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A Multiplex Fluorescence Immunoassay Method for Rapid Differentiation of 6 Avian Respiratory Pathogens

A multiple fluorescence and immunoassay technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve difficult problems such as reducing detection costs, avoiding cross-hybridization, and simple operation

Active Publication Date: 2018-07-06
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. Big

Method used

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  • A Multiplex Fluorescence Immunoassay Method for Rapid Differentiation of 6 Avian Respiratory Pathogens
  • A Multiplex Fluorescence Immunoassay Method for Rapid Differentiation of 6 Avian Respiratory Pathogens
  • A Multiplex Fluorescence Immunoassay Method for Rapid Differentiation of 6 Avian Respiratory Pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Primer Design

[0055] A set of multiple fluorescence immunoassay primers for rapidly distinguishing avian influenza virus, chicken infectious bronchitis virus, chicken Newcastle disease virus, chicken infectious laryngotracheitis virus, mycoplasma gallisepticum and mycoplasma gallisepticum is designed, wherein, the The primer pair for detecting bird flu virus is designed according to the conservative sequence of the M gene of bird flu virus; the primer pair for detecting chicken infectious bronchitis virus is designed according to the N gene conservative sequence of chicken infectious bronchitis virus Designed; the primer pair for detecting chicken Newcastle disease virus is designed according to the conserved sequence of the F gene of chicken Newcastle disease virus; the primer pair for detecting chicken infectious laryngotracheitis virus is designed according to chicken infectious laryngotracheitis virus The conserved sequence design of the gD gene of Mycop...

Embodiment 2

[0082] Example 2 Establishment of Multiplex Fluorescence Immunoassay Kit for Rapidly Distinguishing 6 Kinds of Avian Respiratory Pathogens

[0083] The kit includes the following components:

[0084] (1) The multiplex fluorescent immunoassay primer set designed in Example 1;

[0085] (2) 6 kinds of fluorescently encoded microspheres containing anti-tag sequences encoding different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences on the upstream primers of the multiple fluorescent immunoassay primer set; six kinds of microspheres The balls were purchased from luminex company, and the specific numbers of the fluorescent coded microspheres corresponding to AIV, NDV, IBV, ILT, MS and MG are MTAG-A029, MTAG-A034, MTAG-A036, MTAG-A067, MTAG-A025 and MTAG -042.

[0086] (3) streptavidin-phycoerythrin complex;

[0087] (4) PCR amplification reaction system.

Embodiment 3

[0088] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for 6 Kinds of Poultry Respiratory Tract Pathogens

[0089] 1. Construction of six kinds of avian respiratory pathogenic plasmids

[0090] Extract the RNA / DNA of AIV, NDV, IBV, ILT, MG, and MS pathogens with Tiangen’s automatic nucleic acid extractor, respectively, and perform RT-PCR amplification with the corresponding primers designed in Example 1, and the amplified products were respectively Carry out agarose gel electrophoresis detection and gel cutting purification. The purified cDNA was connected to the pMD-19T vector with a kit from TaKaRa Company, the connected product was transformed into DH5a competent cells, single clones were selected, colony PCR identification was carried out, and the colonies identified as positive bacteria were subjected to plasmid extraction. Send for sequencing.

[0091] 2. Plasmid PCR amplification

[0092] Use specific amplification AIV, NDV, IBV, ILT, MS...

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Abstract

The invention discloses a multi-fluorescent immunoassay method for rapidly distinguishing 6 types of poultry respiratory pathogens. The multi-fluorescent immunoassay method is simple to operate; a target amplified fragment is obtained through a PCR (Polymerase Chain Reaction); then an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized; an MFI (Mean Fluorescence Intensity) value is read through a detector to distinguish viruses of different types. According to the method disclosed by the invention, avian influenza viruses, chicken infectious bronchitis viruses, chicken Newcastle disease viruses, chicken infectious laryngotracheitis viruses, mycoplasma gallisepticum and mycoplasma synoviae can be accurately detected at the same time; the multi-fluorescent immunoassay method has high specificity, high sensitivity and good repeatability. Compared with a traditional detection method, the method disclosed by the invention realizes simultaneous detection of a plurality of types of different target molecules in the same sample; the use amount of the sample is less; the method is simple and rapid to operate and the detection cost can be greatly reduced.

Description

technical field [0001] The invention belongs to the field of virus detection in the breeding industry, and in particular relates to a multiple fluorescence immunoassay method for rapidly distinguishing six kinds of poultry respiratory pathogens. Background technique [0002] As the market demand for poultry continues to increase, the poultry industry has developed rapidly, the degree of intensification of the poultry industry has continued to increase, and the infection of poultry respiratory pathogens has been on the rise year by year. Respiratory diseases of poultry have been a major factor causing significant economic losses in the poultry industry. The pathogenesis of chicken respiratory disease has its complexity. It can be caused by viruses, mycoplasma and bacteria, and various mixed infections will appear clinically. The occurrence of these diseases cannot be ignored in chicken production. Respiratory diseases of chickens occur frequently. Chickens of various ages c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6804C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/149
Inventor 郭鹏举朱余军丛峰黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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