Primer composition for detecting H5, H7, H9 and 9 NA subtype AIVs and application thereof

A primer combination and universal primer technology, applied in the biological field, can solve problems such as difficulty in primer combination

Active Publication Date: 2019-03-19
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, based on the common knowledge of those skilled in the art, it is very difficult to design primer combinations for multiplex PCR that can effectively amplify multiple target fragments in the same system
Currently, there are no reagents or kits that can simultaneously detect H5, H7, H9, N1, N2, N3, N4, N5, N6, N7, N8, and N9 subtypes of AIV based on the GeXP system

Method used

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  • Primer composition for detecting H5, H7, H9 and 9 NA subtype AIVs and application thereof
  • Primer composition for detecting H5, H7, H9 and 9 NA subtype AIVs and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0205] Embodiment 1, acquisition and preparation of primers

[0206] Through extensive sequence analysis, primer design, primer verification and primer screening, 13 primer pairs were obtained. The primer pair AIV-M consists of a forward primer (sequence 1 in the sequence listing) and a reverse primer (sequence 2 in the sequence listing), and is used to identify avian influenza virus. The primer pair H5 consists of a forward primer (sequence 3 in the sequence listing) and a reverse primer (sequence 4 in the sequence listing), and is used to identify H5 subtype avian influenza virus. The primer pair H7 consists of a forward primer (sequence 5 in the sequence listing) and a reverse primer (sequence 6 in the sequence listing), and is used to identify H7 subtype avian influenza virus. The primer pair H9 consists of a forward primer (sequence 7 in the sequence listing) and a reverse primer (sequence 8 in the sequence listing), and is used to identify H9 subtype avian influenza vir...

Embodiment 2

[0212] Embodiment 2, the specificity of each primer pair

[0213] 1. Template preparation

[0214] 1. Viral RNA extraction and cDNA acquisition

[0215] ① Viral RNA extraction

[0216] Use the kit EasyPure Viral DNA / RNA Kit (Beijing Quanshijin Biotechnology Co., Ltd., catalog number ER201) to extract RNA from the chicken embryo allantoic fluid of the following virus strains according to the kit instructions (to extract negative chicken embryo RNA obtained from allantoic fluid is a negative control sample): avian influenza virus strains: A / Sparrow / Guangxi / GXs-1 / 2012 (H1N2 subtype), A / Chicken / GuangXi / DX / 2008 (H9N2 subtype), A / Duck / GuangXi / N42 / 2009 (subtype H3N2), A / Duck / HK / 77 / 76 (subtype H2N3), A / Duck / HK / 876 / 80 (subtype H10N3), A / Turkey / Ontario / 6118 / 68 (subtype H8N4), A / Duck / Guangxi / GXd-1 / 2009 (subtype H6N5), A / Duck / HK / 862 / 80 (subtype H12N5), A / Duck / Guangxi / 070D / 2010 (subtype H4N6), A / Duck / Guangxi / GXd-6 / 2010 (subtype H6N8), A / Duck / PA / 2099 / 12 (subtype H11N9), A / Gull / Md / 704 / ...

Embodiment 3

[0243] Embodiment 3, the specificity of primer combination

[0244] 1. Template preparation

[0245] Same as Step 1 of Example 2.

[0246] 2. Take the cDNA obtained in step 1 as a template, use the primers to be tested for GeXP multiplex PCR, and then use GeXP System (GeXP capillary electrophoresis system) for detection and analysis.

[0247] The primer combination consists of 13 primer pairs designed in Example 1.

[0248] Reaction system (20μL): GenomeLab GeXP Start Kit 5×PCR Buffer 4μL, primer combination solution 2μL, 25mM MgCl 2 Aqueous solution 4μL, Taq DNA Polymerase 1μL, template cDNA 2μL, add dH 2 0 to 20 μL.

[0249] GenomeLab GeXP Start Kit 5×PCR Buffer: containing GeXP universal primer pair, purchased from Shanghai Aibo Caisi Analytical Instrument Trading Co., Ltd., catalog number A85017.

[0250] The primer combination solution provided the 13 primer pairs. In the reaction system, the final concentration of each forward primer is 20nM, the final concentratio...

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Abstract

The invention discloses a primer composition for detecting H5, H7, H9 and 9 NA subtype AIVs and an application thereof. The invention protects the primer combination. The primer combination comprises13 primer pairs (26 primers), and the 26 primers are shown from sequence 1 to sequence 26 in a sequence table in sequence. When used together with the GeXP detection method for identifying H5, H7, H9and 9 NA subtypes, the primer combination has the advantages of high flux, quickness and time-saving and labor-saving effects, provides an effective method for quickly identifying H5, H7, H9 and 9 different NA subtype AIVs and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of primers for detecting H5, H7 and H9 and nine NA subtypes of AIV and an application thereof. Background technique [0002] Avian influenza virus (AIV) belongs to Orthomyxoviridae type A influenza. According to the different antigenicity of HA and NA, 16 HA subtypes and 9 NA subtypes have been found in birds. In recent years, the 17th and 18th subtypes have been found in bats. HA subtypes and 10, 11 NA subtypes. In 1997, a fatal case of human infection with H5N1 subtype AIV appeared in Hong Kong, and an outbreak of the epidemic caused people to "change color when talking about poultry" and caused serious economic losses to the breeding industry. In recent years, cases of AIV-infected people have been reported one after another. In 2013, a case of H7N9-infected and fatal cases was found in the Yangtze River Delta region of my country, and spread to most par...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor谢芝勋罗思思黄娇玲谢志勤谢丽基张民秀李孟王盛李丹曾婷婷张艳芳范晴邓显文
OwnerGUANGXI VETERINARY RES INST