Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR

A real-time fluorescence quantitative and avian influenza virus technology, applied in the field of life sciences and biology, to achieve high accuracy, reduce the fatality rate, control the epidemic and gain time

Active Publication Date: 2013-08-07
杭州艾迪康医学检验中心有限公司
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  • Abstract
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  • Claims
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  • Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR
  • Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR
  • Kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR

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Embodiment 1

[0046] A kit for detecting H7N9 avian influenza virus nucleic acid, including:

[0047] (i) RNA extraction reagent: QIAamp Viral RNA mini Kit;

[0048] (ii) Reagent for reverse transcription: 5×RT-Buffer, 100uM Primer Mix, 100U / ul ReverTra Ace reverse transcriptase, DEPC water;

[0049] (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:

[0050] Primers and probes for detecting H7N9 avian influenza virus envelope surface hemagglutinin H7:

[0051] H7-F: GGGWTTCACMTACAGYGGAATAAG

[0052] H7-R: ACAGGARCCATTTCATCTCTCYGC

[0053] H7-probe: FAM-CAACSAGTGCATGTAGGAGATCAGGWTCTTC-TAMARA

[0054] Primers and probes for detecting H7N9 avian influenza virus neuraminidase N9:

[0055] N9-F: TGARTGCAGGTTCTATGCTCTCA

[0056] N9-R: TGTATACTGTGGGYGGTGATGA

[0057] N9-Probe: HEX-CTCAAACGGAACAATACACGATAGGTCCCA-TAMARA

[0058] The using method of described kit comprises ...

Embodiment 2

[0063] Embodiment 2: detection method

[0064] 1. Extraction of RNA from samples to be tested

[0065] 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).

[0066] 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).

[0067] 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).

[0068] 4) Centrifuge briefly, and throw the liquid on the cap back to the bottom of the tube.

[0069] 5) Add 560ul absolute ethanol (96%-10...

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Abstract

The invention discloses a kit for testing H7N9 bird flu virus using real time fluorescence quantitative PCR, which is characterized in that the kit comprises: (1) a RNA extraction reagent, a reverse transcription reagent and a real time fluorescence quantitative PCR amplification reaction reagent; (2) two pairs of primers H7-F, H7-R and N9-F and N9-R for amplification of sample, and corresponding probes N7-probe and N9-probe. The kit for testing H7N9 bird flu virus nucleic acid of the present invention can test H7N9 bird flu virus by virus RNA extraction, reverse transcription and real time fluorescence quantitative PCR, and has the advantages of high accuracy, high sensitivity, high singularity and short detection window period, etc., and can provide early diagnosis and early treatment of epidemic disease and reduced case fatality rate and time for epidemic situation control.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a kit for detecting H7N9 avian influenza virus. Background technique [0002] 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 cause avian influenza, but not all avian influenza viruses can cause human influenza. Among avian influenza viruses, H3, H5, H7, and H9 can infect humans, and H5 is highly pathoge...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
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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