathogenicity to poultry. Among them, highly pathogenic avian influenza viruses include partial strains of H5 and H7 subtypes. [0003] In 1996, my country isolated the H5N1 subtype highly pathogenic avian influenza virus for the first time from geese in Guangdong. Since late 2003, outbreaks of H5N1 subtype highly pathogenic avian influenza have occurred in poultry in many countries in Southeast Asia, including Vietnam, Thailand, South Korea, Japan, Cambodia and Indonesia. From 2004 to 2015, epidemics broke out in many provinces of my country, and more than 20 million poult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107
Inventor 蒋文明刘华雷
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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