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Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1 avian influenza virus

A bird flu virus and kit technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problems of high cost, time-consuming, unsuitable for grass-roots promotion and use, etc.

Active Publication Date: 2012-08-29
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional chicken embryo isolation method of virus is accurate and simple, but it takes too long, usually takes two days to a week; IFA and ELISA are fast but require special reagents; RT-PCR, RRT-PCR, Gene chips detection technology is fast and sensitive , but all require special instruments and technical personnel, and the cost is relatively high, so it is not suitable for promotion and use at the grassroots level

Method used

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  • Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1 avian influenza virus
  • Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1 avian influenza virus
  • Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1 avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, set of primers

[0051] The loop-mediated isothermal amplification set of primers for detecting or assisting the detection of H1, H1N1 and H1N2 subtype avian influenza viruses in this embodiment is a set of loop-mediated isothermal amplification primers for detecting or assisting the detection of H1 subtype avian influenza viruses, detection The three sets of primers are composed of a set of primers for loop-mediated isothermal amplification or a set of loop-mediated isothermal amplification for auxiliary detection of avian influenza virus of subtype N1 and a set of primers for loop-mediated isothermal amplification for detection or auxiliary detection of avian influenza virus of subtype N2. Among them, the loop-mediated isothermal amplification set of primers for detection or auxiliary detection of H1 subtype avian influenza virus is composed of degenerate primer FIP-H1, degenerate primer BIP-H1, degenerate primer F3-H1, degenerate primer B3-H1 , degenerate ...

Embodiment 2

[0059] Embodiment 2. Optimization of reverse transcription loop-mediated isothermal amplification reagents for detection or auxiliary detection of H1 subtype, N1 subtype, and N2 subtype avian influenza viruses

[0060] In this example, strains A / Duck / Guangxi / 030D / 2009(H1N1) and A / Duck / Guangxi / D / 2011(H1N2) were used as the samples to be tested to test the following detection or auxiliary detection of H1 subtype, N1 subtype and N2 The effect of reverse transcription loop-mediated isothermal amplification reagents of subtype avian influenza virus:

[0061] Methods as below:

[0062] 1. RNA extraction of H1N1, H1N2 subtype avian influenza virus

[0063] Clinical isolates A / Duck / Guangxi / 030D / 2009(H1N1), A / Duck / Guangxi / D / 2011(H1N2) identified by hemagglutination test, hemagglutination inhibition test and sequence determination using TRIZOL method or RNA extraction reagent Box to extract total RNA.

[0064] 2. Optimization of RT-LAMP reaction conditions

[0065] The reagents that...

Embodiment 3

[0074] Example 3. Detection of chicken-derived samples using RT-LAMP kits for detection or auxiliary detection of H1 subtype, N1 subtype, and N2 subtype avian influenza viruses

[0075] A. In this example, the following RT-LAMP kits for detection or auxiliary detection of H1 subtype avian influenza virus are used for detection. The kit is a product obtained by assembling RT-LAMP reaction reagent A, positive control A, negative control and fluorescent reagent for detecting or assisting the detection of H1 subtype avian influenza virus.

[0076] Among them, 19 μl of RT-LAMP reaction reagent for detection or auxiliary detection of H1 subtype avian influenza virus (hereinafter referred to as RT-LAMP reaction reagent A) is composed as follows: 2.5 μl of 10-fold reaction buffer (20mM Tris-HCl, 10mMKCl, 10mM ( NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1%Triton X-100), 6mmol / L MgSO 4 , 1mmol / L betaine, outer primers F3-H1 and B3-H1 each 0.2μmol / L, inner primers FIP-H1 and BIP-H1 each 1.6μmo...

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Abstract

The invention discloses a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for an H1 avian influenza virus. The kit comprises a set of LAMP primers for detecting or assisting to detect the H1 avian influenza virus. The primers consist of a degenerate primer FIP-H1, a degenerate primer BIP-H1, a degenerate primer F3-H1, a degenerate primer B3-H1, a degenerate primer LF-H1 and a degenerate primer LB-H1. The nucleotide sequences of the primers are respectively shown by a sequence 1, a sequence 2, a sequence 3, a sequence 4, a sequence 5 and a sequence 6 in a sequence table. The RT-LAMP visual detection kit for the H1 avian influenza virus has the advantages that the specificity is strong, the sensitivity is high, instrument equipment and operations are simple, and results can be conveniently observed. The RT-LAMP visual detection kit for the H1 avian influenza virus is especially suitable for performing rapid diagnosis and early screening on the H1 avian influenza virus in basic-level veterinary stations, farms and frontier ports.

Description

technical field [0001] The invention relates to a RT-LAMP visual detection kit for H1 subtype avian influenza virus. Background technique [0002] Avian Influenza (AI) is the abbreviation of Avian Influenza, which is an infectious disease of poultry (poultry and wild birds) caused by Avian Influenza A virus of the family Orthomyxoviridae. According to the different antigenicity of influenza A virus surface glycoprotein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA), avian influenza virus (AIV) can be further divided into different subtypes, 16 kinds of HA have been found so far subtypes and 9 NA subtypes. AIV is an enveloped, single-stranded negative-strand, segmented RNA virus, and the viral genome consists of eight segmented negative-strand RNAs. The segmented nature of the AIV genome makes it possible that when two or more influenza viruses of different subtypes co-infect a cell, the nucleic acid fragments of the progeny viruses may undergo gene rearrangement...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 谢芝勋彭宜刘加波谢志勤庞耀珊邓显文谢丽基范晴
Owner GUANGXI VETERINARY RES INST
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