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Primer system and method for amplifying avian influenza virus whole genomes

An avian influenza virus and whole-genome technology, applied in the field of molecular biology, can solve the problem of difficulty in detecting specific HA/NA primers for HI detection and other problems

Inactive Publication Date: 2016-03-02
ZHAOQING DAHUANONG BIOLOGIC PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This frequent variation and recombination brings difficulties to traditional HI detection and specific HA / NA primer detection methods

Method used

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  • Primer system and method for amplifying avian influenza virus whole genomes
  • Primer system and method for amplifying avian influenza virus whole genomes
  • Primer system and method for amplifying avian influenza virus whole genomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the adoption of sick material and the isolation of virus

[0046] 1) Collection of disease materials

[0047] Sterile collection of cloacal swabs and tracheal swabs of sick poultry; mixed samples of tissues such as trachea, lungs, intestinal tract and intestinal contents, liver, spleen, kidney and brain.

[0048] 2) Isolation of virus

[0049] The collected cloacal swabs and tracheal swabs were first immersed in sterilized saline, and the nasal swabs were squeezed repeatedly on the tube wall, then taken out, fully oscillated the liquid in the tube, added an appropriate amount of antibiotics, stood at room temperature for 30m, and centrifuged at 4000rpm for 5min , take the supernatant, quickly extract the total viral RNA with an RNA extraction kit, and store at -70°C.

[0050] The collected disease tissues, such as trachea and lung, were first ground with a sterilized glass grinder under aseptic conditions, made a 10% homogenate with Hanks solution, and...

Embodiment 2

[0053] Embodiment 2: One-step RT-PCR amplification of the whole genome of avian influenza virus

[0054]RT-PCR reaction system: 10.0 μL viral total RNA, 10.0 μL 5*buffer, 10.0 μL 10mMdNTP, 1.0 μL AIV -F, 1.0μLU AIV -R, 1.0 μL Enzyme, DEPCH 2 O to make up 50.0 μL;

[0055] Among them, U AIV -F and U AIV The concentrations of -R were all 10 pmol / μL.

[0056] RT-PCR reaction conditions: 58°C for 30 min, 94°C for 3 min, then add cycle: 94°C for 30 s, 48°C for 30 s, 72°C for 7.0 min; after 35 cycles, 72°C for 10 min. Take 5ul-RT-PCR product in the sample hole of 1.5% agarose gel, electrophoresis at 130V for 30min, there are 7 gradient bands detected, the amplification result of a certain strain of H9N2 subtype is as follows figure 1 shown. Sequencing after cloning or direct sequencing.

[0057] figure 1 Among them, the M1 lane is DL5000DNAMarker;

[0058] Lane 1 is the whole genome of a strain of H9N2 subtype, including PB2 / PB12341bp, PA2233bp, HA1742bp, NP1565bp, NA145...

Embodiment 3

[0063] Embodiment 3: Two-step method PCR amplifies the whole genome of avian influenza virus

[0064] 1) Synthesis of cDNA:

[0065] Use the RT primer AGCRAAAGCAGG as the reverse transcription primer to perform PCR on the total viral RNA to synthesize cDNA;

[0066] Wherein, the reaction liquid of this PCR is as shown in table 1:

[0067] Table 1 Genomic cDNA synthesis reaction solution

[0068]

[0069] After mixing the above reaction solution, put it in a water bath at 70°C for 5 minutes, cool on ice for 2 minutes, and add it to the system described in Table 2 for reaction:

[0070] Table 2 Genomic cDNA synthesis reaction system

[0071]

[0072] Place it at room temperature for 10 minutes, in a water bath at 55°C for 70 minutes, and react at 94°C for 5 minutes. The reaction product is directly amplified by PCR or stored at -20°C for later use to obtain cDNA.

[0073] 2) Amplification of 8 segments: using cDNA as a template, using the primer pairs of the above-me...

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Abstract

The invention provides a primer system and a method for amplifying avian influenza virus whole genomes. Specifically, the primer system comprises a primer pair, namely UAIV-F and UAIV-R, for amplifying the avian influenza virus whole genomes, as well as eight segmental primer pairs; and the primer pair comprises forward sequence: 5'-AGCRAAAGCAGG, and backward sequence: 3'-AGTAGAAACAAG. According to the first method, virus RNA is taken as a template, and one-step RT-PCR amplification is carried out by virtue of the UAIV-F and the UAIV-R, so as to obtain the avian influenza virus whole genomes; and according to the second method, amplification is carried out by taking virus RNA as a template so as to obtain cDNA and then amplification of eight segments of the avian influenza virus whole genomes is carried out respectively by virtue of the eight segmental primer pairs. The primer system and the method disclosed by the invention are applicable to amplification of all avian influenza virus whole genomes, including the genomes of the various subtype avian influenza viruses as well as quite conserved non-coding regions at two ends of the genomes; therefore, the invention is broad in applicability.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a primer system and method for amplifying the whole genome of avian influenza virus. Background technique [0002] Influenza virus, referred to as influenza virus for short, is the most harmful zoonotic pathogen. So far, it has not only brought catastrophic damage to poultry and animal husbandry, causing huge economic losses, but also often causes worldwide human influenza pandemics, seriously endanger human life and health. Influenza viruses are divided into three types: A, B, and C. Among them, type A influenza virus is the most harmful and widely distributed, and poultry is the sensitive host. The genome of type A avian influenza virus is divided into 8 segments PB2 / PB1 / PA / HA / NP / NA / M / NS, expressing 12 kinds of proteins, these 12 kinds of proteins are involved in virus adsorption, fusion, transcription and translation, and the new generation virus respectively. Each...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 陈瑞爱罗琴芳赖汉漳黄文科刘玉鹏张东霞张爱国刘郁夫
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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