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Reagent set and method for detecting avian influenza viruses and chicken parvoviruses

A technology of avian influenza virus and chicken parvovirus, applied in the biological field, can solve the problems of increasing the difficulty of virus diagnosis, difficult and rapid differential diagnosis, and the development hazard of poultry industry, and achieve the effect of good specificity, intuitive detection, and good specificity

Active Publication Date: 2017-05-10
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the diseases caused by these two viruses have caused serious harm to the development of the poultry industry.
[0003] Pathogen isolation and identification is a routine method for the diagnosis of viral diseases, but this method takes a long time, and it is difficult to perform rapid differential diagnosis for mixed infections of multiple viruses
Due to the unique replication mode of ChPV, it is difficult to proliferate normally in chicken embryos and cells, further increasing the difficulty of diagnosis of the virus

Method used

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  • Reagent set and method for detecting avian influenza viruses and chicken parvoviruses
  • Reagent set and method for detecting avian influenza viruses and chicken parvoviruses
  • Reagent set and method for detecting avian influenza viruses and chicken parvoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1, Preparation of a complete set of reagents for detection or auxiliary detection of avian influenza virus and chicken parvovirus

[0072] The complete set of reagents for detection or auxiliary detection of avian influenza virus and chicken parvovirus provided by the present invention consists of a primer pair M-P for detection or auxiliary detection of avian influenza virus and a primer pair ChPV-P for detection or auxiliary detection of chicken parvovirus; M-P consists of The single-stranded DNA (M-F) shown in SEQ ID No.1 and the single-stranded DNA (M-R) shown in SEQ ID No.2 in the sequence listing consist of a band of 658bp that can be amplified from M-P ; ChPV-P consists of single-stranded DNA (ChPV-F) shown in SEQ ID No.3 and single-stranded DNA (ChPV-R) shown in SEQ ID No.4 in the sequence table, ChPV-P can be obtained from chicken A band with a size of 305bp was amplified from the virus. In SEQ ID No.3, S is C or G.

[0073] In the above kit of reagen...

Embodiment 2

[0074] Example 2, Optimization of detection conditions for a complete set of reagents for detection or auxiliary detection of avian influenza virus and chicken parvovirus

[0075] Extract genomic RNA of H1N1 subtype AIV and genomic DNA of chicken parvovirus (ChPV), and reverse transcribe the genomic RNA of H1N1 subtype AIV to obtain cDNA, a complete set of reagents for detection or auxiliary detection of avian influenza virus and chicken parvovirus The detection conditions were optimized.

[0076] 25 μL double PCR amplification system: 2×Easy Taq PCR Super Mix (Beijing Quanshijin Biotechnology Co., Ltd.) 12.5 μL; M-F and M-R; ChPV-F and ChPV-R; template (genomic cDNA of H1N1 subtype AIV 1 μL , Genomic DNA of chicken parvovirus (ChPV) was mixed in equal volumes) 2 μL, and made up to 25 μL with ultrapure water.

[0077] Reaction program: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, a total of 35 cycl...

Embodiment 3

[0090] Example 3, the specificity of the complete set of reagents for detection or auxiliary detection of avian influenza virus and chicken parvovirus

[0091] Extract H1-H16AIV (H1N1 subtype AIV, H2N3 subtype AIV, H3N2 subtype AIV, H4N6 subtype AIV, H5N2 subtype AIV, H6N6 subtype AIV, H7N2 subtype AIV, H8N4 subtype AIV, H9N2 subtype AIV, Genomic RNA of H10N3 subtype AIV, H11 subtype AIV, H12N5 subtype AIV, H13N6 subtype AIV, H14N5 subtype AIV, H15N9 subtype AIV and H16N3 subtype AIV) and Newcastle disease virus (NDV), and reverse transcription Obtain cDNA, extract the genomic DNA of chicken parvovirus (ChPV), and detect the specificity of the kit of reagents in Example 1.

[0092] According to the optimal reaction system in Example 2, the templates were replaced with template 1, template 2, template 3, template 4, ChPV genomic DNA 1, ChPV genomic DNA 2, H1N1 subtype AIV, H2N3 subtype AIV, H3N2 subtype AIV, H4N6 subtype AIV, H5N2 subtype AIV, H6N6 subtype AIV, H7N2 subtype AI...

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Abstract

The invention discloses a reagent set and a method for detecting avian influenza viruses and chicken parvoviruses. The reagent set for detecting or assisting in detecting the avian influenza viruses and the chicken parvoviruses comprises a primer pair named as M-P and a primer pair named as ChPV-P, the M-P comprises two single-chain DNA (deoxyribonucleic acid) shown as SEQ ID No.1 and SEQ ID No.2 in a sequence table, and the ChPV-P comprises two single-chain DNA shown as SEQ ID No.3 and SEQ ID No.4 in the sequence table. Experimental results show that the reagent set and the method for detecting or assisting in detecting the avian influenza viruses and the chicken parvoviruses are good in specificity and accuracy and high in sensibility and can be used for specifically detecting the avian influenza viruses and the chicken parvoviruses, and the sensibility of detecting the avian influenza viruses and the chicken parvoviruses is 100fg.

Description

technical field [0001] The invention relates to a set of reagents and a method for detecting avian influenza virus and chicken parvovirus in the field of biotechnology. Background technique [0002] Avian influenza (Avian influenza, AI) is the abbreviation of avian influenza, which is an infection of poultry (including poultry and wild fowl) caused by type A influenza virus. animal disease. According to the different pathogenicity of avian influenza, it can be divided into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI), among which highly pathogenic avian influenza is the most serious. It spread rapidly, resulting in extremely high mortality rates, and crossed the barrier of species infection, from poultry to humans. With the frequent outbreaks of avian influenza in my country and the world, the scope of the epidemic continues to expand, and the impact of avian influenza outbreaks on the economy and society has become a consensus, and it...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 谢芝勋何莹罗思思李孟谢丽基邓显文奉彬黄娇玲张艳芳
Owner GUANGXI VETERINARY RES INST
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