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h5, h7, h9 subtype avian influenza virus detection kit

An avian influenza virus and detection kit technology, applied in the biological field, can solve problems such as unsatisfactory search results

Inactive Publication Date: 2011-11-30
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although there are many softwares that can help design primers, such as "Premier Primer 5.0", "Olio 6", "Vector N TI Suit", "DNAsis" and "DNAstar" all have primer automatic search functions, but the search results are not very ideal

Method used

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  • h5, h7, h9 subtype avian influenza virus detection kit
  • h5, h7, h9 subtype avian influenza virus detection kit
  • h5, h7, h9 subtype avian influenza virus detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Standard strains of H5, H7 and H9 subtypes of avian influenza virus are preserved by Harbin State Key Laboratory of Veterinary Biotechnology. Chicken Infectious Bronchitis Virus (IBV) H52, Chicken Infectious Bursal Virus (IBDV) B87, Chicken Infectious Laryngotracheitis Virus (ILTV) Vaccine Strain, Chicken Newcastle Disease Virus (NDV) Lasota Strain, Chicken Pullorum Salmonella (SP) CVCC528, preserved by the Preventive Veterinary Department of Jilin Agricultural University.

[0028] Trizol was used to extract 1 to 4 g of total RNA from virus or suspected avian influenza disease materials (lung, lymph node, spleen). During the extraction process, all utensils and consumables were treated with DEPC water.

[0029] (1) Centrifuge the collected allantoic fluid at 4000rpm for 30min at 4°C, discard the precipitate, transfer the supernatant to a new Eppendorf tube for ultracentrifugation at 10,000rpm at 4°C for 2h, collect the precipitate, and use 1 / 20 volume of the original al...

Embodiment 2

[0054] Artificially synthesized primers in Table 4;

[0055] Table 4 H9 subtype avian influenza virus nested PCR internal primers

[0056]

[0057] Get H9 subtype AIV and amplify by the PCR method of embodiment 1, get reaction product, press table 4 reaction system;

[0058] Table 5H9 subtype AIV nested PCR reaction system components

[0059]

[0060] According to the reaction conditions: after pre-denaturation at 94°C for 3 min, denaturation at 94°C for 45 s, annealing at 53°C for 1 min, extension at 72°C for 1 min, and a total of 30 cycles, then extension at 72°C for 10 min, and PCR amplification was terminated at 4°C. Take 5 μL of the PCR product, and perform electrophoresis on a 0.8 kg / L agarose gel to observe the amplification result. For electrophoresis results, see figure 1 .

Embodiment 3

[0061] Embodiment 3 specificity test

[0062] The RNA of H5, H7 and H9 subtype AIV and chicken infectious bronchitis virus (IBV) H52, chicken infectious bursal virus (IBDV) B87, chicken infectious laryngotracheitis virus (ILTV) vaccine strain, chicken Newcastle disease virus (NDV) Lasota strain, Salmonella pullorum (SP) CVCC528, and RNA and DNA from healthy poultry tissues were respectively amplified according to the methods in Examples 1 and 2 under the same conditions. Recover and purify the amplified fragments, send them to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequencing, analyze the sequencing results, and test the specificity of RT-PCR.

[0063] The cDNA of H5, H7 and H9 subtype AIV was amplified by PCR. As a result, a cDNA amplification band appeared at the position of 427 bp in the H5 subtype AIV, a cDNA amplification band appeared in the position of 312 bp in the H7 subtype AIV, and a cDNA amplification band appeared in the position of H9 sub...

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Abstract

The invention discloses an H5, H7 and H9 subtype avian influenza virus detection kit. In the GenBank, three kinds of subtype HA gene sequences are searched, and specific primers are designed. By many tests and actual detections, three pairs of primers and reaction systems thereof are screened out and optimized, and the sizes of the amplified cDNA (complementary deoxyribonucleic acid) fragments are respectively 427bp, 312bp and 826bp. The result shows that by optimizing multiple RT-PCR (reverse transcription-polymerase chain reaction) amplification conditions, a multiple RT-PCR detection kit capable of detecting three kinds of subtype avian influenza viruses at the same time is prepared, and the minimum detectable quantity is 10pg. The detection kit does not have cross reaction to various kinds of chicken infectious diseases and normal structures, has strong specificity, and has consistent sensibility to three kinds of subtype detections. The avian influenza can be diagnosed in a shorttime in a primary laboratory and can be positioned in a subtype so as to gain the time for preventing and controlling the avian influenza, thus the spread of the avian influenza can be controlled in a small range as much as possible by adopting measures in time.

Description

technical field [0001] The invention belongs to the field of biological technology, specifically a detection kit for H5, H7, H9 subtype avian influenza virus Background technique [0002] Avian influenza (Avian influenza, AI) is an acute and severe infectious disease of poultry caused by type A influenza virus of Orthomyxoviridae family. OIE is listed as a class A infectious disease. Avian influenza virus (AIV) belongs to type A influenza virus. Avian influenza virus (AIV) mutates frequently and has many serotypes. According to the difference of HA antigenicity, it is divided into 16 subtypes of HA. Strong antigen variability, wide range of hosts, and no cross-protection between subtypes lead to frequent outbreaks of avian influenza and become a devastating disease in the poultry industry. [0003] Highly pathogenic avian influenza is mainly caused by H5 or H7 subtype AIV, with a very short incubation period, sudden outbreak, and sudden death without any clinical symptoms...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 钱爱东张文惠李影王铁峰张旺
Owner JILIN AGRICULTURAL UNIV
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