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A Biotin Labeling Method for Detecting Genome-Vrna Interaction of H7N9 Subtype Avian Influenza Virus

A biotin-labeled, avian influenza virus technology, applied in the field of virus molecular biology, can solve the problems that have not been explored in vitro to rearrange the genome of H7N9 subtype influenza virus

Active Publication Date: 2021-05-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no effective technical system for exploring the vRNA-vRNA interaction of rearranged H7N9 subtype influenza virus genome in vitro

Method used

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  • A Biotin Labeling Method for Detecting Genome-Vrna Interaction of H7N9 Subtype Avian Influenza Virus
  • A Biotin Labeling Method for Detecting Genome-Vrna Interaction of H7N9 Subtype Avian Influenza Virus
  • A Biotin Labeling Method for Detecting Genome-Vrna Interaction of H7N9 Subtype Avian Influenza Virus

Examples

Experimental program
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Embodiment 1

[0050] Embodiment 1 quantitatively detects the method for H7N9 subtype avian influenza virus genome vRNA-vRNA interaction

[0051] This embodiment provides a method for quantitative detection of H7N9 subtype avian influenza virus genome vRNA-vRNA interaction (brief process as follows figure 1 shown), specifically include the following steps:

[0052] (1) Preparation of templates for in vitro transcription

[0053] 1. Primer design: 8 pairs of amplification primers were designed for the highly conserved regions of 8 genes (HA, NA, PB2, PB1, PA, NP, M, NS) of the H7N9 subtype influenza virus. The primers were all synthesized by entrusting merchants. The specific amplification primers for each gene are listed in Table 1.

[0054] Table 1 Specific primers used in PCR amplification of 8 genes

[0055]

[0056] 2. Extraction and reverse transcription of viral RNA

[0057] Viral RNA Extraction (Roche Viral RNA Extraction Kit)

[0058] (1) Add 200 μL virus liquid to 400 μL Bi...

experiment example 1

[0142] Utilize the method for the quantitative detection H7N9 subtype avian influenza virus genome vRNA-vRNA interaction that embodiment 1 provides to carry out the interaction analysis of the vRNA of NP gene and the vRNA of NA gene, the result is as follows image 3 Shown; Utilize the method for the quantitative detection H7N9 subtype avian influenza virus genome vRNA-vRNA interaction that comparative example 1 and comparative example 2 provide to carry out the interactive analysis of the vRNA of NP gene and the vRNA of NA gene, the result is as follows Figure 4 and Figure 5 shown. The results show that the method for quantitatively detecting the vRNA-vRNA interaction of the H7N9 subtype avian influenza virus genome provided by Example 1 is used to analyze the interaction between the vRNA of the NP gene and the vRNA of the NA gene, and the band detected by the vRNA is clear and complete, and the hybridization background It is clean and pollution-free, and can analyze the i...

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Abstract

The invention relates to the technical field of virus molecular biology, in particular to a biotin labeling method for detecting the interaction between vRNA-vRNA of H7N9 subtype avian influenza virus genome. The invention adopts biotin-labeled RNA pairs of H7N9 subtype avian influenza virus to be tested, and after the RNA pairs to be tested are denatured, hybridized and gel migrated, the interaction of the RNA pairs to be tested is analyzed by streptavidin binding reaction . The method of the present invention can obtain biotin-labeled genomic RNA fragments with higher purity and concentration, and can effectively ensure the specificity of hybridization and the clarity of vRNA hybridization bands, thereby significantly improving the detection efficiency of vRNA-vRNA binding and interaction Accuracy, to achieve quantitative detection of vRNA-vRNA interaction strength.

Description

technical field [0001] The invention relates to the technical field of virus molecular biology, in particular to a biotin labeling method for detecting H7N9 subtype avian influenza virus genome vRNA-vRNA interaction. Background technique [0002] Animal influenza virus is a major public health security problem that threatens human health. The generation and prevalence of H7N9 subtype avian influenza virus are closely related to the mutation and natural rearrangement of influenza virus. Previous studies have shown that the packaging of the eight vRNAs of influenza virus into the progeny virus genome is a specific selective packaging process, which is prone to mutation and rearrangement, resulting in new viral genotypes. Direct vRNA-vRNA interactions affect the selective packaging of eight gene segments in progeny viruses. [0003] Biotin labeling technology was initially used to label nucleic acids, and on this basis, a molecular hybridization technology for non-radioactivel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6816C12R1/93
CPCC12Q1/6816C12Q1/701C12Q2563/131C12Q2565/125
Inventor 蒲娟祖之鹏高卫华佟琪刘金华
Owner CHINA AGRI UNIV
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