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Method for detecting H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as H9-subtype avian influenza virus

An avian influenza virus, highly pathogenic technology, applied in the field of virus detection, can solve the problems of specificity, decreased sensitivity, false negative, difficult to distinguish, etc., to achieve the effects of high sensitivity, high hardware requirements, and short detection time

Active Publication Date: 2019-05-07
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The rapid variation of HA nucleotides increases the difficulty of detection methods using nucleotides as target genes, resulting in a decrease in the specificity and sensitivity of detection methods using HA genes as target genes, resulting in false negative results
[0004]After the H7N9 virus mutated into a highly pathogenic strain, it caused huge economic losses to the poultry industry
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
Although the H9 subtype avian influenza virus is a low pathogenic strain, single infection will not directly kill poultry, but mixed infection with other pathogens can increase the mortality of infected poultry

Method used

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  • Method for detecting H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as H9-subtype avian influenza virus
  • Method for detecting H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as H9-subtype avian influenza virus
  • Method for detecting H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as H9-subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Determination of conserved sequence regions and screening of primer probes

[0040] 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:

[0041] ATGGAAAAARAACGTYACTGTWACRCATGCYMAAGACATACTGGARAAGACACAYAAYGGGARGCTYTG;

[0042] nt210–nt242 region, the sequence is

[0043] ATTGYAGTGTRGCWGGATGGCTHCTYGGRAAYCC has a higher degree of conservation; wherein, R=A or G, Y=C or T, M=or C, W=A or T.

[0044] Table 1: H5 virus strain information table used in the design of primers and probes in the present invention

[0045]

[0046]

...

Embodiment 2

[0082] Embodiment 2: Establish detection method

[0083] After establishing the detection reaction system and reaction conditions, 25 μl of 2×RT-PCR reaction buffer (containing dNTPs, Mg 2+ ), the upstream primer 2.0 μl (concentration is 10 μmol / L) formed in the first step, the downstream primer 2.0 μl (concentration is 10 μmol / L) formed in the first step, and the probe 1.5 μl formed in the first step μl (concentration 10μmol / L), enzyme mixture (reverse transcriptase, RNase inhibitor, Taq enzyme with 5'→3'exocutting activity) 2.5μl, viral nucleic acid to be detected 6.0μl (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 collected during the annealing extension of the...

Embodiment 3

[0089] Embodiment 3: the effect detection of primer and probe

[0090] 1. Using the primer probes and methods established by the above screening, the sensitivity test of H5 and H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus was carried out, including the following steps:

[0091] The first step: extract viral RNA, and measure the viral RNA content with a micro-nucleic acid analyzer. The RNA was diluted 10 times, and 6.0 μl of the diluted RNA template was taken and added to 44.0 μl of qRT-PCR master mix;

[0092] The second step: use the established real-time fluorescent quantitative RT-PCR method to detect and determine its sensitivity;

[0093] The result shows that the real-time fluorescence 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, can detect the 0.01fg H9 virus RNA template ( Figure 5 ).

[0094] 2. The ...

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Abstract

The invention provides a rapid detection method capable of synchronously detecting and differentiating H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as H9-subtype avianinfluenza virus. By analyzing nucleotide sequences of HA genes of an H5-subtype highly pathogenic avian influenza virus, an H7N9-subtype highly pathogenic avian influenza virus and an H9 subtype avian influenza virus isolated in recent years, as well as designing primers and probes in conservative regions, a rapid real-time fluorescence quantitative RT-PCR nucleic acid detection method for the H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as the H9-subtype avian influenza virus is established. The rapid real-time fluorescence quantitative RT-PCR nucleic acid detection method for the H5-subtype and H7N9-subtype highly pathogenic avian influenza viruses as well as the H9-subtype avian influenza virus provided by the invention has the advantages of being highin sensitivity, short in detection time, and free of open links such as electrophoresis and the like; moreover, only one fluorescent PCR instrument is required for accomplishing detection on the threesubtypes of the avian influenza viruses in a closed reaction tube. In addition, quick judgment on results is allowed for real-time viewing of response curve is feasible during the detection process.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a method for simultaneously rapidly detecting and distinguishing H5 and H7N9 subtype highly pathogenic avian influenza viruses and H9 subtype avian influenza virus. 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. H9 subtype avian influenza virus is a low pathogenic strain. [0003] However, it was found in the breeding process that vaccine immunization accelerated virus mutation, making it necessary to accelerate the replacement of vaccines. With the evolution of the virus, the H5 subtype avian influenza virus has been mutating rapidly; from the initial N28, Re-1 to Re-4, Re-5, then to Re-6, Re-8, to the latest Re-11, Re-12. Vi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 蒋文明刘华雷
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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