Specific primers for detecting swine infuenza virus subtype H5 by using DHPLC (denaturing high performance liquid chromatography) and application thereof

A swine influenza virus, specific technology, applied in microorganisms, recombinant DNA technology, microorganism-based methods, etc., to achieve high-throughput screening, improve detection efficiency, and shorten detection time.

Inactive Publication Date: 2014-12-03
徐超
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a specific primer and application thereof for detecting swin

Method used

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  • Specific primers for detecting swine infuenza virus subtype H5 by using DHPLC (denaturing high performance liquid chromatography) and application thereof
  • Specific primers for detecting swine infuenza virus subtype H5 by using DHPLC (denaturing high performance liquid chromatography) and application thereof
  • Specific primers for detecting swine infuenza virus subtype H5 by using DHPLC (denaturing high performance liquid chromatography) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The steps of embodiment 1 (detection of the DHPLC method specificity of swine influenza H5 subtype) are as follows:

[0022] (1) Sequence analysis of SIV H5 subtype and design and synthesis of primers for swine influenza H5 subtype

[0023] Look up the HA gene sequence of swine influenza virus H5 subtype (ie the target gene sequence of SIV H5 subtype) on NCBI, the HA gene sequence is as follows:

[0024] Agtgaattggaatatggtaactgcaacaccaagtgtcaaactccaataggggcgataaactctagtatgccattccacaacatccaccctctcaccatcggggaatgccccaaatatgtgaaatcaaacagattagtccttgcgactgggctcagaaatagccctcaaggagagagaagaagaaaaaagagaggactatttggagctatagcaggttttatagagggaggatggcagggaatggtagatggttggtatgggtaccaccatagcaacgagcaggggagtgggtacgctgcagacaaagaatccactcaaaaggcaatagatggagtcaccaataaggtcaactcgatcattgacaaaatgaacactcagtt

[0025] BLAST analysis and comparison of gene sequences, using the primer design software Primer 5.0 to design a pair of primers, the specific sequence is as follows: the upstream primer SEQ ID ...

Embodiment 2

[0034] Embodiment 2 (detection of the sensitivity analysis of the DHPLC method of swine influenza H5 hypotype) step is as follows:

[0035] (1) Construction of SIV-H5 subtype recombinant plasmid

[0036] Extract the nucleic acid of the standard strain of swine influenza H5 subtype, perform PCR amplification with reference to the method of Example 1, recover the target PCR product, TA clone it in the pMD18-T plasmid, select positive recombinants, and use EcoR Ⅰ and Hind Ⅲ to carry out single, Double-enzyme digestion identification and sequence determination.

[0037] (2) Sensitivity test

[0038] Determination of swine influenza virus H5 subtype recombinant plasmid concentration with a micro-nucleic acid protein analyzer, diluted to 10 6 ,10 5 ,10 4 ,10 3 ,10 2 10 and 1 copies nucleic acid, carry out PCR amplification and DHPLC detection with reference to the method for embodiment 1, the sensitivity of research method, the result shows that DHPLC method can detect minimum...

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Abstract

The invention relates to specific primers for detecting swine infuenza virus subtype H5 by using DHPLC (denaturing high performance liquid chromatography) and application thereof. The specific primers are nucleotide sequences disclosed as SEQ ID NO.1 and SEQ ID NO.2. By adopting the PCR (polymerase chain reaction) amplification-nondenatured DHPLC combined analysis mode, the invention establishes a new method for quickly screening swine infuenza viruses. The method can quickly and automatically screen swine infuenza viruses at high flux, greatly enhances the detection efficiency, shortens the detection time, and has important clinical meanings for preventing and controlling outbreak of swine infuenza subtype H5.

Description

technical field [0001] The invention relates to the technical field of animal virus detection, in particular to a specific primer for detecting swine influenza virus H5 subtype by DHPLC and application thereof. Background technique [0002] Swine influenza is an acute, febrile, highly contagious respiratory infectious disease caused by swine influenza virus (SIV) of the Orthomyxoviridae family Influenzavirus A. Under the pressure of immune selection, swine influenza virus has many serotypes and frequent genetic variation. According to serological and etiological research reports, there are nine subtypes of H1N1, H1N2, H1N7, H3N1, H3N2, H3N6, H4N6, H5N1, H9N2 in the world, and H1N1, H1N2, H3N2, H5N1, H6N6 have been reported in my country and H9N2 and other subtypes, among which the H5 subtype swine influenza is more virulent and may lead to the prevalence of highly pathogenic avian influenza, and has become the main swine influenza subtype concerned by the animal husbandry in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6827C12Q1/701C12Q2600/112C12Q2565/137
Inventor 徐超袁萍张子宏杨娜李珂苗丽
Owner 徐超
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