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Combination of primers and probes for detecting avian influenza viruses and kit and detection method

A bird flu virus and kit technology, applied in the field of primer and probe combinations for detection of bird flu virus, can solve the problem of high reaction conditions

Pending Publication Date: 2020-06-19
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem of high reaction conditions of existing PCR diagnostic methods, the present invention provides a combination of primers and probes for detecting avian influenza virus

Method used

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  • Combination of primers and probes for detecting avian influenza viruses and kit and detection method
  • Combination of primers and probes for detecting avian influenza viruses and kit and detection method
  • Combination of primers and probes for detecting avian influenza viruses and kit and detection method

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Effect test

Embodiment 1

[0031] The embodiment of the present invention provides a combination of primers and probes for detecting avian influenza virus, the sequences are respectively:

[0032] Upstream primer: 5′-ACAATGGTGATGGAACTGATTCGGATGATAAA-3′;

[0033] Downstream primer: 5′-TCCCAGGATTTCTGCTCTCTCGCACTTGAT-3′;

[0034] Probe: 5'-AACATCCTCAAAGGGAAATTCCAAACAGCA-THF-CACAAAGGGCAATGA-3'.

Embodiment 2

[0036] Embodiments of the present invention provide a kit for detecting avian influenza virus, specifically comprising:

[0037] (1) Isothermal amplification primers and probes: the sequences are respectively:

[0038] Upstream primer: 5′-ACAATGGTGATGGAACTGATTCGGATGATAAA-3′;

[0039] Downstream primer: 5′-Biotin-TCCCAGGATTTCTGCTCTCTCGCACTTGAT-3′;

[0040] Probe: 5'-FAM-AACATCCTCAAAGGGAAATTCCAAACAGCA-THFCACAAAGGGCAATGA-P-3'.

[0041] (2) Base buffer, magnesium acetate (both are RT-RAA nucleic acid amplification reagents from Jiangsu Qitian Gene Biotechnology Co., Ltd.).

Embodiment 3

[0043] The embodiment of the present invention provides a method for detecting avian influenza virus, which is operated with the kit in embodiment 2. AIV (subtypes H5, H7, and H9) were preserved in the laboratory of Hebei Agricultural University. The steps are as follows:

[0044] (1) Recombinase-mediated isothermal nucleic acid amplification reaction

[0045] Use the viral genome DNA / RNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to extract the genome of the test object, use the cDNA obtained by reverse transcription as a template, and perform recombinase-mediated isothermal amplification according to the following reaction system and reaction conditions. Increased reaction:

[0046]

[0047] The reaction conditions are: 39°C for 30 minutes.

[0048] (2) Amplification product detection

[0049] Take 10 μL of the amplified product obtained in step (1) and add it dropwise to the detection area of ​​the LFD test strip (purchased from Hangzhou...

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Abstract

The invention relates to the technical field of virus detection and in particular relates to a combination of primers and probes for detecting avian influenza viruses and a kit and a detection method.A determinand with an avian influenza virus RNA (ribonucleic acid) segment can be massively amplified by using the primers and the probes through reverse transcription recombinase mediated isothermalnucleic acid amplification techniques under isothermal conditions, the reaction conditions are low, good specificity and high sensitivity are achieved, and convenience in clinical use can be achieved.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a combination of primers and probes for detecting avian influenza virus, a kit and a detection method. Background technique [0002] Avian influenza (Avian influenza, AI) is a viral infectious disease caused by avian influenza virus (AIV), which is generally susceptible to poultry, and has always caused huge economic losses to the poultry breeding industry in my country. AIV is a single-stranded negative-strand RNA virus with 8 independent RNA segments and many subtypes. The most common virus subtypes are H5, H7, and H9. AI is widely distributed, and the pathogen of the disease has been isolated all over the world. As a zoonosis, the disease not only causes serious harm to the poultry industry, but also poses a potential threat to human health. [0003] There are many laboratory diagnostic methods for AI, such as diagnosis by observing chicken embryo hemorrhage, growth r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q1/6804C12Q2521/507C12Q2565/625C12Q2563/131
Inventor 张铁王文静王春光张鹏翟向和孟凡国
Owner HEBEI AGRICULTURAL UNIV.
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