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Fluorescent RT-RAA primer for detecting A(H1N1) virus, and probe and detection method

A detection method and probe technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of complex primers, inability to directly clone and sequence, and achieve strong specificity and meet the epidemic situation. Rapid diagnosis and whole-process monitoring, the effect of reducing the fatality rate

Inactive Publication Date: 2018-01-12
宁波国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are real-time RT-PCR detection methods, semi-nested PCR detection methods, LAMP detection methods, etc., which have been established at home and abroad for the detection of influenza A (H1N1) virus, but the PCR method needs to use complex instruments and well-equipped laboratories, and The primers of the LAMP method are complex and must be designed with special software, and the amplification products obtained by the LAMP method are fragments of various sizes, which cannot be directly cloned and sequenced, and can only be used to determine the existence of the target gene

Method used

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  • Fluorescent RT-RAA primer for detecting A(H1N1) virus, and probe and detection method
  • Fluorescent RT-RAA primer for detecting A(H1N1) virus, and probe and detection method
  • Fluorescent RT-RAA primer for detecting A(H1N1) virus, and probe and detection method

Examples

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Embodiment 1

[0052] Embodiment 1, be used to detect the design of the fluorescent RT-RAA primer of influenza A H1N1 virus, probe

[0053] Aiming at the specific conserved region of influenza A H1N1 virus as the target region, design specific primers and fluorescent RAA probes, the sequences of which are:

[0054] Forward primer, such as the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing:

[0055] 5'-GCGAACAATTCAACAGACACTGTAGACACAGTAC-3'

[0056] Reverse primer, such as the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing:

[0057] 5'-CTAGGTGTTTCCACAATGTAGGACCATGAGC-3'

[0058] Oligonucleotide probe, such as the nucleotide sequence shown in SEQ ID No. 3 in the sequence listing:

[0059] 5'-CATTTGGGTAAATGTAACATTGCTGGCTGGA(FAM-dT)(THF)C(BHQ-dT)GGGAAATCCAGAGTG-3';

[0060] in:

[0061] FAM-dT represents a thymidine deoxynucleotide carrying a fluorescein group FAM (6-Carboxyfluorescein);

[0062] THF represents a tetrahydrofuran (tetrahydrofuran) linker...

Embodiment 2

[0064] Embodiment 2. Establishment of a fluorescent RT-RAA method for detecting influenza A (H1N1) virus

[0065] (1) Fluorescence RT-RAA reaction

[0066] 1) Extraction of sample RNA: The sample RNA can be extracted by using the viral RNA nucleic acid extraction kit from Kaiser Bioengineering Co., Ltd., Germany, according to the kit instructions.

[0067] 2) Using the forward primer, reverse primer and probe designed in Example 1, and the RAA reaction kit (this example specifically uses the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.), with this example Step 1) The prepared sample RNA to be detected is used as a template to perform an amplification reaction, wherein the reaction system is as follows:

[0068] 25 μl RAA reaction buffer (provided by the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.);

[0069] 2 μL each of the forward primer and reverse primer (10 μM) designed in Example 1;

[0070] 0.5 μL of the probe designed in Example 1 (10...

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Abstract

The invention provides a fluorescent RT-RAA primer for detecting an A(H1N1) virus by using a reverse transcription recombinase mediated nucleic acid amplification technique, a fluorescent probe, and an application of the fluorescent RT-RAA primer and the fluorescent probe in detecting the A(H1N1) influenza virus. By adopting the fluorescent RT-RAA primer and the fluorescent probe, the A(H1N1) influenza virus in a throat swab sample can be rapidly and quantitatively detected, detection can be implemented at normal temperature of 39 DEG C, diagnosis results can be obtained within 20 minutes, andthe detection time is greatly shortened; the fluorescent RT-RAA primer and the fluorescent probe are respectively good in specificity and relatively high in sensitivity, rapid diagnosis and whole monitoring on epidemic situations can be met, and time for diagnosing and treating epidemic situations at an early stage, reducing the case fatality rate and controlling the epidemic situations is achieved.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a fluorescent RT-RAA primer, a probe and a detection method for detecting influenza A (H1N1) virus in throat swab samples. Background technique [0002] Influenza A (H1N1) is an acute respiratory infectious disease, and its pathogen is a new type of influenza A (H1N1) virus that spreads among people. Unlike previous or current seasonal influenza viruses, this virus strain contains gene segments from three influenza viruses: swine flu, bird flu and human flu. The crowd is generally susceptible to influenza A (H1N1) virus and can be transmitted from person to person. The early symptoms of human infection are similar to common influenza, including fever, cough, sore throat, body pain, headache, chills and fatigue. Diarrhea or vomiting, muscle pain or tiredness, red eyes, etc. may also occur. Beginning in 2009, influenza A (H1N1) became widespread worldwid...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 周冬根罗洁应清界俞雪钧
Owner 宁波国际旅行卫生保健中心
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