Detection kit and detection method of reverse transcription polymerase chain reaction (RT-PCR) of avian influenza virus N9

An RT-PCR, avian influenza virus technology, applied in the field of laboratory emergency detection of influenza virus N9 type outbreaks, can solve the problems of insufficient detection speed, complicated operation, etc. simple steps

Active Publication Date: 2014-12-17
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Detection kit and detection method of reverse transcription polymerase chain reaction (RT-PCR) of avian influenza virus N9
  • Detection kit and detection method of reverse transcription polymerase chain reaction (RT-PCR) of avian influenza virus N9
  • Detection kit and detection method of reverse transcription polymerase chain reaction (RT-PCR) of avian influenza virus N9

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Embodiment 1

[0038] 1 Materials and methods

[0039] Virus strains and clinical specimens:

[0040] 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 come from the throat swabs and environmental samples of patients suspected of the A(H7N9) virus outbreak in Zhejiang Province. After the samples are collected, they are transported to the laboratory with ice.

[0041] 1.2 Primers and probes

[0042] Avian influenza 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 N9 virus-specific primers and specific probes were designed in the N9 gene region of all avian influenza virus genomes. The sequences are as follows:

[0043] Upstream primer N9-FP: 5'-ACAATACACGATAGRTCCCAGTA-3'

[0044]Downstream primer N9-RP: 5'-CTAGTACTTGACCACCCAATGC-3'

[0045] Specific pr...

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Abstract

The invention relates to a detection kit and a detection method of reverse transcription polymerase chain reaction (RT-PCR) of an avian influenza virus N9, and aims at providing the detection kit and the detection method, wherein the provided detection kit has the characteristics of being convenient to use and accurate to detect; and the provided method has the characteristics of being high in detection accuracy, and simple and convenient to detecting process. The technical scheme is as follows: the detection kit of fluorescent quantitative RT-PCR of the avian influenza virus N9 comprises deoxy triphosphate nucleoside, MgCl2, RT-PCR buffer solution, an avian influenza virus N9 genetic standard, a ribonucleic acid (RNA) enzyme inhibitor, moloney murine leukemia virus (MMLV) reverse transcriptase and deoxyribonucleic acid (DNA) polymerase. The detection kit of fluorescent quantitative RT-PCR is characterized by also comprising an upstream primer, a downstream primer and a specific probe. The detection method of fluorescent quantitative RT-PCR of the avian influenza virus N9 is carried out according to the following steps of: (1) extracting the RNA; (2) carrying out RT-PCR; and (3) carrying out fluorescence detection on an RT-PCR reaction product.

Description

technical field [0001] The invention relates to an influenza virus N9 type nucleic acid fluorescent quantitative RT-PCR detection kit and a detection method, which can be applied to laboratory emergency detection of influenza virus N9 type outbreaks. 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 infection with H7N...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor陈直平张严峻李辉
OwnerZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION