Primers and method for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR

A real-time fluorescence quantitative technology for avian influenza virus, applied in the field of life sciences and biology, to achieve the effect of reducing the fatality rate, high accuracy and high specificity

Inactive Publication Date: 2015-03-04
杭州艾迪康医学检验中心有限公司
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  • Abstract
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Problems solved by technology

No definitive evidence of human-to-human transmission

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  • Primers and method for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR
  • Primers and method for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR
  • Primers and method for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR

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Effect test

Embodiment 1

[0047] A kit for detecting H7N9 avian influenza virus nucleic acid, including:

[0048] (i) RNA extraction reagent: QIAamp Viral RNA mini Kit;

[0049] (ii) Reagent for reverse transcription: 5×RT-Buffer, 100uM Primer Mix, 100U / ul ReverTra Ace reverse transcriptase, DEPC water;

[0050] (iii) Real-time fluorescence quantitative PCR reagents: qPCR Mix, 50×ROX, 2 pairs of primers and corresponding probes for amplifying sample cDNA, sterile water, of which:

[0051] Primers and probes for detecting H7N9 avian influenza virus envelope surface hemagglutinin H7:

[0052] H7-F: GGGWTTCACMTACAGYGGAATAAG

[0053] H7-R: ACAGGARCCATTTCATCTCTCYGC

[0054] H7-probe: FAM-CAACSAGTGCATGTAGGAGATCAGGWTCTTC-TAMARA

[0055] Primers and probes for detecting H7N9 avian influenza virus neuraminidase N9:

[0056] N9-F: TGARTGCAGGTTCTATGCTCTCA

[0057] N9-R: TGTATACTGTGGGYGGTGATGA

[0058] N9-Probe: HEX-CTCAAACGGAACAATACACGATAGGTCCCA-TAMARA

[0059] The using method of described kit comprises ...

Embodiment 2

[0064] Embodiment 2: detection method

[0065] 1. Extraction of RNA from samples to be tested

[0066] 1) Pipette 560ul of prepared buffer AVL (containing carrier RNA) into a 1.5ml centrifuge tube. (Adjust the buffer AVL-carrier RNA proportionally according to the actual amount of the sample).

[0067] 2) Add 140ul plasma, serum, urine, cultured cell supernatant or cell-free body fluid to the centrifuge tube containing buffer AVL-carrier RNA. Mediate for 15 seconds and mix well. (To ensure lysis efficiency, it must be mixed thoroughly to form a homogeneous solution. Samples that have been dissolved only once can still be used).

[0068] 3) Leave at room temperature for 10 minutes. (10 minutes is enough, prolonging the time will not increase the quality of the product. Buffer AVL can inactivate potential contamination and RNase).

[0069] 4) Centrifuge briefly, and throw the liquid on the cap back to the bottom of the tube.

[0070] 5) Add 560ul absolute ethanol (96%-10...

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Abstract

The invention discloses primers for detecting H7N9 avian influenza virus by using real-time fluorescence quantitative PCR. The primers include: (1) primers and a probe which are used for detecting avian influenza virus envelope hemagglutinin H7; and (2) primers and a probe which are used for detecting H7N9 avian influenza virus neuraminidase N9. By virtue of the primers disclosed by the invention, H7N9 avian influenza virus can be detected through the extraction of the virus RNA, reverse transcription and real-time fluorescence quantitative PCR. The primers and the method, disclosed by the invention, have the advantages of high accuracy, high sensitivity, high specificity and short detection window phase and can be used for buying more time for early diagnosis, early treatment, reduction of case fatality rate and epidemic situation control.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 201310129302.4 and the application date on April 12, 2013. technical field [0002] The invention belongs to the field of life science and biotechnology, in particular to a kit for detecting H7N9 avian influenza virus. Background technique [0003] Influenza viruses belong to the Orthomyxoviridae family (orthomyxoviridae), single negative-sense RNA viruses with envelopes and segmented genomes. According to the antigenicity of the nucleoprotein and the M1 protein on the inner side of the envelope, influenza viruses are divided into three types: A (A), B (B) and C (C). Influenza A virus is further divided into several subtypes according to the antigenicity of the two spikes on the envelope surface, hemagglutinin (HA) and neuraminidase (neuraminidase, NA), currently including subtypes H1 to H16, and NA includes N1 ~ N9 subtype. All human influenza viruses can caus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/112
Inventor 陈奕磊李文静王淑一黎飒徐建成
Owner 杭州艾迪康医学检验中心有限公司
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