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Reagent kit for detecting H4 subtype avian influenza virus based on nested RT-PCR

A technology of RT-PCR and avian influenza virus, which is applied in the field of kits for detecting H4 subtype avian influenza virus based on nested RT-PCR, can solve the problems such as the unreported rapid detection method of H4 subtype AIV, and achieve specific detection Methods, Effects of Sensitive Detection Methods

Inactive Publication Date: 2016-04-06
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the rapid detection methods established in China are for other subtypes of AIV, such as H1, H3, H5, H6, H7, and H9 subtypes, but no rapid detection method for H4 subtype AIV has been reported.

Method used

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  • Reagent kit for detecting H4 subtype avian influenza virus based on nested RT-PCR
  • Reagent kit for detecting H4 subtype avian influenza virus based on nested RT-PCR
  • Reagent kit for detecting H4 subtype avian influenza virus based on nested RT-PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, design and synthesis of primers

[0059] According to the conserved region of the nucleotide sequence of the HA gene of the H4 subtype avian influenza virus, two pairs of specific primers were designed and screened using the biological software Primer Premier 5.0, that is, outer primers and inner primers, as shown in Table 1. Wherein, the target fragment amplified by the outer primer is 342bp (sequence 5), and the target fragment amplified by the inner primer is 199bp (position 54-252 of sequence 5). Primers were synthesized by Invitrogen.

[0060] Table 1 Outer and inner primer sequences

[0061]

Embodiment 2

[0062] Embodiment 2, the establishment of the reaction system of nested RT-PCR identification of H4 subtype avian influenza virus

[0063] 1. Extraction and reverse transcription of viral RNA

[0064] Extraction of viral RNA: Extract the RNA of the H4N2 subtype avian influenza virus A / duck / Guangxi / 125D17 / 2012 (H4N2) strain according to the instruction manual of the DNA / RNA extraction kit.

[0065] Reverse transcription: Add 2.0 μL of random primers, 70°C for 10 min; ice bath for 5 min; add reverse transcription system: 10 μL 5×AMVBuffer, 4 μL dNTP, 0.5 μL RNase inhibitor, 1 μLAMV reverse transcriptase, centrifuge briefly, place at 42°C for 90 min, cDNA was prepared. All extracted DNA and cDNA obtained by reverse transcription were stored at -30°C for future use.

[0066] 2. Optimization of nested RT-PCR conditions

[0067] The two PCRs were 25 μL reaction system, and the other system components were unchanged (2×TapPCRMix 12.5 μL, template 2 μL), respectively optimized for pr...

Embodiment 3

[0074] Embodiment 3, the specificity experiment of nested RT-PCR

[0075] 1. Extraction and reverse transcription of viral RNA

[0076] The tested viruses are as follows:

[0077] H4N2 subtype avian influenza virus A / duck / Guangxi / 125D17 / 2012 (H4N2) strain,

[0078] H4N5 subtype avian influenza virus A / Duck / HK / 668 / 79 (H4N5) strain,

[0079] H4 subtype avian influenza virus A / Duck / Guangxi / 027D / 09(H4) strain,

[0080] Two other strains of H4N2 subtype avian influenza virus;

[0081] H1N2 subtype avian influenza virus A / Sparrow / Guangxi / GXs-1 / 2012 (H1N2) strain;

[0082] H3N2 subtype avian influenza virus A / Duck / Guangxi / N42 / 2009 (H3N2) strain;

[0083] H5N2 subtype avian influenza virus A / Turkey / GA / 209092 / 02 (H5N2) strain;

[0084] H6N2 subtype avian influenza virus A / duck / Guangxi / GXd-3 / 2009 (H6N2) strain;

[0085] H7N2 subtype avian influenza virus A / Chicken / NY / 273874 / 03 (H7N2) strain;

[0086] H9N2 subtype avian influenza virus A / turtledove / Guangxi / 49B6 / 2013 (H9N2) strain...

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Abstract

The invention discloses a reagent kit for detecting an H4 subtype avian influenza virus based on nested RT-PCR. The reagent kit comprises a primer pair set composed of an outer primer pair and an inner primer pair, wherein the outer primer pair is composed of two pieces of single-chain DNA shown in the sequence 1 and sequence 2 in a sequence table, and the inner primer pair is composed of two pieces of single-chain DNA shown in the sequence 3 and sequence 4 in the sequence table. It is proved through experiments that by means of the primer pair set provided in the reagent kit, the H4 subtype avian influenza virus can be obtained through specific amplification while other common poultry disease pathogens are not amplified, and the lower detection limit for the H4 subtype avian influenza virus is 360 fg / micro L. By means of the nested RY-PCR method established through the reagent kit, a quick, specific and sensitive detection method is provided for early diagnosis and effective prevention and treatment of the H4 subtype avian influenza virus.

Description

technical field [0001] The invention belongs to the field of virus detection and relates to a kit for detecting H4 subtype avian influenza virus based on nested RT-PCR. Background technique [0002] Avian influenza (Avian Influenza, AI) is an infection and / or disease syndrome of poultry caused by avian influenza A virus (AIV) of the family Orthomyxoviridae, mainly causing systemic or respiratory diseases of poultry [ Fu. Poultry Diseases [M]. Beijing: China Agricultural Press, 2005.]. There are many subtypes of AIV. According to the antigenic differences of surface glycoproteins hemagglutinin (HA) and neuraminidase (Neuraminidase, NA), 16 subtypes of HA (H1-H16) and 9 subtypes of NA ( N1-N9) subtype YMSaif. Poultry Diseases (Twelfth Edition) [M]. Beijing: China Agricultural Press, 2012: 176-178.]; AIV can be divided into highly pathogenic according to the difference in pathogenicity Avian influenza virus (HighlyPathogenicAvianInfluenzaVirus, HPAIV) and low pathogenicity of...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谢芝勋吴嫒琼谢丽基罗思思邓显文谢志勤黄莉黄娇玲曾婷婷
Owner GUANGXI VETERINARY RES INST
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