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NASBA-ELISA (Nucleic Acid Sequence Based Amplification-Enzyme-Linked Immuno Sorbent Assay) detection primer and probe for detecting porcine epidemic diarrhea

A porcine epidemic diarrhea and probe technology, applied in the field of zoonotics, to achieve good specificity and large detection volume

Inactive Publication Date: 2020-12-01
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purpose is to overcome the deficiencies of the existing detection technology, and provide a fast, simple, high sensitivity and specificity that can solve the problem of difficult diagnosis and prevention and control of PEDV for grassroots practitioners, reduce the morbidity and mortality of pig herds, Improve farming efficiency

Method used

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  • NASBA-ELISA (Nucleic Acid Sequence Based Amplification-Enzyme-Linked Immuno Sorbent Assay) detection primer and probe for detecting porcine epidemic diarrhea
  • NASBA-ELISA (Nucleic Acid Sequence Based Amplification-Enzyme-Linked Immuno Sorbent Assay) detection primer and probe for detecting porcine epidemic diarrhea
  • NASBA-ELISA (Nucleic Acid Sequence Based Amplification-Enzyme-Linked Immuno Sorbent Assay) detection primer and probe for detecting porcine epidemic diarrhea

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Experimental program
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Effect test

Embodiment 1

[0023] First, establish a NASBA reaction method for PEDV

[0024] (1) NASBA-ELISA reaction methods are designed to synthesize NASBA-ELISA reaction methods, based on separate PEDV FJ-FQ 2012 strain gene sequences (login number: KJ646581), respectively design the NASBA-ELISA reaction method, respectively, NASBA, a primer NASBA The segment size of the pre-expansion is 216 bp, the upstream primer is PEDV-UP1: 5'-AACAAATCCAGGGCCCACTTC-3 'downstream primers for PEDV-LOW1: 5'- AattctaATAGGACTCACTATAGGGAGAAGGGGGGGA AAACTGGCGATCTGAGCATAG-3 ', wherein the scribe portion is a T7 promoter sequence; capturing biotin probe 1 is BIO-5'-agcgcagcttgctTCGG-3',

[0025] Capture biotin probe 2 is Bio-5'acatcccagagTGGGGAGGAG-3 '.

[0026] The fragment of B primer pre-expansion is 173 bp, upstream primers are PEDV-UP2: 5'-TccagagTGGGAGAATTC-3 ', downstream primers PEDV-LOW2: 5'- AattctaATAGGACTCACTATAGGGAAG G AAACTGGCGATCTGAGCATAG-3 ', wherein the scribe portion is a T7 promoter sequence; the capture b...

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Abstract

The invention relates to an NASBA-ELISA (Nucleic Acid Sequence Based Amplification-Enzyme-Linked Immuno Sorbent Assay) detection primer and probe for detecting porcine epidemic diarrhea. According toan N gene segment of a variant porcine epidemic diarrhea virus, a synthetic primer and a biotin probe are designed, DIG-11-UTP nucleotide is adopted to replace a digoxin detection probe, and an NASBA-ELISA detection method is established. The NASBA-ELISA detection method established by adopting the primer and the probe has no cross reaction with porcine reproductive and respiratory syndrome virus,swine fever virus, transmissible gastroenteritis virus, porcine circovirus type 2 and porcine pseudorabies virus, 4.3 pg of virus nucleic acid can be detected, and good specificity, sensitivity and repeatability are achieved. The method disclosed by the invention is large in one-time detection amount, does not need special expensive instruments and high-requirement technical content, is suitablefor rapid diagnosis of the porcine epidemic diarrhea disease by grassroots employees, and has important significance for effectively diagnosing, preventing and controlling the porcine epidemic diarrhea virus.

Description

Technical field [0001] The present invention relates to NASBA-ELISA detection primers and probes for detecting swine epidemic diarrhea, belonging to the field of animal infectious diseases. Background technique [0002] Pig Fluid Diarrhea (PED) is a pig acute intestinal infectious disease characterized by a coronavirus PEDV. Since the second half of 2010, due to PEDV variation, the disease has led to the disease again, and a major economic loss is caused to the pig industry. PEDV belongs to the Nitro virus, the coronary virus coronary virus member, encoding S protein, E protein, M protein and N protein and other four major structural proteins. Where N protein consists of 441 amino acids, it has a highly conserved, including 6-7 potential phosphorylated sites. N protein is one of the main immunoproteins of viruses, which can induce specific antibody immunity and cellular immunity in the body, and pigs in early infection of PEDV, produce high level antibodies of anti-N protein, whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/6844G01N33/543G01N33/535
CPCC12Q1/6844C12Q1/701C12Q2600/156G01N33/535G01N33/543
Inventor 王隆柏周伦江陈秋勇吴学敏车勇良陈如敬刘玉涛
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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