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Avian influenza virus H7 type RT-PCR (reverses transcription-polymerase chain reaction) detecting kit and detecting method

A technology of RT-PCR and avian influenza virus, which is applied in the field of RT-PCR detection kit and detection of avian influenza virus H7, can solve the problems of complicated operation and unsatisfactory detection speed, and achieve high sensitivity and convenient operation , strong specific effect

Inactive Publication Date: 2013-09-04
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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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 virus H7 type RT-PCR (reverses transcription-polymerase chain reaction) detecting kit and detecting method
  • Avian influenza virus H7 type RT-PCR (reverses transcription-polymerase chain reaction) detecting kit and detecting method
  • Avian influenza virus H7 type RT-PCR (reverses transcription-polymerase chain reaction) detecting kit and detecting method

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Embodiment 1

[0038] 1 Materials and methods

[0039] Virus strains and clinical specimens:

[0040] A H1N1, seasonal H1N1, A H3N1, avian influenza H5N1, and avian influenza H9N1 strains were isolated from Zhejiang Center for Disease Control and Prevention. The clinical samples were obtained from the throat swabs of patients suspected of the recent A(H7N9) virus outbreak in Zhejiang Province. The samples were collected and transported to the laboratory with ice.

[0041] 1.2 Primers and probes

[0042] Avian influenza H7 virus sequences from around the world were downloaded from the NCBI GenBank in the United States. Homology comparisons were carried out, and avian influenza H7 virus-specific primers and specific probes were designed in the HA gene region of all avian influenza virus genomes. The sequences are as follows:

[0043] Upstream primer H7-FP: 5'-CACTGCARAATAGAATACAGATWGAC-3'

[0044] Downstream primer H7-RP: 5'-GTGCACYGCATGTTTCCATT-3'

[0045] Specific probe H7-P: 5'-FAM-TTATG...

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Abstract

The invention relates to an avian influenza virus H7 type RT-PCR (reverses 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 virus H7 type fluorescence-quantitative RT-PCR detecting kit comprises deoxynucleotide triphosphate, MgCl2, an RT-PCR buffer solution, an avian influenza virus H7 gene 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 fluorescence-quantitative RT-PCR detecting kit also comprises an upstream primer, a downstream primer and a specific probe. The avian influenza virus H7 type fluorescence-quantitative RT-PCR detecting method comprises the following steps of: (1) extracting RNA of a sample; (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 a detection method for H7 type nucleic acid of avian influenza virus, which can be applied to laboratory emergency detection of outbreaks caused by H7 type of avian influenza virus. 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 pr...

Claims

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Application Information

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