Multi-linkage probe amplification identification kit capable of detecting a plurality of avian respiratory pathogens

A technology for multiple ligation of probes, avian respiratory tract, applied in microorganism-based methods, microbial determination/examination, microorganisms, etc., can solve the problems of difficult pathogenic causes, diagnosis, mixed infection of different pathogens, etc. good effect

Active Publication Date: 2019-05-03
ZHEJIANG FORESTRY UNIVERSITY
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Problems solved by technology

[0003] Since there are more than one disease causing poultry respiratory symptoms, the clinical manifestations are relatively similar, and mixed infection with different pathogens is common, making it diffic

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  • Multi-linkage probe amplification identification kit capable of detecting a plurality of avian respiratory pathogens
  • Multi-linkage probe amplification identification kit capable of detecting a plurality of avian respiratory pathogens
  • Multi-linkage probe amplification identification kit capable of detecting a plurality of avian respiratory pathogens

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preparation example Construction

[0099] Preparation of in vitro transcribed RNA of Newcastle disease virus M gene: by sequence comparison in the database, the relatively conserved 499bp sequence in the Newcastle disease virus M gene was selected for gene synthesis in vitro, cloned into the pGEM-5zf vector (purchased from Promega Company), Named pGEM-NDV-M. Using the purified plasmid as a template, linearize the plasmid with MluI enzyme, and use Ambion’s MEGAscript T7 kit for in vitro transcription. The in vitro transcription product is precipitated with LiCl, washed with 70% ethanol and dissolved in RNase-free ddH 2 In O, the integrity and correctness of RNA synthesis were detected by nucleic acid electrophoresis, and the RNA concentration was determined. The in vitro transcribed RNA of Newcastle disease virus M gene was named NDV-M-RNA. The Newcastle disease virus M gene is shown as SEQ ID NO: 34 in the sequence listing.

[0100] Preparation of in vitro transcribed RNA of avian metapneumovirus N gene: by s...

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Abstract

The invention relates to the field of inspection and quarantine, and aims to provide a multi-linkage probe amplification identification kit capable of detecting a plurality of avian respiratory pathogens. The kit comprises: a pre-amplification primer mixed liquid containing pre-amplification primers with a sequence as shown in SEQ ID NO: 1-14; a probe mixed liquid containing a left probe and a right probe with sequences as shown in SEQ ID NO: 15-28; MLPA buffer solution; ligation buffer solution A; ligation buffer solution B; Ligase-65; PCR reaction mixed liquid containing a universal primer with a sequence as shown in SEQ ID NO: 29 and 30; SALSA polymerase; negative control; and positive control. The kit of the invention has the characteristics of high flux, can simultaneously detect 7 pathogens, and can automatically detect 96 samples at a time by using a fully automatic nucleic acid analyzer, and provides a high-flux detection technology for differential diagnosis and emergency diagnosis of pathogens. In addition, the kit has the advantages of good specificity and high sensitivity, and can detect at least 1 copy of target genes by increasing a RT-PCR step to enrich target genes.

Description

technical field [0001] The invention provides a multiple connection for detecting avian influenza virus H9, avian influenza virus H5, avian influenza virus H7, Newcastle disease virus, avian metapneumovirus, avian infectious bronchitis virus and avian infectious laryngotracheitis virus The probe amplification identification detection kit, primers and probes can realize the simultaneous detection of 7 pathogens that cause poultry respiratory diseases, and belong to the field of inspection and quarantine. Background technique [0002] Avian influenza virus H9, avian influenza virus H5, avian influenza virus H7, Newcastle disease virus, avian metapneumovirus, avian infectious bronchitis virus and avian infectious laryngotracheitis virus are common pathogens that cause respiratory diseases in poultry. [0003] Since there are more than one disease causing poultry respiratory symptoms, the clinical manifestations are relatively similar, and mixed infection with different pathogen...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
Inventor 周莹珊邵春艳宋厚辉王晓杜陈琳姜胜宋泉江周彬杨杨杨永春孙静程昌勇
Owner ZHEJIANG FORESTRY UNIVERSITY
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