Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof

A technology for influenza virus and fluorescence quantification, applied in the direction of fluorescence/phosphorescence, microbiological-based methods, biochemical equipment and methods, etc., can solve the problems of increasing biosafety risks by detecting positive references, and achieve highly specific effects

Active Publication Date: 2010-02-17
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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Problems solved by technology

[0009] According to the relevant regulations, the cell culture of the influenza A (H1N1) virus should be carried out in the BSL-3 laboratory, which increases the biosafety risk for the positive reference of the production influenza network laboratory.

Method used

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  • Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof
  • Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof
  • Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof

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Embodiment 1

[0045] 1 Materials and methods

[0046] Virus strains and clinical specimens:

[0047] Influenza A H1N1, SWH1N1, seasonal H1N1, and seasonal H3N1 virus strains were isolated from Hong Kong and Zhejiang Center for Disease Control and Prevention. The clinical samples were obtained from the throat swabs of suspected patients with the recent outbreak of H1N1 virus in Zhejiang Province. The samples were collected and transported to the laboratory with ice.

[0048] 1.2 Primers and probes

[0049] A(H1N1), seasonal H1N1, porcine H1N1 influenza virus strains from all over the world were downloaded from the NCBI gene bank in the United States. Homology comparisons were carried out, and influenza A H1N1-specific primers and Taqman probes were designed in the HA gene region of all influenza virus genomes. The sequences are as follows:

[0050] Upstream primer Type A H1N1-FP: 5'-AGGTTTGAGATATTCCCCAAGACA-3'

[0051] Downstream Primer Type A H1N1-RP: 5'-AATTTTTGTAGAAGCTTTTTGCTCC-3'

...

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Abstract

The invention provides a nucleic-acid fluorescent quantitative RT-PCR detection kit of H1N1 influenza virus A and a detection method thereof. The detection kit mainly comprises a specific primer, a fluorescent probe, a PCR buffer solution, a deoxidized nucleoside triphosphate compound, a DNA polymerase, a reverse transcriptase and an RNA enzyme inhibitor, wherein the specific primer and the fluorescent probe have the following sequences: an upstream primer H1N1 A-FP:5'-AGGTTTGAGATATTCCCCAAGACA-3'; a downstream primer H1N1 A-RP:5'-AATTTTTGTAGAAGCTTTTTGCTCC-3'; and a specific probe H1N1 A-P:5'-F-CATGGCCCAATCATGACTCGAACA-Q-3', wherein F is a fluorescent reporter group; and Q is a fluorescent quenching group. The invention has the advantages that the nucleic-acid fluorescent quantitative RT-PCR detection kit of the H1N1 influenza virus A and the detection method thereof can be applied to the emergent detection and the monitoring of a lab where the H1N1 influenza virus A causes an outbreakepidemic.

Description

(1) Technical field [0001] The invention relates to a nucleic acid fluorescent quantitative RT-PCR detection kit and a detection method of type A (H1N1) influenza virus, which can be applied to laboratory emergency detection of outbreaks caused by type A (H1N1) influenza virus. (2) Background technology [0002] Since March 18, 2009, there has been an outbreak of human infection with H1N1 influenza virus in Mexico. Since the first death case of human infection with influenza A H1N1 virus appeared in Mexico on April 13, 2009, as of May 5, 21 countries around the world have confirmed the existence of influenza A (H1N1) influenza cases through laboratory testing. The number of cases rose to 1,124, including 26 deaths. In addition to Mexico and the United States, many countries have reported confirmed cases of influenza A (H1N1). To this end, the World Health Organization (WHO) raised the global influenza pandemic warning level to level 4, and further raised it to level 5 on A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 张严峻
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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