Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for H7N9 avian influenza virus

A RT-PCR and avian influenza virus technology, applied in the field of H7N9 avian influenza virus triple RT-PCR detection kit, can solve the problems of lack of correction function of RNA polymerase, high frequency of gene mutation of avian influenza virus, etc.

Active Publication Date: 2013-08-21
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Because the genome of avian influenza virus replicates in segments, it relies on RNA polymerase to complete the replication, and RNA polymerase lacks the correction function, so the frequency of gene mutation of avian influenza virus is extremely high

Method used

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  • Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for H7N9 avian influenza virus
  • Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for H7N9 avian influenza virus
  • Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for H7N9 avian influenza virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The design of embodiment 1 primer and the preparation of kit

[0017] 1. Design of primers

[0018] According to the conserved sequences of 3 genes including the HA gene of H7 subtype avian influenza virus, the NA gene of N9 subtype avian influenza virus and the M gene of all subtype avian influenza viruses published in GenBank, three genes were designed and synthesized through Blast verification. Pair-specific primers (Table 1).

[0019] Table 1 Primer Information

[0020]

[0021] AIV H7-1 and H7-2 primer pairs are used to detect whether it contains H7 subtype avian influenza virus;

[0022] AIV N9-1 and N9-2 primer pairs are used to detect whether it contains N9 subtype avian influenza virus;

[0023] The AIV M-1 and M-2 primer pairs are universal for the detection of all subtypes of avian influenza viruses.

[0024] 2. Sample preparation

[0025] According to the instructions of the TIANamp virus genome DNA / RNA extraction kit, avian influenza virus (H1N2, H3...

Embodiment 2 3

[0031] The specificity test of embodiment 2 triple RT-PCR

[0032] The cDNA of avian influenza virus (H7N2, H11N9, H7N9, H1N2, H1N7, H2N3, H3N2, H4N5, H5N3, H6N1, H8N4, H9N2 and H10N3), Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus / DNA were added to the triple RT-PCR reaction system for amplification, and the specificity was tested. Applying optimal reaction conditions, RT-PCR amplification of avian influenza virus, Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus nucleic acid was performed. Such as figure 1 As shown, viral nucleic acid templates containing H7 subtype AIV, N9 subtype AIV, H7N9AIV, and other subtypes of AIV can amplify amplified bands that match the size of the experimental design, while other common respiratory diseases in the same position do not. There are no amplified bands. The results showed that the detection of H7 subtype avian influenza virus, N9 subtype avian ...

Embodiment 3 3

[0033] Therefore, the primer pair set and method provided by the present invention can be applied to identify whether the test sample is infected with H7 subtype avian influenza virus, N9 subtype avian influenza virus, H7N9 avian influenza virus and other subtype avian influenza viruses. If a fragment of 330bp is obtained, the sample to be tested contains H7 subtype avian influenza virus, otherwise there is no; if a fragment of 207bp is obtained, the sample to be tested contains N9 subtype avian influenza virus, otherwise there is no; , 330bp and 632bp fragments, the sample to be tested contains H7N9 subtype avian influenza virus, and vice versa; Types of avian influenza virus, and vice versa. The sensitivity test of embodiment 3 triple RT-PCR

[0034] Use the full-length primers of H7 subtype avian influenza virus HA gene, N9 subtype avian influenza virus NA gene and avian influenza virus M gene full-length primers to carry out PCR amplification with the corresponding cDNA t...

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Abstract

The invention discloses a triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for an H7N9 avian influenza virus, as well as a primer pair or a primer pair group for identifying an H7 subtype avian influenza virus and an N9 avian influenza virus and generally detecting all the avian influenza viruses, and a kit thereof. Three pairs of specific primers which specially aim at the conserved sequences of an H7 subtype avian influenza virus HA gene, an N9 subtype avian influenza virus NA gene and all the avian influenza virus M genes are designed by the inventor and are combined to be the triple fluorescence quantitative RT-PCR detection kit for the H7N9 avian influenza virus, and the triple fluorescence quantitative RT-PCR detection kit can be used for determining whether H7N9 avian influenza virus infection exists or not through one-pipe detection. The primer pairs, the primer pair groups or the detection kit can be used for detecting whether samples to be detected are infected by the H7N9 subtype avian influenza virus, the H7 subtype avian influenza virus, the N9 subtype avian influenza virus and other subtype avian influenza viruses, have important significance in effectively preventing and controlling avian influenza and cutting off the transmission route of the avian influenza, and has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of RT-PCR detection kits, in particular to a triple RT-PCR detection kit for H7N9 avian influenza virus, identification of H7 subtype avian influenza virus, N9 subtype avian influenza virus, and universal detection of all subtypes of poultry Primer pair or primer pair set for influenza virus and kit thereof. Background technique [0002] Avian influenza virus (Avian Influenza Virus, AIV) belongs to Orthomyxoviridae Influenza A virus genus, single-stranded negative-sense RNA, polymorphic enveloped virus. The genome of avian influenza virus is divided into 8 segments, encoding HA, NA, NP, NS, MP, PA, PB1 and PB2 proteins respectively, and the filaments on the virus envelope have hemagglutinin (HA) and neuramin Acidase (NA) activity. These two genes are the specific antigens of the virus. According to the different antigenicity of HA and NA proteins, they can be divided into 16 H subtypes (H1-H16) and 9 N sub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谢芝勋罗思思刘加波庞耀珊邓显文谢志勤谢丽基范晴
Owner GUANGXI VETERINARY RES INST
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