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GeXP rapid detection kit capable of identifying H5 subtype avian influenza virus and four different NA subtypes thereof at the same time

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

Active Publication Date: 2015-12-30
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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  • GeXP rapid detection kit capable of identifying H5 subtype avian influenza virus and four different NA subtypes thereof at the same time
  • GeXP rapid detection kit capable of identifying H5 subtype avian influenza virus and four different NA subtypes thereof at the same time
  • GeXP rapid detection kit capable of identifying H5 subtype avian influenza virus and four different NA subtypes thereof at the same time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 : Multiplex RT-PCR Primer Design for Avian Influenza Virus

[0024] Refer to relevant literature to download the sequences of avian influenza virus M, H5, N1, N2, N6 and N86 genes from the GenBank database, use DNAStar to analyze and compare the nucleotide sequences of each gene, and find out the suitable ones for the design of specific primers. Conserved region, utilize GeXPexpressprofiler tool to design the specific primer (seeing table 1) for 6 genes of avian influenza virus, the designed primer adopts PrimerPremier5.0, NCBIPrimerBlast and Oligo7.0 to analyze and screen, then in all forward primer and A non-homologous unique sequence was added to the 5' end of the reverse primer as a universal primer (Uni-Primer), and the 5' end of the upstream universal primer was labeled with a fluorescent dye Cy5, namely Cy5-Tag-F, synthesized by Shanghai Invitrogen Company, HPLC purification.

[0025] Table 1 Primer information

[0026]

[0027]

[0028] In Ta...

Embodiment 2

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

[0030] 2.1 Preparation of template and monoclonal plasmid standard containing target gene

[0031] According to the instructions of TaKaRa Company MiniBESTViralRNA / DNAExtractionKitVer.5.0 (catalogue number DV819A), extract nucleic acids of different subtypes (H1-16 and N1-9) of avian influenza virus and other poultry viruses, obtain 50 μL nucleic acid samples, and divide them into devices at -80°C save. The RT reaction system was carried out according to the instructions of TaKaRa reverse transcriptase (catalogue number D2639A), and the obtained RNA samples were reverse-transcribed according to the following reaction system and reaction conditions to obtain cDNA; DEPC water was used as the control of total RNA.

[0032] Reaction system (25 μL): 5×Reverse Transcriptase Buffer 5 μL, 50 mmol / L RandomPrimer (9mer) 1 μL, dNTPMixture (10 mM / L) 2 μL, 40URibonuclease Inhibitor 0.5 μL, 5U / μL MLVReverse Transcriptase 0...

Embodiment 3

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

[0048] Extract HA (1-16) and NA (1-9) subtype avian influenza virus nucleic acids from allantoic fluid according to MiniBESTViralRNA / DNAExtraction KitVer. In the GeXP multiple detection system established in Example 2, the specificity of the method was tested. After the multiplex PCR was completed, the PCR products were analyzed by GeXP capillary electrophoresis, and the results showed that only specific signals appeared in each reaction, and there was no cross-reaction. HA gene except H5 subtype, NA gene subtypes except N1, N2, N6 and N8 subtypes, as well as NDV, IBV, ILTV and blank control all had no reaction signal, suggesting that the established method has strong specificity, which is consistent with There was no cross-reaction with other test objects.

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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 poultry virus detection, and in particular relates to a GeXP primer set, a kit and an application thereof for simultaneously identifying H5 subtype avian influenza virus and four different NA subtypes thereof. Background technique [0002] Avian influenza is an acute infectious disease of poultry caused by avian influenza virus, and some subtypes of avian influenza virus can also infect humans. Avian influenza viruses can be divided into 16 HA subtypes and 9 NA subtypes according to the differences in the antigenic relationship between their 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. Especially in recent years, the H5 subtype avian influenza outbreaks have caused huge economic losses to the poultry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/70
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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