A gexp rapid detection kit for simultaneous identification of h5 subtype avian influenza virus and its four different na subtypes

A bird flu virus and kit technology, applied in the direction of recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as high mortality, unfavorable prevention and control and emergency measures, and acute onset, and achieve high sensitivity , fast sensitivity and strong specificity

Active Publication Date: 2018-11-09
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the outbreaks of different NA subtypes of H5AI have the common characteristics of acute onset, rapid transmission, similar clinical symptoms, and high mortality, it is difficult to make accurate judgments in a short period of time with existing diagnostic techniques. Conducive to the formulation of its prevention and control and emergency measures
At present, most of the nucleic acid detection methods for detecting H5 subtype AIV are aimed at its HA gene, and the technology of simultaneously detecting and genotyping H5, N1, N2, N6 and N8 genes has not been reported yet

Method used

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  • A gexp rapid detection kit for simultaneous identification of h5 subtype avian influenza virus and its four different na subtypes
  • A gexp rapid detection kit for simultaneous identification of h5 subtype avian influenza virus and its four different na subtypes
  • A gexp rapid detection kit for simultaneous identification of h5 subtype avian influenza virus and its four different na subtypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 :Design of multiple RT-PCR primers for avian influenza virus

[0024] Refer to the relevant literature to download the 6 gene sequences of avian influenza virus M, H5, N1, N2, N6 and N8 from the GenBank database, and use DNAStar to analyze and compare the nucleotide sequences of each gene to find suitable specific primers. Use the GeXP express profiler tool to design specific primers for the 6 genes of avian influenza virus (see Table 1). The designed primers are analyzed and screened using Primer Premier 5.0, NCBI PrimerBlast and Oligo 7.0, and then all A non-homologous unique sequence is added to the 5'end of the forward primer and reverse primer as a universal primer (Uni-Primer), and the 5'end of the upstream universal primer is labeled with a fluorescent dye Cy5, namely Cy5-Tag-F, produced by Shanghai Invitrogen Synthesized by the company and purified by HPLC.

[0025] Table 1 Primer information

[0026]

[0027]

[0028] In Table 1, the merged base code R=A / ...

Embodiment 2

[0029] Example 2: Establishment of multiplex PCR detection system

[0030] 2.1 Preparation of templates and monoclonal plasmid standards containing target genes

[0031] According to TaKaRa’s MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (Cat. No. DV819A) instructions, a 50μL nucleic acid sample was obtained by extracting nucleic acids from different subtypes (H1-16 and N1-9) avian influenza viruses and other avian viruses. The device is stored at -80°C. The RT reaction system was carried out according to the instructions of TaKaRa company reverse transcriptase (catalog number D2639A). The RNA samples obtained were subjected to reverse transcription according to the following reaction system and reaction conditions to obtain cDNA; DEPC water was used as a control for total RNA.

[0032] Reaction system (25μL): 5×Reverse Transcriptase Buffer 5μL, 50mmol / L RandomPrimer (9mer) 1μL, dNTP Mixture (10mM / L) 2μL, 40U Ribonuclease Inhibitor 0.5μL, 5U / μL MLV Reverse Transcriptase 0.5μL, tem...

Embodiment 3

[0047] Example 3: GeXP system multiple detection system specific detection

[0048] According to the MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 instructions, the HA (1-16) and NA (1-9) subtypes of avian influenza virus nucleic acid were extracted from the allantoic fluid, and the common avian viruses such as IBV, NDV and ILTV were also extracted. The nucleic acids were added to the GeXP multiple detection system established in Example 2 to test the specificity of the method. After the multiplex PCR was completed, the PCR products were analyzed by GeXP capillary electrophoresis on the machine, and the results showed that only specific signals appeared in each reaction without cross-reaction. In addition to the H5 subtype of the HA gene, NA subtypes other than the N1, N2, N6 and N8 subtypes of the NA gene, as well as NDV, IBV, ILTV and blank control, there is no response signal, indicating that the established method has strong specificity. No cross-reaction with other test s...

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Abstract

The invention belongs to the technical field of avian virus detection, and discloses a GeXP primer group and a kit capable of identifying an H5 subtype avian influenza virus and four different NA subtypes thereof at the same time. The inventor researches and designs six pairs of specific primers and one pair of universal primers based on a GeXP system, and builds the GeXP kit capable of identifying the H5 subtype avian influenza virus and four different NA subtypes thereof at the same time accordingly. Through adoption of the GeXP rapid detection kit, the H5 subtype avian influenza virus and the four different NA subtypes thereof can be identified at the same time, and the sensitivity is 102 copies per microliter. The GeXP rapid detection kit has the advantages of high flux, high specificity, high sensitivity, rapidness and the like, and has important significance to epidemiological investigation and differential diagnosis of the H5 subtype avian influenza virus.

Description

Technical field [0001] The invention belongs to the technical field of avian virus detection, and in particular relates to a GeXP primer set, kit and application for simultaneously distinguishing H5 subtype avian influenza virus and four different NA subtypes. Background technique [0002] Avian influenza is an acute infectious disease of poultry caused by avian influenza viruses, and certain subtypes of avian influenza viruses can also infect humans. Avian influenza viruses can be divided into 16 HA subtypes and 9 NA subtypes based on the difference in the antigenic relationship between the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). According to its pathogenicity, it can be divided into highly pathogenic avian influenza and low pathogenic avian influenza. Highly pathogenic avian influenza includes H5 and H7 subtypes. In particular, the frequent outbreaks of H5 subtype avian influenza in recent years have caused huge economic losses to the poultry industry ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/686C12Q1/70
Inventor 谢芝勋李孟谢志勤罗思思谢丽基黄莉邓显文黄娇玲张艳芳曾婷婷王盛
Owner GUANGXI VETERINARY RES INST
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