A-type H5N6 subtype avian influenza virus dual-channel real-time fluorescence PCR (polymerase chain reaction) detection kit and detection method

Abstract

The invention discloses a detection kit for detecting A-type H5N6 subtype avian influenza virus by utilizing dual fluorescence PCR (polymerase chain reaction). The detection kit comprises a PCR primer for amplifying H5 and N6 genes and a probe, the nucleotide sequence is shown in Seq ID No: 1-6, a fluorescence reporter group is marked at the 5' terminal of the probe, and a fluorescence quenching group is marked at 3 terminal; a reaction system comprises an RT-PCR reaction liquid, an enzyme mixing liquid, a positive standard substance and a negative standard substance. The invention further discloses a method for detection by utilizing the kit. The method comprises steps such as extraction of sample nucleic acid, preparation of a reaction system, amplification through fluorogenic quantitative PCR, result reading and the like. The detection kit can rapidly detect the A-type H5N6 subtype avian influenza virus and has the advantages of simplicity in operation, high sensitivity, good specificity and the like, and an efficient detection means can be provided for monitoring and clinical diagnosis of the A-type H5N6 subtype avian influenza virus.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a type A H5N6 subtype avian influenza virus dual-channel real-time fluorescent PCR detection kit and detection method. Background technique [0002] Influenza virus is the pathogen of influenza, belongs to Orthomyxoviridae, and is an RNA virus. According to the difference in antigenicity of influenza virus nucleoprotein (Nucleoprotein, NP) and matrix protein 1 (Matrixprotein1, M1), influenza virus can be divided into A, Types B and C, of ​​which influenza virus type A is the only genus of Orthomyxoviridae, according to the antigenicity of hemagglutinin (HA) and neuraminidase (neuraminidase, NA) on the surface of influenza virus, influenza virus type A It can be divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). protein. Therefore, in theory, 198 subtypes of influenza viruses can be combined based on the currently known 18 HA and 11 NA subtypes. Human...

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Problems solved by technology

In 2004, JurgenA.Richt et al. used real-timeRT-PCR technology to carry out the molecular diagnosis of North American swine influenza virus type and subtype. This system can only diagnose one type or subtype at a time. In 2006, multiplexPCR technology was adopted Molecular diagnosis of respiratory viruses. In 2007, someone used 2 sets of multiplexPCR to carry out molecular diagnosis of 12 kinds of respiratory viruses, but both methods rely on gel electrophoresis for results analysis, and they have their own shortcomings such as poor sensitivity and easy cross-contamination

In 2007, real-time PCR was used for simultaneous molecular diagnosis of human avian influenza virus H5N1 subtype. In 2008, multiple real-time PCR began to be used for molecular diagnosis of common respiratory viruses. In 2009, multi-tube multiplex fluorescent quantitative PCR was used for detection New type A H1N1 influenza virus and seasonal influenza virus. In 2010, single-tube multiplex fluorescent quantitative PCR began to be used for influenza virus subtyping. However, due to the variability of influenza virus, there is no specific method for the newly discovered type A H5N6 influenza. Report on the single-tube multiplex fluorescent quantitative PCR kit and diagnostic method for viruses.

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