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Brucellosis rapid detection method

A technology for rapid determination of brucellosis, applied in the field of determination, can solve problems such as low success rate, harsh conditions for isolation and identification of pathogens, cumbersome CFT operation, etc., shorten detection time, reduce operation steps and labor intensity, and overcome detection The long-term effect

Inactive Publication Date: 2017-05-31
BEOSON JIANGSU FOOD SAFETY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional methods require harsh conditions for the isolation and identification of pathogens, and are laborious, time-consuming, high risk, and low success rate
PBT and SAT have low specificity and low sensitivity; CFT is cumbersome to operate, requires high experimental conditions and technical level, and is extremely inconvenient for practical application
The PCR detection method is much faster and more accurate than the previous ones, but it requires complex instruments and equipment, and the cost is high, so it is not suitable for grassroots and on-site detection
The LAMP method has the advantages of high sensitivity, good specificity, short reaction time, convenient determination of results, and no need for expensive instruments. Analyze the final result of the LAMP reaction, and there is a risk of aerosol contamination of the laboratory. Due to the lack of real-time monitoring of the reaction, it is difficult to eliminate these interference factors, and it is impossible to make an accurate judgment on the test results

Method used

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Effect test

Embodiment 1

[0011] The rapid assay method for brucellosis of the present invention comprises the following steps: 1) expression fragment cloning: boil the S2 live bacteria vaccine in boiling water for 30 minutes, and then take 1 μL-2 μL as a template for PCR amplification of the target gene expression fragment, Use primers LFGCGAACCGGCAATACCAG and LBGCCGCGTTGAGTACCGT as front and rear primer pairs to carry out PCR reaction, use DNA gel recovery kit to purify and recover the target fragment, and recover the lysate of Brucella Omp19 outer membrane protein gene expression fragment; 2) Escherichia coli prokaryotic expression vector The construction of the method: take the recovered Brucella Omp19 outer membrane protein gene expression fragment lysate and the Escherichia coli pET-28a plasmid vector lysate in step (1) and carry out double enzyme digestion treatment with endonuclease EcoRI and XhoI respectively, and then , T4 DNA ligase ligation in a metal bath at 16°C for 12 hours to obtain the ...

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PUM

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Abstract

The invention discloses a brucellosis rapid detection method. The detection method comprises the following steps: boiling a S2 live vaccine in a boiling water bath for 30 to 60 minutes, then fetching 1 to 2 [mu]L of the S2 live vaccine as the template of expression fragments of a target gene in PCR amplification, utilizing primers namely LFGCGAACCGGCAATACCAG and LBGCCGCGTTGAGTACCGT as the forward primer pair and reverse primer pair, carrying out PCR reactions, purifying and recovering target segments by utilizing a DNA gel recovering kit, and recovering and obtaining a Brucella Omp19 outer membrane protein gene expression fragment dissolved solution. The shortages of conventional detection method such as long detection period, tedious steps, labor waste, time waste, and the like, are overcome. The detection time is largely shortened. The operation steps and labor strength are largely reduced. The time and labor are saved. The detection is quick and sensitive.

Description

technical field [0001] The invention relates to an assay method, in particular to a rapid assay method for brucellosis. Background technique [0002] Brucellosis is caused by Brucella, and is one of the most prevalent and harmful zoonotic diseases in the world, causing miscarriage, infertility and local lesions of various tissues. Brucella can be infected through the nose, pharynx, and oral cavity, mainly through the infiltration of mucosal epithelial tissues. At present, there are more than 10 species of Brucella in the genus Brucella. Different species of Brucella have obvious host hazard tendencies, but most of them have the ability of cross-infection between different hosts, which can cause serious diseases. public health issues. With the rapid development of animal husbandry in my country in recent years, brucellosis has become one of the serious diseases that limit the development of animal husbandry in my country. Brucella includes provenances such as Brucella floc...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/68
CPCG01N33/68G01N33/543G01N2333/23
Inventor 周合张根义张进周朱晨杨敏胡彬吴念绮
Owner BEOSON JIANGSU FOOD SAFETY TECH CO LTD
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