A rapid detection method for distinguishing between strong and weak viruses of h7n9 subtype avian influenza virus

A bird flu virus, strong and weak technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the economic loss of poultry industry and other problems, achieve short detection time, high hardware requirements, long-term Effect

Active Publication Date: 2022-05-17
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Huge economic losses to the poultry industry after the H7N9 virus mutated into a highly pathogenic strain
Although the H7N9 attenuated virus has low pathogenicity to poultry, the virus can spread to the human population and can mutate into a highly pathogenic strain, posing a potential threat

Method used

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  • A rapid detection method for distinguishing between strong and weak viruses of h7n9 subtype avian influenza virus
  • A rapid detection method for distinguishing between strong and weak viruses of h7n9 subtype avian influenza virus
  • A rapid detection method for distinguishing between strong and weak viruses of h7n9 subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Determination of conserved sequence regions and screening of primer probes

[0031] The applicant studied the HA and NA nucleotide sequences (Table 1) of the virulent and attenuated H7N9 subtype avian influenza virus by molecular biology methods, and compared them with the HA sequences of the H7N9 strains isolated and preserved in the applicant's laboratory. Determine its conserved region ( figure 1 , figure 2 ). Sequence comparison found that the HA sequence of the strong and weak virus of H7N9 subtype avian influenza virus is in

[0032] nt907–nt956 region, the sequence of this region is as follows:

[0033] TTTCAGAACATWGAYAGCAGRRCARTTGGAAAATGYCCRAGATAKGTTAA;

[0034] nt964–nt995 region, the sequence is

[0035] AGTCTKCTGCTKGCWACAGGRATGAAGAATGT;

[0036] nt1028–nt1076 region, the sequence is

[0037] GAGGCCTRTTTGGTGCTATAGCDGGTTTCATTGAAAATGGATGGGAAGG is relatively conservative; wherein, R=A or G, Y=C or T, M=A or C, W=A or T, K=G or T.

[0038] Comp...

Embodiment 2

[0060] Embodiment 2: Establish detection method

[0061] After establishing the detection reaction system and reaction conditions, 25 μl of 2×RT-PCR reaction buffer (containing dNTPs, Mg 2+ ), 2.0 μl of upstream primers synthesized in the first step (concentration is 10 μmol / L), 2.0 μl of downstream primers synthesized in the first step (concentration is 10 μmol / L), 1.5 μl of probes synthesized in the first step (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 was 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 e...

Embodiment 3

[0067] Embodiment 3: the effect detection of primer and probe

[0068] 1. The sensitivity test of the H7N9 subtype avian influenza virus was carried out using the primer probes and methods established by the above screening, including the following steps:

[0069] Step 1: Extract the RNA of H7N9 strong and weak strains respectively, 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;

[0070] The second step: use the established real-time fluorescent quantitative RT-PCR method to detect and determine its sensitivity;

[0071] The results show that the real-time fluorescence quantitative RT-PCR method established by the present invention has a detection limit of 0.004fg RNA template to the H7N9 strong poisonous HA gene, and a detection limit of 0.1fg RNA template to the H7N9 attenuated HA gene. The lower limit of detection is 0.04fg RNA template...

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Abstract

The invention provides a rapid detection method for distinguishing the strong and weak viruses of H7N9 subtype avian influenza virus. By comparing the nucleotide sequences of the HA and NA genes of the H7N9 subtype avian influenza virus isolated in recent years, they are respectively designed in their conserved regions. Primers and probes were identified, and a real-time fluorescence quantitative RT-PCR nucleic acid rapid detection method that can distinguish between strong and weak H7N9 subtype avian influenza virus was established. The real-time fluorescent RT-PCR method for distinguishing the strong and weak viruses of the H7N9 subtype avian influenza virus provided by the present invention can detect and distinguish the strong and weak viruses of the H7 subtype avian influenza virus in the same reaction tube, and can also detect the N9 subtype bird flu virus. It has high sensitivity, short detection time, and does not require open links such as electrophoresis. It can be completed in a closed reaction tube with only one fluorescent PCR instrument, and the reaction curve can be viewed in real time during the detection process to make a quick judgment on the result.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a rapid detection method for distinguishing strong and weak viruses of H7N9 subtype avian influenza virus. Background technique [0002] In 2013, H7N9 virus was detected from human cases and poultry in my country, and animal experiments proved that the H7N9 virus isolated from poultry had no pathogenicity or low pathogenicity to poultry. However, with the evolution of the H7N9 virus, after more than three years, since the end of 2016, the H7N9 avian influenza virus has mutated from a weak strain to a strong strain, and H7N9 highly pathogenic avian influenza outbreaks have broken out in some areas. [0003] After the H7N9 virus mutated into a highly pathogenic strain, it caused huge economic losses to the poultry industry. Although the H7N9 attenuated virus has low pathogenicity to poultry, the virus can spread to humans and can mutate into a highly pathogenic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 蒋文明刘华雷
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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