Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses

A bird flu virus and kit technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of high cost, unsuitable for grass-roots promotion and use, and long time consumption

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

Method used

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  • Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses
  • Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses
  • Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, complete set of primers

[0046]The set of primers for loop-mediated isothermal amplification for detection or auxiliary detection of H1N2 subtype avian influenza virus in this embodiment is composed of the complete set of primers for detection or auxiliary detection of H1 subtype avian influenza virus and the set of primers for detection or auxiliary detection of N2 Primer sets for loop-mediated isothermal amplification of subtypes of avian influenza virus. Among them, the set of primers for loop-mediated isothermal amplification for detection or auxiliary detection of H1 subtype avian influenza virus consists of degenerate primer FIP-H1, degenerate primer BIP-H1, degenerate primer F3-H1, degenerate primer B3-H1 , degenerate primer LF-H1 and degenerate primer LB-H1, and their nucleotide sequences are respectively Sequence 1, Sequence 2, Sequence 3, Sequence 4, Sequence 5 and Sequence 6 in the sequence listing (Table 1). Among them, F3-H1 and B3-H1 are ou...

Embodiment 2

[0052] Example 2. Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification Reagent for Detection or Assisted Detection of H1 Subtype and N2 Subtype Avian Influenza Virus

[0053] In this embodiment, strains A / Duck / Guangxi / 030D / 2009 (H1N1) and A / Duck / Guangxi / D / 2011 (H1N2) are used as samples to be tested for the following detection or auxiliary detection of H1 subtype and N2 subtype avian influenza The viral reverse transcription loop mediates the effect of isothermal amplification reagents:

[0054] Methods as below:

[0055] 1. RNA extraction of H1N2 subtype avian influenza virus

[0056] Total RNA was extracted from clinically isolated strain A / Duck / Guangxi / D / 2011 (H1N2) identified by hemagglutination test, hemagglutination inhibition test and sequence determination using TRIZOL method or RNA extraction kit.

[0057] 2. Optimization of RT-LAMP reaction conditions

[0058] Optimize the reagents that affect the efficiency of the amplification reaction wi...

Embodiment 3

[0065] Embodiment 3, utilize the RT-LAMP kit of detection or auxiliary detection H1 subtype, N2 subtype avian influenza virus to detect chicken source sample

[0066] A. In this example, the following RT-LAMP kits for detection or auxiliary detection of H1 subtype avian influenza virus were used for detection. The kit is a product obtained by assembling together RT-LAMP reaction reagent A, positive control A, negative control and fluorescent reagent for detection or auxiliary detection of H1 subtype avian influenza virus.

[0067] 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 10-fold reaction buffer (20mM Tris-HCl, 10mM KCl, 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 1.6μmol / L, loop prime...

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Abstract

The invention discloses a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses. The kit comprises a set of LAMP primers for detecting or assisting to detect the H1 avian influenza viruses and N2 avian influenza viruses. 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, a degenerate primer LB-H1, a degenerate primer FIP-N2, a degenerate primer BIP-N2, a degenerate primer F3-N2, a degenerate primer B3-N2, a degenerate primer LF-N2 and a degenerate primer LB-N2. The nucleotide sequences of the primers are shown by a sequence 1, a sequence 2, a sequence 3, a sequence 4, a sequence 5, a sequence 6, a sequence 7, a sequence 8, a sequence 9, a sequence 10, a sequence 11 and a sequence 12 in a sequence table. The RT-LAMP visual detection kit for the H1N2 avian influenza viruses 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 H1N2 avian influenza viruses is especially suitable for performing rapid diagnosis and early screening on the H1 avian influenza viruses and the H1N2 avian influenza viruses in basic-level veterinary stations, farms and frontier ports.

Description

technical field [0001] The invention relates to an H1N2 subtype avian influenza virus RT-LAMP visual detection kit. Background technique [0002] Avian influenza (Avian Influenza, AI) is the abbreviation of avian influenza, which is an infectious disease of poultry (poultry and wild birds) caused by the Orthomyxoviridae influenza virus type A avian influenza virus. According to the antigenicity of the surface glycoprotein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) of type A influenza virus, avian influenza virus (AIV) can be further divided into different subtypes, and 16 types of HA have been found so far. subtype and nine 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 different subtypes of influenza viruses co-infect a cell, the nucleic acid fragments of the progeny vi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
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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