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Primer pair combination and application thereof for identifying or helping identifying avian influenza virus and H6N1 subtype thereof

A bird flu virus and auxiliary identification technology, applied in the direction of microbes, recombinant DNA technology, microbe-based methods, etc., can solve the problem of long detection cycle

Inactive Publication Date: 2015-01-28
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Virus isolation and identification is a classic influenza detection method, that is, the sample is first inoculated with SPF chicken embryo proliferation virus for 2-5 days, and then the chicken embryo allantoic fluid is collected for hemagglutination test and hemagglutination inhibition test. The results are accurate and reliable, but there are detection Disadvantages of long cycle
Enzyme-linked immunosorbent assay can detect antigens of avian influenza, but requires specific standard positive sera

Method used

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  • Primer pair combination and application thereof for identifying or helping identifying avian influenza virus and H6N1 subtype thereof
  • Primer pair combination and application thereof for identifying or helping identifying avian influenza virus and H6N1 subtype thereof
  • Primer pair combination and application thereof for identifying or helping identifying avian influenza virus and H6N1 subtype thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1, prepare the primer pair composition of identifying or assisting in identifying avian influenza virus

[0075] The primer pair composition of identification or auxiliary identification avian influenza virus (AIV), is made up of the PCR primer pair that name is H6, the PCR primer pair that name is N1 and the PCR primer pair that name is M; Said H6 is made up of SEQ in the sequence table Composition of single-stranded DNA (H6-F) shown in ID No.1 and single-stranded DNA (H6-R) shown in SEQ ID No.2, can be amplified from H6 subtype avian influenza virus with a size of 447bp The band; said N1 is made up of the single-stranded DNA (N1-F) shown in SEQ ID No.3 and the single-stranded DNA (N1-R) shown in SEQ ID No.4 in the sequence table, which can be obtained from N1 In the avian influenza virus of type, amplify the band that size is 325bp; Described M is by the single-stranded DNA (M-F) shown in SEQ ID No.5 and the single-stranded DNA shown in SEQ ID No.6 in the s...

Embodiment 2

[0077] The identification of embodiment 2, embodiment 1 or the primer of auxiliary identification avian influenza virus sensitivity experiment of composition

[0078] According to the instructions of the MiniBEST Viral RNA / DNA Extraction kit, the total RNA of the H6N1 subtype avian influenza virus (referred to as H6N1RNA) was extracted from the H6N1 subtype avian influenza virus at a concentration of 10 ng / μL.

[0079] HA, NA and M genes in Hoffmann E, Stech J, Guan Y, et al. Universal primer set for the full-length amplification of all influenza A viruses [J]. Arch Virol, 2001, 146:2275–2289, respectively Primers, H6N1RNA as template for RT-PCR amplification, the reaction system is as follows: 2×1Step Buffer 12.5μL, PrimeScript 1Step Enzyme Mix 1μL, HA or NA or M gene upstream primer (25μM) 0.5μL, HA or NA or M Gene downstream primer (25 μM) 0.5 μL, H6N1 RNA (10 ng / μL) 1 μL, make up 25 μL with RNase-free ultrapure water; the reaction program is as follows: 94 ° C for 5 min, 9...

Embodiment 3

[0088] The identification of embodiment 3, the specificity experiment of the primer of embodiment 1 or the primer pair composition of auxiliary identification avian influenza virus

[0089] 1. Preparation of test samples

[0090] The viruses used in the experiment are H6N1 subtype avian influenza virus, H6N2 subtype avian influenza virus, H6N5 subtype avian influenza virus, H6N6 subtype avian influenza virus, H6N8 subtype avian influenza virus, H1N1 subtype avian influenza virus, H2N3 subtype avian influenza virus, H3N2 subtype avian influenza virus, H4N5 subtype avian influenza virus, H5N1 subtype avian influenza virus, H7N2 subtype avian influenza virus, H8N4 subtype avian influenza virus, H9N2 subtype avian influenza virus, H10N3 Subtype avian influenza virus, H11N9 subtype avian influenza virus, H12N5 subtype avian influenza virus and H13N5 subtype avian influenza virus, Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus...

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Abstract

The invention discloses a primer pair combination and an application thereof for identifying or helping identifying an avian influenza virus and an H6N1 subtype thereof. The primer pair combination and the application thereof for identifying or helping identifying the avian influenza virus and the H6N1 subtype thereof, which are provided by the invention, refer to a combination 1, a combination 2 or a combination 3. The combination 1 consists of a PCR (Polymerase Chain Reaction) primer pair of which the name is H6, a PCR primer pair of which the name is N1 and a PCR primer pair of which the name is M; the H6 consists of two single-chain DNAs shown as SEQ ID No.1 and SEQ ID No.2 in a sequence list; N1 consists of two single-chain DNAs shown as SEQ ID No.3 and SEQ ID No.4 in the sequence list; M consists of two single-chain DNAs shown as SEQ ID No.5 and SEQ ID No.6 in the sequence list; the combination 2 consists of the H6 and the M; the combination 3 consists of the N1 and the M.

Description

technical field [0001] The invention relates to the primer pair composition and application for identifying or assisting in identifying avian influenza virus and its H6N1 subtype in the field of biomedicine. Background technique [0002] Avian Influenza Virus (AIV) is divided into different subtypes according to the surface glycoprotein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) antigenic differences, and 16 HA subtypes (H1 -H16) and 9 NA subtypes (N1-N9), the combination of HA and NA can produce at least 135 subtypes, of which, some combinations of H5 and H7 subtypes (H5N1, H7N7 and H7N9, etc.) are called high Pathogenic AIV, which can infect and kill humans. Although low-pathogenic AIV infection has no symptoms or shows mild and mild atypical symptoms, it is not a threat to the health of livestock and humans, but when different subtypes of low-pathogenic AIV strains mix to infect livestock and poultry , can exchange genes in animals, thereby producing new ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q2600/112
Inventor 谢芝勋罗思思谢志勤邓显文刘加波黄莉黄娇玲曾婷婷
Owner GUANGXI VETERINARY RES INST
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