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Avian influenza H7N9 virus RT-PCR (reverse transcription-polymerase chain reaction) detecting kit and detecting method

An RT-PCR and detection kit technology is applied in the field of avian influenza virus H7N9 nucleic acid fluorescence quantitative RT-PCR detection kits, which can solve the problems of complicated operation and unsatisfactory detection speed, and achieve high sensitivity and meet disease prevention and control requirements. Demand, detect fast effects

Inactive Publication Date: 2013-09-04
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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AI Technical Summary

Problems solved by technology

The 3 cases are all sporadic cases, and no epidemiological relationship between the 3 cases has been found so far
The disadvantage of the above detection method is that the detection speed cannot meet the requirements (usually takes about 5 days); and the operation is more complicated

Method used

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  • Avian influenza H7N9 virus RT-PCR (reverse transcription-polymerase chain reaction) detecting kit and detecting method
  • Avian influenza H7N9 virus RT-PCR (reverse transcription-polymerase chain reaction) detecting kit and detecting method
  • Avian influenza H7N9 virus RT-PCR (reverse transcription-polymerase chain reaction) detecting kit and detecting method

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Embodiment 1

[0055] 1 Materials and methods

[0056] Virus strains and clinical specimens:

[0057] 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 throat swabs and related environmental samples of patients suspected of the outbreak of H7N9 avian influenza virus in Zhejiang Province recently. The samples were collected and transported to the laboratory with ice.

[0058] 1.2 Primers and probes

[0059] Avian influenza H7 and N9 virus sequences from around the world were downloaded from the NCBI GenBank in the United States. Homology comparisons were carried out, and avian influenza H7N9 virus-specific primers and specific probes were designed in the HA and NA gene regions of all avian influenza virus genomes. The sequences are as follows:

[0060] Upstream primer H7-FP: 5'-CACTGCARAATAGAATACAGATWGAC-3'

[0061] Downstream primer H7-RP: 5'...

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Abstract

The invention relates to an avian influenza H7N9 virus RT-PCR (reverse transcription-polymerase chain reaction) detecting kit and a detecting method, and aims at providing the detecting kit which has the characteristics of convenience in use and accuracy in detection, and the detecting method which has the characteristics of high accuracy, simplicity and convenience in detection. The technical scheme is as follows: the avian influenza H7N9 virus fluorescence-quantitative RT-PCR detecting kit comprises deoxynucleotide triphosphate, MgCl2, an RT-PCR buffer solution, an avian influenza H7N9 virogene standard product, an RNA (Ribonucleic Acid) enzyme inhibitor, an MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and a DNA (Deoxyribonucleic Acid) polymerase, and is characterized in that the detecting kit also comprises an upstream primer, a downstream primer and a specific probe. The avian influenza H7N9 virus fluorescence-quantitative RT-PCR detecting method comprises the following steps of: (1) extracting RNA of a sample to be detected; (2) carrying out RT-PCR reaction; and (3) carrying out fluorescence detection on the RT-PCR reaction product.

Description

technical field [0001] The invention relates to a fluorescent quantitative RT-PCR detection kit and detection method of avian influenza virus H7N9 nucleic acid, which can be applied to laboratory emergency detection of outbreaks caused by avian influenza virus H7N9. Background technique [0002] Avian influenza virus belongs to Orthomyxoviridae Influenza A virus genus. Avian influenza A virus particles are pleomorphic, with a spherical diameter of 80-120nm and a capsule. The genome is segmented single-stranded negative-sense RNA. According to the antigenicity of the outer membrane hemagglutinin (H) and neuraminidase (N) proteins, it can be divided into 16 H subtypes (H1-H16) and 9 N subtypes (N1-N9). In addition to infecting poultry, avian influenza A virus can also infect humans, pigs, horses, mink and marine mammals. Previous studies have found that the subtypes of avian influenza viruses that can infect humans are H5N1, H9N2, H7N7, H7N2, and H7N3. At present, human in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 张严峻李辉茅海燕
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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