Primer pair composition for identification or assisted identification of H6N1 subtype of avian influenza virus and application of primer pair composition

A bird flu virus and auxiliary identification technology, applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of long detection cycle

Active Publication Date: 2015-02-04
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 composition for identification or assisted identification of H6N1 subtype of avian influenza virus and application of primer pair composition
  • Primer pair composition for identification or assisted identification of H6N1 subtype of avian influenza virus and application of primer pair composition
  • Primer pair composition for identification or assisted identification of H6N1 subtype of avian influenza virus and application of primer pair composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Preparation of a primer pair composition for identification or auxiliary identification of H6N1 subtype avian influenza virus

[0068] A primer pair composition for identifying or assisting in the identification of H6N1 subtype avian influenza virus (AIV), consisting of a PCR primer pair named H6 and a PCR primer pair named N1; the H6 is represented by SEQ ID No. 1 in the sequence table The single-stranded DNA (H6-F) shown in the figure and the single-stranded DNA (H6-R) shown in SEQ ID No. 2 can be amplified from the H6 subtype avian influenza virus with a size of 447 bp; The N1 is composed 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 derived from the N1 subtype of avian influenza virus A band with a size of 325 bp was amplified.

[0069] In the above primer pair composition for identifying or assisting in identifying the H6N1 subtype avian influenza virus, th...

Embodiment 2

[0070] Example 2, The identification of Example 1 or the auxiliary identification of H6N1 subtype avian influenza virus primers to the composition sensitivity test

[0071] According to the MiniBEST Viral RNA / DNA Extraction kit instructions, the H6N1 subtype avian influenza virus was extracted from the H6N1 subtype avian influenza virus at a concentration of 10ng / μl to obtain the total RNA of the H6N1 subtype avian influenza virus (referred to as H6N1RNA).

[0072] Use 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 in the HA and NA gene primers, respectively, Using H6N1RNA as a template for RT-PCR amplification, the reaction system is as follows: 2×1Step Buffer 12.5μL, PrimeScript 1Step Enzyme Mix 1μL, HA or NA gene upstream primer (25μM) 0.5μL, HA or NA gene downstream primer (25μM) ) 0.5μL, H6N1RNA (10ng / μL) 1μL, make up 25μL with RNase-free ultrapure water; the reaction proc...

Embodiment 3

[0081] Example 3, the identification or auxiliary identification of avian influenza virus in Example 1 and the specificity experiment of the primer to the composition

[0082] 1. Preparation of test samples

[0083] 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, H13N5 subtype avian influenza virus and H15N8 subtype avian influenza virus, Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and Infectious lary...

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Abstract

The invention discloses a primer pair composition for identification or assisted identification of H6N1 subtype of avian influenza virus and application of the primer pair composition. The primer pair composition for identification or assisted identification of H6 and/or N1 subtype of avian influenza virus is composed of two primer pairs which are independently packaged, namely a polymerase chain reactor (PCR) primer pair named as H6 and a PCR primer pair named as N1; the H6 is composed of two single-stranded DNA as shown in SEQ ID NO. 1 and SEQ ID NO. 2; and the N1 is composed of two single-stranded DNA as shown in SEQ ID NO. 3 and SEQ ID NO. 4.

Description

Technical field [0001] The invention relates to a primer pair composition for identifying or assisting in identifying the H6N1 subtype avian influenza virus in the field of biomedicine and its application. Background technique [0002] Avian influenza virus (Avian Influenza Virus, AIV) is divided into different subtypes according to the antigenic difference of surface glycoprotein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA). At present, 16 HA subtypes (H1 -H16) and 9 NA subtypes (N1-N9). The combination of HA and NA can produce at least 135 subtypes. Among them, some combinations of H5 and H7 subtypes (H5N1, H7N7, H7N9, etc.) are called high Pathogenic AIV can infect humans and cause death. 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 low pathogenic AIV strains of different subtypes are mixed infecting livestock and poultry , Can exchange genes in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q2600/16
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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