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Fluorescent quantitative PCR detection method for influenza viruses

A fluorescent quantitative and influenza virus technology, applied in the field of quantitative PCR detection of influenza virus, can solve the problems of RNA extraction with many steps, unfavorable accurate analysis, unstable and easy to degrade RNA, etc.

Pending Publication Date: 2020-06-12
GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the detection of influenza virus, it is first necessary to use an RNA kit to extract RNA from the tissue standard sample (such as throat swab) to be tested. Due to the many steps of RNA extraction, and the RNA itself is unstable and easy to degrade, it is not conducive to accurate analysis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Extraction of RNA from tissue samples:

[0045] 1. Preparation of immunomagnetic beads:

[0046] 1) Preparation of influenza A virus immune magnetic beads: Weigh 8 mg of silica magnetic beads containing carboxyl groups, then add 12 mg of EDC and 7 mg of NHS in sequence, and stir for 16 hours at room temperature in the dark to form activated ester magnetic beads. Dissolve 50ug of anti-influenza A virus antibody in 6mL PBS, slowly add the above-mentioned activated ester magnetic beads into the anti-influenza A virus antibody solution, stir and react overnight at 4°C, and obtain the immune magnetic beads of influenza A virus antibody .

[0047] 2) Preparation of influenza B immune magnetic beads: Weigh 8 mg of silica magnetic beads containing carboxyl groups, then add 12 mg of EDC and 7 mg of NHS in sequence, and stir at room temperature for 16 hours in the dark to form activated ester magnetic beads. Take 50mg of anti-influenza virus antibody and dissolve it in 6mL PBS,...

Embodiment 2

[0055] Real-time fluorescent quantitative PCR:

[0056] 1. Design of primers and probes: Download multiple gene sequences covering domestic and foreign influenza A viruses and influenza B viruses from the NCBI gene bank, compare homology, and use Primer Express 3.0 software to design highly specific genes in their conserved regions The specific primers and Taqman probes, upstream primers and downstream primers and the corresponding specific probe sequences are as follows:

[0057] Upstream primer Influenza A H1-F: 5'-GAGCTAAGAGAGCAATTGA-3'

[0058] Downstream primer Influenza A H1-R: 5'-GTAGATGGATGGTGAATG-3'

[0059] Probe for Influenza A H1 P: 5'-Fam-TTGCTGAGCTTTGGGTATGA-3'-BHQ-1

[0060] Upstream primer Influenza A N1-F: 5'-TCCACGCCCTAATGATAA-3'

[0061] Downstream primer Influenza A N1-R: 5'-TTCTCCCTATCCAAACAC-3'

[0062] Probe Influenza A N1 P: 5'-Fam-ATCCTTTTACTCCATTTGCTCC-3'BHQ-1

[0063] Upstream primer Influenza A H3-F: 5'-AGCAAAAGCCTACAGCAA-3'

[0064] Downstrea...

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PUM

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Abstract

The invention discloses an fluorescent quantitative PCR detection method for influenza virus. The immunomagnetic beads and DEAE magnetic beads are mixed to obtain virus nucleic acid extraction magnetic beads; viral nucleic acid is extracted in a sample to be detected by using the obtained viral nucleic acid extraction magnetic beads; a high-resolution dissolution curve method combining a Taqman probe and a fluorescent quantitative PCR technology is adopted to perform fluorescent quantitative PCR amplification on extracted viral nucleic acid, so that typing detection of influenza viruses in a to-be-detected sample is realized. The method can be used for clinical rapid differential diagnosis, prevention and control during outbreak of epidemic situations, epidemiological monitoring of virusesand the like, and the method overcomes the defects that RNA extraction steps of tissue samples are various, sample virus RNA is caused, and the detection sensitivity is influenced.

Description

technical field [0001] The invention relates to the field of influenza virus quantitative PCR detection, more specifically, the invention relates to an influenza virus fluorescent quantitative PCR detection method for directly detecting type A and B influenza viruses. Background technique [0002] Influenza is abbreviated as influenza, which is a common, highly contagious, and fast-spreading acute respiratory infectious disease caused by influenza virus; contact with or with contaminated items. Clinically, it is characterized by systemic poisoning symptoms such as high fever, fatigue, headache, and body aches. [0003] Influenza virus belongs to the Orthomyxoviridae family and is an enveloped negative-strand RNA virus. Its viral genome includes 8 segments, which are single-stranded and negative-strand RNAs, encoding at least 10 or more proteins. Influenza viruses include three types: A (A), B (B), and C (C). Among them, type A is the most likely to mutate and can infect hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/6806C12R1/93
CPCC12Q1/701C12Q1/6851C12Q1/6806C12Q2531/113C12Q2561/101C12Q2563/143C12Q2563/149
Inventor 刘凤鸣马运国师凤华谭木秀
Owner GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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