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PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Lolymorphism) primers for differentiating different genetic evolutionary branches of NA gene of avian influenza virus and assay method and application thereof

A technology of PCR-RFLP and avian influenza virus, which is applied in the field of zoonotic diseases, can solve the problems that molecular biological methods are not easy to distinguish, and achieve the effect of high accuracy and simple identification method

Active Publication Date: 2018-02-06
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the H5N6 subtype avian influenza virus containing these two subbranch NAs is prevalent, but the homology between the strains of the neuraminidase N6 gene is as high as 90%, and conventional molecular biology methods (such as common PCR) are not easy Distinguish, urgently need a method to identify the two sub-branch N6 genes

Method used

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  • PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Lolymorphism) primers for differentiating different genetic evolutionary branches of NA gene of avian influenza virus and assay method and application thereof
  • PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Lolymorphism) primers for differentiating different genetic evolutionary branches of NA gene of avian influenza virus and assay method and application thereof
  • PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Lolymorphism) primers for differentiating different genetic evolutionary branches of NA gene of avian influenza virus and assay method and application thereof

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Experimental program
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preparation example Construction

[0030] (2) Preparation of cDNA: Reverse transcribe the RNA extracted in step (1) into cDNA respectively;

[0031] (3) PCR amplification reaction:

[0032] Utilize upstream primer N6F: 5'-ACAGATGGTCCGGCAAACAAC-3' and downstream primer N6R: 5'-CATTCCTTGTTTGCCCAGTAGT-3' to carry out PCR amplification reaction to the cDNA obtained in step (2);

[0033] (4) Digestion of PCR products:

[0034] The PCR product of step (3) amplification utilizes RFLP to carry out EcoR I digestion reaction;

[0035] (5) Agarose gel electrophoresis:

[0036] Carry out agarose gel electrophoresis to the product of step (4) enzyme digestion gained; Observation agarose gel electrophoresis, the PCR product of subbranch 2 neuraminidase N6 gene amplification can be cut into 2 fragments, and a size is 351 bp, the size of the other one is 179bp; while the PCR product amplified by the subbranch 1 neuraminidase N6 gene has not changed after digestion with the restriction endonuclease EcoRI, that is, it is stil...

Embodiment 1

[0045] 1. Materials

[0046] 1.1 Experimental reagents and consumables: 200 μL PCR tubes were purchased from Corning; One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., Premix Taq TM (TaKaRa Taq TM Version 2.0plus dye)PCR kit and QuickCut TM EcoRI restriction endonuclease was purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0047] 1.2 Strains: subclade 1 neuraminidase N6 gene virus (ZZ03 strain), subclade 2 neuraminidase N6 gene virus (XZ16 strain), avian Tembusu virus (ATV), duck hepatitis A virus (DAHV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), duck reovirus (DRV), and egg drop syndrome virus (EDSV) were all preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0048] 2. Genetic evolution analysis of N6 gene, restriction site selection and primer design

[0049] 2.1 Genetic evolution analysis...

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Abstract

The invention relates to PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Lolymorphism) primers for differentiating different genetic evolutionary branches of the NA gene of avian influenza virus and an assay method and an application thereof. The sequences of the primers are: forward primer N6F: 5'-ACAGATGGTCCGGCAAACAAC-3'; and reverse primer N6R: 5'-CATTCCTTGTTTGCCCAGTAGT-3'. Theprimers are utilized to establish the PCR-RFLP assay method. The primers can amplify both the subbranch 1 neuraminidase N6 gene and the subbranch 2 neuraminidase N6 gene, restriction enzyme digestionis carried out by restriction endonuclease EcoRI digestion on amplified PCR products and whether restriction enzyme digestion products have one or two bands is observed according to agarose gel electrophoresis, and the two subbranch neuraminidase N6 genes are specifically differentially diagnosed. The assay method disclosed by the invention is simple and highly accurate, and can be applied to thedifferentiation of the two types of subbranch neuraminidase N6 genes.

Description

technical field [0001] The invention belongs to the field of animal epidemiology, and in particular relates to a PCR-RFLP primer for distinguishing different genetic evolutionary branches of the NA gene of avian influenza virus, a detection method and application thereof. Background technique [0002] Avian influenza (AI) is an acute and highly contagious disease caused by type A influenza virus that mainly affects poultry and wild birds. Syndrome or recessive infection and other disease types. At present, bird flu spreads all over the world, causing huge economic losses to the poultry industry of various countries. [0003] Type A influenza virus not only causes serious harm to the poultry industry, but also has important public health significance. Some subtypes of avian influenza virus can cause human infection, but usually this infection does not cause human-to-human infection continuous transmission. Avian influenza virus (AIV) belongs to the Orthomyxoviridae family ...

Claims

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Application Information

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IPC IPC(8): C12Q1/683C12N15/11
CPCC12Q1/683C12Q2531/113
Inventor 陈翠腾万春和黄瑜陈珍朱春华刘斌琼
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI