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Freeze-dried microchip for identifying H9 and H6 subtype low-pathogenicity avian influenza viruses, kit and method thereof

A bird flu virus, low pathogenicity technology, applied in the field of virus molecular biology detection, can solve the problems of easy pollution, cumbersome operation of ordinary PCR, long detection time and so on

Active Publication Date: 2020-03-17
BEIJING YISEN BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ordinary PCR still has disadvantages such as cumbersome operation, long detection time and easy contamination.

Method used

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  • Freeze-dried microchip for identifying H9 and H6 subtype low-pathogenicity avian influenza viruses, kit and method thereof
  • Freeze-dried microchip for identifying H9 and H6 subtype low-pathogenicity avian influenza viruses, kit and method thereof
  • Freeze-dried microchip for identifying H9 and H6 subtype low-pathogenicity avian influenza viruses, kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0127] Experimental example 1 H9 and H6 subtype low pathogenic avian influenza virus freeze-dried microchip fluorescence RT-PCR detection kit opposite sex verification

[0128] 1. Design primers and Taqman-MGB probes

[0129] According to the detection of H9 and H6 subtype low-pathogenic avian influenza viruses found in China, find out the HA gene sequence, the specific conserved sequence of the low-pathogenicity H9 and H6 subtype low-pathogenic avian influenza virus HA gene, and design Multiple pairs of primers and probes. After comparison and screening, a set of optimal primers and a Taqman-MGB probe were finally determined.

[0130] Avian influenza H9 subtype virus upstream primer: 5'-CCACCCGCCGATGCT-3',

[0131] Avian influenza H9 subtype virus downstream primer: 5'-TGTTGCCACACTCGTTGTTGT-3',

[0132] Avian influenza H9 subtype virus Taqman probe:

[0133] 5'FAM-AGATCTGTACACGAGAACC-MGB-3';

[0134] Avian influenza H6 subtype virus upstream primer: 5'-GCCAGACTACTGAA...

experiment example 2

[0151] Experimental example 2, H9 and H6 subtype low pathogenic avian influenza virus freeze-dried microchip fluorescent RT-PCR detection kit and Sensitivity Verification and Comparison of Conventional Fluorescent RT-PCR Reagents

[0152] Set the concentration to 1 x 10 7 TCID 50 / mL of avian influenza H9N2 subtype virus liquid was subjected to 10-fold serial dilution. Extract 1×10 6 TCID 50 / mL~1×10 0 TCID 50 / mL Genomic RNA of avian influenza H9N2 subtype virus at each gradient concentration is used as a template, nuclease-free water is used as a negative control, and the freeze-dried microchip fluorescent RT-PCR reagent and conventional fluorescent RT-PCR reagent are used to detect avian influenza H9N2 subtype virus. Sensitivity testing.

[0153] The freeze-dried microchip fluorescent RT-PCR system was prepared according to Example 1. Add 10× buffer dilution to 24 μL of nuclease-free water for shaking and mixing, then add 0.6 μL to each well, add 600 μL of mineral...

experiment example 3

[0159] Experimental Example 3: Development of H9 and H6 Subtype Low Pathogenic Avian Influenza Virus Freeze-dried Microchip Fluorescent RT-PCR Detection Kit Preparation and detection

[0160] 1. Preparation of the kit:

[0161] Preparation of lyophilized microchips:

[0162] Prepare the freeze-dried microchip RT-PCR system according to the following reaction system:

[0163]

[0164]

[0165] The whole freeze-dried microchip sample hole of the AriaYSB microchip (Beijing Yisenbao Biotechnology Co., Ltd.) of fluorescent quantitative PCR is 30, and the above-mentioned fluorescent quantitative PCR system (comprising H9 and H6 subtype low pathogenic avian influenza virus 2 sets of primer probes), add 1.2 μL to each microchip well.

[0166] The microchips coated with PCR reagents were frozen at -80°C for 1 h. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the clapboard drops to -55°C, and the holding time is 1...

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Abstract

The invention discloses a freeze-dried microchip for identifying H9 and H6 subtype low-pathogenicity avian influenza viruses, a kit and a method thereof, which belong to the field of molecular detection. The freeze-dried microchip is characterized in that a fluorescent PCR reaction system used for identifying H9 and H6 subtype low-pathogenicity avian influenza viruses is fixed on the microchip through freeze drying; wherein the fluorescent PCR reaction system comprises the following primers and probes: an avian influenza H9 subtype virus upstream primer, an avian influenza H9 subtype virus downstream primer and an avian influenza H9 subtype virus Taqman probe; an avian influenza H6 subtype virus primer, an avian influenza H6 subtype virus upstream primer, an avian influenza H6 subtype virus downstream primer and an avian influenza H6 subtype virus Taqman probe. According to the invention, H9 and H6 subtype low-pathogenicity avian influenza viruses are simultaneously detected, and the detection method of the detection kit is high in accuracy, specificity and sensitivity and short in detection time.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of viruses, in particular to a freeze-dried microchip, a kit and a method for identifying H9 and H6 subtype low pathogenicity avian influenza viruses. Background technique [0002] The full name of avian influenza virus infection is avian influenza virus infection, which is an acute respiratory infectious disease caused by certain subtypes of influenza A virus. The genome is segmented single-stranded negative-sense RNA. According to the antigenicity of the outer membrane hemagglutinin (H) / neuraminidase (N) protein, it can be divided into 17 H subtypes (H1-H17) and 9 N subtypes (N1-N9) . Avian influenza viruses can be divided into highly pathogenic avian influenza viruses, low pathogenic avian influenza viruses and apathogenic avian influenza viruses. Low pathogenic avian influenza, also known as pathogenic avian influenza, non-highly pathogenic avian influenza and mild avian...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2521/107
Inventor 王新杰高姗姗孙晓明胡祥钰刘玉良韩焘王传彬杨林
Owner BEIJING YISEN BIOTECH
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