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Rapid detection method for distinguishing strong and weak toxicities of H7N9 subtype bird flu 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: 2019-04-26
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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  • Rapid detection method for distinguishing strong and weak toxicities of H7N9 subtype bird flu virus
  • Rapid detection method for distinguishing strong and weak toxicities of H7N9 subtype bird flu virus
  • Rapid detection method for distinguishing strong and weak toxicities of H7N9 subtype bird flu 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 strong and weak toxicities of H7N9 subtype bird flu virus. A real-time fluorescent quantitation RT-PCR nucleic acid rapid detection method for distinguishing strong and weak toxicities of H7N9 subtype bird flu virus is established by comparing nucleotide sequences of HA and NA genes of H7N9 subtype bird flu virus separated in recent years and respectively designing a primer and a probe in a conservation zone thereof. According to the real-time fluorescent quantitation RT-PCR nucleic acid rapid detection method for distinguishing strong and weak toxicities of H7N9 subtype bird flu virus provided by the invention, strong and weak toxicities of H7N9 subtype bird flu virus can be detected and distinguished in a same reaction tube and the H7N9 subtype bird flu virus can be detected; the rapid detection method has high sensitivity and short detection time; open links, such as electrophoresis, are not required; only a fluorescent PCR instrument is required for finishing the detection in an airtight reaction tube; during the detection process, a reaction curve can be checked in real time and a result 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 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 Applications(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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