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Multiplex fluorescence quantitative PCR detection kit for avian influnza virus N subtype genetic typing and application thereof

A technology of multiple fluorescence quantification and avian influenza virus, which is applied in the determination/testing of microorganisms, methods based on microorganisms, microorganisms, etc., can solve the problems of poor specificity and low sensitivity

Active Publication Date: 2017-10-24
广东省疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to overcome the defects of existing avian influenza virus N subtype genotyping detection kits such as poor specificity and low sensitivity, and provide a multiplex kit for avian influenza virus N subtype genotyping. Fluorescent quantitative PCR detection kit, which has the advantages of high throughput, easy operation, strong repeatability, rapid and objective detection results, etc., has a good application prospect for the subtype detection of avian influenza virus N subtype

Method used

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  • Multiplex fluorescence quantitative PCR detection kit for avian influnza virus N subtype genetic typing and application thereof
  • Multiplex fluorescence quantitative PCR detection kit for avian influnza virus N subtype genetic typing and application thereof
  • Multiplex fluorescence quantitative PCR detection kit for avian influnza virus N subtype genetic typing and application thereof

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Embodiment 1

[0066] Embodiment 1 Preparation of a nucleic acid typing detection kit for rapid detection of N subtype of avian influenza virus

[0067] The kit includes nucleic acid amplification reaction solution, enzyme mixed solution, avian influenza virus N subtype reaction solution A, avian influenza virus N subtype reaction solution B, avian influenza virus N subtype reaction solution C, positive control A, positive Control B, positive control C and blank control;

[0068] Wherein, the nucleic acid amplification reaction liquid includes Tris, potassium chloride, magnesium chloride, and nucleotide mixture;

[0069] Avian Influenza Virus N Subtype Reaction Solution A contains the following components:

[0070] Component (1): consists of six primers for detecting the N1-3 subtype of avian influenza virus; wherein, the base sequences of the six primers are SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, SEQ ID No.3, SEQ ID No. Shown in ID No.4, SEQ ID No.5 and SEQ ID No.6; The primer concentr...

Embodiment 2

[0087] Application of the kit of the present invention in the genotyping of N subtype of avian influenza virus of embodiment 2

[0088] 1. Extract sample RNA

[0089] 1.1 Take 200ul of clinical samples, add 400ul of Poly(A)-added Binding Buffer, mix thoroughly, transfer to a high-purification filter tube, centrifuge at 8000rmp for 15s, and discard the waste liquid in the collection tube.

[0090] 1.2 Add 500ul Inhibitor Removal Buffer to the filter tube, centrifuge at 8000rmp for 1min, and discard the waste liquid in the collection tube.

[0091] 1.3 Add 450ul Washing Buffer to the filter tube, centrifuge at 8000rmp for 1min, and discard the waste liquid in the collection tube.

[0092] 1.4 Repeat step 1.3, and then centrifuge at high speed for 10s. Be sure to remove the waste liquid in this step.

[0093] 1.5 Add 50ul Elution Buffer to the filter tube, let stand at room temperature for 2min, centrifuge at 8000rmp for 1min, and the solution obtained by centrifugation is the ...

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Abstract

The invention discloses a multiplex fluorescence quantitative PCR detection kit for avian influnza virus N subtype genetic typing and the application thereof. The kit comprises a nucleic acid amplified reaction liquid, an enzyme mixed liquid, an avian influnza virus N subtype reaction liquid A, an avian influnza virus N subtype reaction liquid B, an avian influnza virus N subtype reaction liquid C, a positive control A, a positive control B, a positive control C and a blank control. The kit overcomes the deficiencies in the prior art that most of avian influnza virus N subtype nucleic acid typing detection kits are poor in specificity and low in sensitivity, has the advantages of high throughput, high convenience in operation, high repeatability and rapid and objective detection results, and has a great application prospect in terms of clinical diagnosis and disease surveillance of avian influnza virus N subtype.

Description

technical field [0001] The invention relates to a nucleic acid detection kit and an application thereof, in particular to a multiple fluorescence quantitative PCR detection kit for subtype N genotyping of avian influenza virus and an application thereof, belonging to the field of biotechnology. Background technique [0002] Avian Influenza virus (AIV) is an RNA virus belonging to the family Orthomyxoviridae. Since it was discovered in 1997 that it can infect humans and cause death, it has attracted great attention in the world. The genome of avian influenza virus is composed of 8 independent single-strand negative strands, each encoding different proteins, and has a high degree of recombination. Among them, NA and HA have the strongest variability and are also the basis for dividing different subtypes of viruses. Typical symptoms after human infection with AIV are sudden high fever, dizziness and headache, cough (usually dry cough), fatigue, loss of appetite, muscle and jo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 柯昌文袁润余武婕王峥邹丽容杨静李秀林刘中华王国强
Owner 广东省疾病预防控制中心
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