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GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases

A technology for respiratory tract and pathogens, which is applied in the field of GeXP rapid detection kits to achieve promising results

Active Publication Date: 2014-01-15
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 based on the GeXP system that can simultaneously detect multiple respiratory infectious disease pathogens in poultry

Method used

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  • GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases
  • GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases
  • GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the design of PCR primer pair

[0040] Using the primer sequences published by GenBank as a reference, the known sequences were downloaded from NCBI and sequence alignment was performed using DNASTAR software, and highly conserved and specific gene fragments for each target gene were selected, and 10 specific primers were designed by Premier 5.0 software, and Add the GeXP universal primer (underlined sequence) at the 5' end of the primer, and the primer direction is from the 5'-3' end, as follows:

[0041] 1) Primer pair A with the M gene of avian influenza virus (AIV) as the target gene:

[0042] AIV-F1: AGGTGACACTATAGAATA AGCCGAGATCGCGCAGA (Sequence Listing Sequence 1);

[0043] AIV-R1: GTACGACTCACTATAGGGA CGCTCACTGGGCACGGT (Sequence Listing Sequence 2);

[0044] The expected length of the amplified product (i.e., the target peak position) is 190bp;

[0045] The target sequences are positions 88-104 and 224-240 of Genbank DQ485227.

[0046] 2) Pr...

Embodiment 2

[0092] Embodiment 2, the specific detection of PCR primer pair

[0093] 1. Template preparation

[0094] 1. Viral RNA extraction and cDNA acquisition

[0095] 1) Viral RNA extraction

[0096] Use the kit MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 (Dalian TaKaRa Company, product number DV819A) to extract RNA from the chicken embryo allantoic fluid of the following virus strains according to the kit instructions (to extract negative chicken embryo urine The sample obtained from cyst fluid is a negative control sample): Avian influenza virus strains: Duck / HK / 717 / 79-d1 (H1N3 subtype), Duck / HK / 77 / 76 (H2N3 subtype), Duck / HK / 526 / 79 / 2B (subtype H3N6), 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 / 147 / 77 (subtype H9N6), Duck / HK / 876 / 80 (subtype H10N3), Duck / HK / 661 / 79 (subtype H11N3), Duck / HK / 862 / 80 (subtype H12N5), Gull / MD / 704 / 77 (subtype H13N5) Literature: ZhixunXie, Yao-shan Pang, Ji...

Embodiment 3

[0177] Embodiment 3, the sensitivity detection of PCR primer pair

[0178] 1. Preparation of monoclonal plasmid standards containing target genes

[0179] The avian influenza virus, H5 subtype avian influenza virus, H7 subtype avian influenza virus, H9 subtype avian influenza virus, Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis respectively obtained in step 1 in Example 2 The cDNA or DNA samples of viruses Mycoplasma gallisepticum, Mycoplasma synoviosus and Haemophilus paragallinarum are used as templates to amplify the M gene of avian influenza virus obtained by PCR, the HA gene of H5 subtype avian influenza virus, and the H7 subtype avian influenza virus HA gene, H9 subtype avian influenza virus HA gene, Newcastle disease virus ND gene, infectious bronchitis virus N gene and infectious laryngotracheitis virus TK gene, Mycoplasma gallisepticum 16rRNA gene, Mycoplasma synovialis VLHA gene and parachicken The full-length cDNA or DNA fragmen...

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Abstract

The invention discloses a GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases. The kit is used based on a GeXP system and comprises ten polymerase chain reaction (PCR) primer pairs; the kit is used for identifying and detecting avian influenza virus, H5, H7 and H9 subtype avian influenza virus, newcastle disease virus, infectious bronchitis, infectious laryngotracheitis, mycoplasma gallisepticum, bursa synovialis mycoplasma and haemophilus paragallinarum; and the kit is good in specificity, high in sensitivity and can detect 100 copy / mu l. Compared with an identifying result of the conventional experiment method of a pathogen separation and hemagglutination inhibition experiment or a serology experiment and the like, the GeXP rapid detection kit has the advantage that the coincidence rate reaches 100 percent. The kit is generally used for detecting the main chicken respiratory tract diseases and the pathogens thereof, so that a simple and high-flux detection kit and a detection system are provided, an actual requirement is met, and the application prospect is wide.

Description

technical field [0001] The invention relates to a GeXP rapid detection kit for simultaneously identifying nine kinds of chicken respiratory disease pathogens. Background technique [0002] H5, H7 and H9 subtype avian influenza virus, Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, mycoplasma gallisepticum, mycoplasma synovialum and infectious rhinitis are the nine main respiratory infections that seriously harm chickens disease. The main clinical manifestations are: rales, cough, wheezing, neck stretching, mouth breathing, head shaking and dyspnea. These infectious diseases infect chickens of different ages and have high morbidity and mortality, but their clinical symptoms are very similar and cannot be identified. The traditional methods for differential diagnosis of these respiratory diseases mainly include pathogen isolation and identification and serological tests, etc., but these methods are often limited by the freshness of c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11
Inventor 谢芝勋罗思思谢丽基邓显文谢志勤庞耀珊刘加波范晴
Owner GUANGXI VETERINARY RES INST
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