PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms) method for distinguishing clade2.3.2.1 from clade2.3.4 H5 AIVs (Avian Influenza Virus)

A technology of RT-PCR and amplification products, applied in the field of rapid diagnosis of veterinary infectious diseases, can solve problems such as difficult identification by molecular biology methods, and achieve the effects of rapid and accurate rapid differential diagnosis and detection, high accuracy, and simple identification methods.

Pending Publication Date: 2017-05-31
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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AI Technical Summary

Problems solved by technology

[0004] However, the gene sequence nucleotide homology rate between clade 2.3.2.1 and clade 2.3.4 is greater than 94.3% (taking the homology rate of classic strains as an example), it is difficult to pass conventional molecular biology methods ( as conventional RT-PCR method) for identification

Method used

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  • PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms) method for distinguishing clade2.3.2.1 from clade2.3.4 H5 AIVs (Avian Influenza Virus)
  • PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms) method for distinguishing clade2.3.2.1 from clade2.3.4 H5 AIVs (Avian Influenza Virus)
  • PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms) method for distinguishing clade2.3.2.1 from clade2.3.4 H5 AIVs (Avian Influenza Virus)

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Embodiment 1

[0026] 1. Extract the nucleic acid RNA of the virus;

[0027] (1) Virus source:

[0028] H5 subtype influenza virus clade 2.3.4 branch (CX1 strain) and clade 2.3.2.1 branch (DY1 strain) were both preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0029] (2) Extract viral RNA:

[0030] The nucleic acid RNAs of clade 2.3.4 branch (CX1 strain) and clade 2.3.2.1 branch (DY1 strain) H5AIV were extracted by conventional methods for RT-PCR amplification.

[0031] 2. RT-PCR amplification

[0032] (1) Design and selection of amplification primers:

[0033] According to the HA gene characteristics of clade 2.3.4 H5 AIV and clade 2.3.2.1 H5 AIV, the upstream primer P1 and downstream primer P2 were designed, wherein the sequences of the primers are as follows:

[0034] Upstream primer P1: 5'-ATGCAAACAACTCGACAG-3',

[0035] Downstream primer P2: 5'-ATCTCTGGTTTAGTGTTGATGT-3'.

[0036] Such as figure 1Shown: there are nuc...

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Abstract

The invention discloses a PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms) method for distinguishing clade2.3.2.1 from clade2.3.4 H5 AIVs (Avian Influenza Virus). The method comprises the following steps: 1) extracting a nucleic acid RNA of a virus from a detection sample; 2) using primers P1 and P2 to conduct RT-PCR amplification on the clade2.3.2.1 and clade2.3.4 viruses to obtain an HA gene segment; 3) conducting enzyme digestion on an RT-PCR amplification product through Dra I, and then conducting RFLP analysis. The method only needs the upstream primer P1 and the downstream primer P2, only the conventional restriction enzyme Dra I is needed in RFLP enzyme digestion to distinguish, no complex condition optimization process is needed, the method is convenient and accurate in the rapid differential diagnosis and detection of the clade2.3.2.1 and clade2.3.4 H5 subtype avian influenza viruses, and fills the research gap in the related fields at home and abroad.

Description

technical field [0001] The invention relates to the technical field of rapid diagnosis of veterinary infectious diseases, in particular to a PCR-RFLP method for distinguishing between clade 2.3.2.1 and clade 2.3.4 H5 subtype avian influenza virus (AIV). Background technique [0002] Avian influenza (AI) is an acute, highly contagious infectious disease caused by type A influenza virus that mainly affects poultry and wild birds. The infection caused by the highly pathogenic H5 subtype avian influenza virus (H5 AIV) is currently listed as an animal infectious disease that must be reported by the World Organization for Animal Health (OIE), and is listed as a first-class animal disease in my country. [0003] The earliest records about influenza can be traced back to a report from Italy in 1878, followed by outbreaks and epidemics of influenza in various parts of the world. In 1996, the first H5N1 subtype HPAIV (A / Goose / Guangdong / 1 / 96(H5N1), GS / GD / 96) was isolated in my country...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/683C12Q1/701C12Q2531/113
Inventor 万春和施少华黄瑜程龙飞傅光华陈红梅傅秋玲刘荣昌
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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