GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases

一种鸭圆环病毒、鸭肝炎病毒的技术，应用在微生物的测定/检验、DNA / RNA片段、重组DNA技术等方向，达到应用前景广阔、高通量检测试剂盒和检测体系的效果

Active Publication Date: 2014-05-07
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no reagent or kit that can simultaneously detect multiple infectious disease pathogens from ducks based on the GeXP system

Method used

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  • GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases
  • GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases
  • GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the design of primer pair

[0053] Using the primer sequence published by GenBank as a reference, download the known sequence from NCBI and use DNASTAR software for sequence alignment, select highly conserved and specific gene fragments for each target gene, and design 12 specific primers by primer premier5.0 software , and add a GeXP universal primer (underlined sequence) at the 5' end of the primer, and the primer direction is from the 5'-3' end, as follows:

[0054] 1) Primer pair A with the M gene of avian influenza virus (AIV) as the target gene:

[0055] AIV-F: AGGTGACACTATAGAATA CAGAAACGGATGGGAGTGC (SEQ ID NO: 1);

[0056] AIV-R: GTACGACTCACTATAGGGA TATCAAGTGCAAGATCCCAATGAT (SEQ ID NO: 2);

[0057] The expected length of the amplified product (i.e., the target peak position) is 122bp;

[0058] 2) Primer pair B targeting the HA gene of H5 subtype avian influenza virus (AIV-H5):

[0059] AIV-H5-F: AGGTGACACTATAGAATA CTTCAGGCATCAAAATGCACA(seq...

Embodiment 2

[0103] Embodiment 2, the specific detection of primer pair

[0104] 1. Template preparation

[0105] 1. Viral RNA extraction and cDNA acquisition

[0106] 1) Viral RNA extraction

[0107] Using DNA / RNA extraction kits (both purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number ER201), according to the kit instructions, the RNA of the virus was extracted as follows (the samples obtained by extracting negative duck embryo allantoic fluid were: negative control sample): avian influenza virus strains: Duck / HK / 717 / 79-d1 (subtype H1N3), Duck / HK / 77 / 76 (subtype H2N3), Duck / HK / 526 / 79 / 2B (subtype H3N6 subtype), Duck / HK / 668 / 79 (subtype H4N5), Duck / HK / 531 / 79 (subtype H6N8), Turkey / ont / 6118 / 68 (subtype H8N4), Duck / Guangxi / 1 / 00 (subtype H9N2), Duck / HK / 876 / 80 (subtype H10N3), Duck / HK / 661 / 79 (subtype H11N3), Duck / HK / 862 / 80 (subtype H12N5), Gull / MD / 704 / 77 (subtype H13N5); duck hepatitis virus, duck yellow virus, Newcastle disease virus, muscovy duck reovirus.

[0108]...

Embodiment 3

[0214] Embodiment 3, the sensitivity detection of primer pair

[0215] 1. Preparation of monoclonal plasmid standard containing target gene and H7 subtype avian influenza virus

[0216] H5 subtype avian influenza virus Duck / HK / 313 / 78 (H5N3 subtype), H7 subtype avian influenza virus Duck / HK / 47 / 76 (H7N2 subtype), H9 subtype obtained in step 1 of Example 2, respectively Avian influenza virus Duck / Guangxi / 1 / 00 (subtype H9N2), duck hepatitis virus, duck plague virus, duck yellow virus, Newcastle disease virus, egg drop syndrome virus, muscovy duck reovirus, muscovy duck parvovirus With the cDNA or DNA sample of duck circovirus as a template, the M gene of avian influenza virus obtained by PCR amplification, the HA gene of H5 subtype avian influenza virus, the HA gene of H7 subtype avian influenza virus, and the HA gene of H9 subtype avian influenza virus Gene, duck hepatitis virus 5'UTR region gene, duck plague virus UL6 gene, duck yellow virus E gene, Newcastle disease virus L ge...

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Abstract

The invention discloses a GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases. The invention provides a GeXP detection primer group for identifying or assisting to identify duck infectious disease pathogens, wherein the primer group consists of a primer pair A, a primer pair B, a primer pair C, a primer pair D, a primer pair E, a primer pair F, a primer pair G, a primer pair H, a primer pair I, a primer pair J, a primer pair K and a primer pair L. According to the GeXP detection kit, shown by experiments, the primer group, a PCR (Polymerase Chain Reaction) reagent and the primer pairs, provided by the invention, are used for simultaneously differentiating and detecting avian influenza viruses, H5, H7 and H9 subtype avian influenza viruses, duck hepatitis viruses, duck plague viruses, duck flaviviruses, newcastle disease viruses, egg drop syndrome viruses, muscovy duck reoviruses, muscovy duck parvoviruses and duck circoviruses and are good in specificity and high in sensitivity. The detection kit, which is simple and convenient and is high in flux, and a detection system are provided for the detection on common major duck infectious disease pathogens, so that the practical needs are better met, and application prospects are broad.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a GeXP detection kit for identifying 11 duck virus diseases. Background technique [0002] Avian influenza virus of H5, H7 and H9 subtypes, duck hepatitis virus, duck plague virus, duck yellow virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus are 11 major infectious diseases that seriously endanger ducks. With the development of duck industry, the incidence of duck infectious virus disease is gradually increasing, which has become a major factor limiting the development of duck industry. The traditional methods for differential diagnosis of duck infectious diseases mainly include pathogen isolation and serological tests, etc., but these methods are often limited by the freshness of clinical disease materials, degree of pollution or course of disease, and the operation is also very cumbersome and time-consuming...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q2600/156C12Q2600/158C12Q2600/16C12Q1/70C12Q1/686
Inventor 谢芝勋张艳芳谢丽基刘加波庞耀珊范晴罗思思邓显文谢志勤
Owner GUANGXI VETERINARY RES INST
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