Target sequence for detecting RNA of influenza A H1N1 viruses and kit

A technology of influenza virus and kit, which is applied in the field of detecting the target sequence and kit of influenza A H1N1 influenza virus RNA, and can solve the problems of low detection fluorescence value, missed detection, insufficient sensitivity, etc.

Inactive Publication Date: 2011-05-25
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with these methods, fluorescent reverse transcription-polymerase chain reaction (RT-PCR) technology has the advantages of accuracy, sensitivity, simplicity and speed. Therefore, the current World Health Organization and the Ministry of Health recommend the use of fluorescent RT-PCR technology to detect influenza A (H1N1) Virus (2009) also announced the primer probe sequence. Due to the high variability of the virus and the lack of published sequences of virus strain genes that can be used for reference at the beginning of the design, the recommended primer probes have sequences and existing published sequences. The problem of gene sequence mismatch of most virus strains, resulting in low detection fluorescence value, insufficient sensitivity or even missed detection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of type A H1N1 influenza virus (2009) nucleic acid detection kit (PCR-fluorescent probe method)

[0038] A fluorescent RT-PCR kit for rapid detection of influenza A (H1N1) virus (2009) RNA was prepared by a conventional method, and the kit included: PCR buffer, RT / Taq mixed enzyme, negative control, and positive control. in:

[0039] (1) PCR buffer contains Tris-HCl, KCl, dNTPs, MgCl 2 , two pairs of primers (sequences such as SEQ ID No.3 and 4, SEQ ID No.6 and 7), two fluorescent probes (sequences such as SEQ ID No.5 and 8, a 5' end labeled FAM fluorescent group, The other 5' end is labeled with a HEX fluorescent group, and the 3' ends of both probes are labeled with a quenching group such as TAMRA or BHQ-1), sterile deionized water.

[0040] Specifically, the fluorescent RT-PCR reaction solution prepared by PCR buffer and RT / Taq mixed enzyme contains 10mM Tris-HCl (pH8.3), 50mM KCl, 3mM MgCl 2 , two pairs of primers at 0.35 μM, two pro...

Embodiment 2

[0049] Embodiment 2: the application of type A H1N1 influenza virus (2009) nucleic acid detection kit (PCR-fluorescent probe method)

[0050] (1) Sample processing

[0051] The samples used the influenza A (2009) influenza A virus (2009) RNA reference products distributed by the China Institute for the Control of Pharmaceutical and Biological Products (including positive reference products, negative reference products, minimum detection amount reference materials, etc., and the minimum detection amount reference products have 5 tubes containing 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 and 1×10 5 copy / ml concentration of influenza A H1N1 virus (2009) chicken embryo culture) and clinically collected throat swabs from patients with viral influenza symptoms. Pharyngeal swab samples collected clinically need to be pretreated, that is, they are first vortexed and mixed in the transport (preservation) solution, and the virus and virus-containing cells adhered to the swab are washed to o...

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Abstract

The invention provides a target sequence for detecting the RNA of influenza A H1N1 viruses (2009) and a kit. The invention is characterized in that: specific primer probes designed on the basis of the two nucleotide target sequences of influenza A H1N1 viruses are adopted to detect the RNA of the influenza A H1N1 viruses (2009) by a one-step fluorescent reverse transcription-polymerase chain reaction (RT-PCR) in a single reaction tube, and detect the existence of the RNA of the influenza A H1N1 viruses in a sample by the fluorescent signal intensity and circulating threshold of an amplification template. The kit comprises the following components: PCR buffer solution, RT / Taq mixed enzymes, a negative reference and a positive reference. The kit is used by two steps, namely, a sample processing step and an amplification detection step. The operation of the kit is simple, convenient and quick, and the sensitivity of the kit is high. The kit can be widely used disease control and quick detection of influenza A H1N1 viruses (2009) in clinic.

Description

technical field [0001] The invention belongs to a target sequence and a kit for detecting RNA of influenza A (H1N1) virus. Background technique [0002] Influenza A virus (Influenza A virus) is a linear single-stranded negative-sense RNA virus of the Orthomyxoviridae family. It is divided into many subtypes according to the virus surface hemagglutinin (HA) and neuraminidase (NA). There are 16 subtypes of lectin (H1~16), and 9 subtypes of neuraminidase (N1~9). Influenza A (H1N1) virus is one of the virus subtypes. Virus particles are polymorphic, generally spherical, with a diameter of 80-120nm and a capsule. There are many radially arranged protruding glycoproteins on the capsule, that is, fibrils and spikes, which are columnar hemagglutinin (HA), mushroom-shaped neuraminidase (NA) and M2 matrix protein on the membrane. Inside the virus particle is the nucleocapsid, which is helical and symmetrical, with a diameter of 10nm and a ring structure at both ends. It exists in th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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