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Human and animal brucella antibody immunochromatography test paper and preparation method thereof

A Brucella and antibody technology, applied in the field of immunological diagnosis and detection, can solve the problems of long detection time, difficult to popularize and use, and difficult to give detection results, and achieve the effects of long shelf life, high sensitivity and strong sensitivity.

Inactive Publication Date: 2013-05-29
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there is a lack of equipment and professional technicians for molecular biology testing and ELISA testing in grass-roots medical institutions and animal husbandry and veterinary stations, and these two methods have a long testing time, and it is difficult to give test results in a short time. It is difficult to popularize and use in areas, especially areas with high incidence of brucellosis

Method used

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  • Human and animal brucella antibody immunochromatography test paper and preparation method thereof
  • Human and animal brucella antibody immunochromatography test paper and preparation method thereof
  • Human and animal brucella antibody immunochromatography test paper and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Prokaryotic expression and purification of Brucella outer membrane protein BP26

[0028] 1.1 Design and synthesis of primers

[0029] With reference to the BP26 gene sequence of the Brucella melitensis reference strain 16M published on GenBank (accession number: U45996), according to the characteristics of the pGM-T cloning vector and the pET-28a prokaryotic expression vector, the design contains restriction internal Primer for the cleavage site of Dicer Xho I / EcoR I. The primer sequences are shown in Table 1-1.

[0030] Table 1-1 Primer sequences

[0031] Table 1-1 Primer sequences

[0032]

[0033] 1.2 PCR amplification of the target fragment

[0034] PCR amplification was carried out with Brucella 16M genome as a template, and the PCR reaction system is shown in Table 1-2. The reaction conditions were as follows: pre-denaturation at 94 °C for 5 min; 95 °C for 50 s, 58 °C for 45 s, 72 °C for 1 min, 30 cycles; extension at 72 °C for 7 min; 4 °C∞. Af...

Embodiment 2

[0065] Example 2: Prokaryotic expression and purification of Brucella outer membrane protein OMP31

[0066] 1.1 Design and synthesis of primers

[0067] With reference to the OMP31 gene sequence of the 16M Brucella sheep reference strain that has been published on GenBank, the 78bp signal peptide is removed. Primer for the cleavage site of Dicer Xho I / Nco I. The primer sequences are shown in Table 1-1.

[0068] Table 1-1 Primer sequences

[0069]Table 1-1 Primer sequences

[0070]

[0071] 1.2 PCR amplification of the target fragment

[0072] PCR amplification was carried out using 16M bacteria liquid of Brucella melis inactivated at 80°C for 30 minutes as a template, and the PCR reaction system is shown in Table 1-2. The following reaction conditions were used: pre-denaturation at 95°C for 4 min; 30 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min; extension at 72°C for 7 min; 4°C∞. After the reaction, the PCR product was analyzed on a 1.2% agarose gel, and...

Embodiment 3

[0104] Embodiment 3: the colloidal gold immunochromatography test strip of Brucella antibody in the preparation serum

[0105] 1.1 Preparation of colloidal gold

[0106] Clean the triangular flask for burning colloidal gold and then soak it in aqua regia overnight; take it out and rinse it with clean water 20 times, then wash it with distilled water for 3 to 5 times, then rinse it with ultrapure water for 3 times, and let it dry After drying, siliconize with 5% dichlorodimethylsilane in chloroform solution for 1 min, rinse with ultrapure water for 3 times after drying, and dry the triangular flask for gold burning.

[0107] Add 49ml of chloroauric acid with a concentration of 0.01% into a silanized amphorae, heat and stir on a magnetic stirrer, and quickly add 1ml of trisodium citrate aqueous solution with a concentration of 1.05% after the solution boils, and the color of the solution is about 5min The left and right sides turn into a stable purple-red color. After cooling, ...

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Abstract

The invention relates to the field of zoonosis immune diagnosis and discloses a brucellosis antibody detection immunochromatography test strip based on colloidal gold as a marking material and a preparation method of the test strip. In a quick brucellosis antibody detection technology, the 40-nm colloidal gold labeled with staphylococcus aureus protein A (SPA) is sprayed on glass fibers to form a gold labeled pad. Genes OMP31 and BP26 are cloned from a brucella genome, form prokaryotic expression recombinant plasmids and are transformed in escherichia coli to express proteins omp31 and bp26, the two proteins as coating antigens are respectively coated on a nitrocellulose membrane to serve as detection lines, and the detection lines, the gold labeled pad, a specially treated sample loading pad and water absorption paper are assembled into an immunochromatography detection device. The test strip has the characteristics of strong specificity, high sensitivity, convenience, simplicity, economy and the like, can be applied to typing detection of brucellosis antibodies of sheep and cattle, and has very important meaning and practical application value for brucellosis monitoring and prevalence control.

Description

technical field [0001] The invention belongs to the technical field of immunological diagnosis and detection, and specifically relates to a detection test paper for detecting Brucella infection and a preparation method thereof. The invention also relates to a method for preparing genetically engineered antigens of Brucella outer membrane proteins bp26 and omp31, and the application of the two proteins in the detection of Brucella antibodies. technical background [0002] Brucellosis is a zoonotic infectious disease caused by bacteria of the genus Brucella invading the body. Brucella is a facultative intracellular parasite. After human infection, arthritis, meningitis, endocarditis, etc. will be manifested as persistent infection, which cannot be cured at present; after animal infection, male animals cause orchitis and epididymitis, and female animals Frequent miscarriages, premature births, and stillbirths are one of the main causes of heavy losses in animal husbandry (Zhao...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 陈创夫张辉王远志石峰鲁之子孟茹
Owner SHIHEZI UNIVERSITY
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