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Colloidal gold test strip for detecting Leucocytozoon caulleryi antibody and preparation method of colloidal gold test strip

A technology of colloidal gold test paper and white blood cells, applied in the fields of botanical equipment and methods, biochemical equipment and methods, biological testing, etc. High, unable to adapt to epidemiological investigations and other issues, to achieve the effect of easy mass production, saving manpower and time, hardware costs and labor costs

Active Publication Date: 2020-08-11
FOSHAN STANDARD BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the blood smear inspection method is simple and feasible, it has great limitations in terms of detection efficiency, detection rate, and identification of insect species. It is increasingly unsuitable for the current prevention and control work, especially for large-scale epidemics. scientific survey
Nucleic acid detection is mainly through ordinary PCR method and fluorescent quantitative PCR method. This kind of method has good sensitivity and relatively high accuracy, but it needs professional equipment for detection, and has high requirements for personnel and equipment.
Serological detection methods include agar diffusion test, immunofluorescence antibody test, latex agglutination test, etc. These methods have problems such as low sensitivity, complicated operation, and lack of standardization
The indirect ELISA detection method based on the recombinant R7 protein of Leukocystis carinii has good sensitivity and specificity, but it requires professional equipment and operators, high requirements for test conditions, and the detection takes a long time

Method used

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  • Colloidal gold test strip for detecting Leucocytozoon caulleryi antibody and preparation method of colloidal gold test strip
  • Colloidal gold test strip for detecting Leucocytozoon caulleryi antibody and preparation method of colloidal gold test strip
  • Colloidal gold test strip for detecting Leucocytozoon caulleryi antibody and preparation method of colloidal gold test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A preparation method for recombinant R7 protein (rR7 protein), comprising the following steps:

[0061] (1) Carrying out PCR amplification of the Leukocystis carinii R7 gene to obtain the recombinant R7 gene of the Leukocystis carinii;

[0062] (2) Digest the psYNO-1 plasmid with restriction enzymes, connect the recombinant R7 gene of Leukocystis carinii to the digested psYNO-1 plasmid with ligase, and transform it into E.coli TOP10 competent cells Medium culture, then carry out PCR to identify positive clones, obtain PCR-identified positive bacterial fluid, extract PCR-identified positive bacterial fluid plasmid, and obtain positive recombinant plasmid psYNO-R7 through double enzyme digestion identification;

[0063] (3) Transform the positive recombinant plasmid psYNO-R7 into E.coli BL21 expression bacteria, add isopropyl-β-D-thiogalactoside to induce expression, and obtain psYNO-R7 recombinant protein;

[0064] (4) Purify the psYNO-R7 recombinant protein after induc...

Embodiment 2

[0069] A preparation method of colloidal gold solution, comprising the following steps:

[0070] Prepare colloidal gold by trisodium citrate reduction method, put 100mL ultrapure water in a clean Erlenmeyer flask, add 1mL of 1% chloroauric acid aqueous solution, heat and boil for 1-2min, then quickly add 1% trisodium citrate aqueous solution 1mL, continue to boil for 10min, and stir evenly while heating on a magnetic heating stirrer during the whole process. After natural cooling, add ultrapure water to make the volume to 100mL to obtain a colloidal gold solution, and store it in the dark at 4°C for later use.

Embodiment 3

[0072] A preparation method of gold-labeled rR7 protein:

[0073] (1) rR7 protein pretreatment

[0074] After diluting the rR7 protein with 0.015mol / L PBS (pH 7.4), place it in an ultrafiltration centrifuge tube, centrifuge at 4°C and 5000r / min for 25min, then centrifuge at 4°C and 10000r / min for 1h, and dilute to obtain 1 mg / mL rR7 protein solution.

[0075] (2) Determination of the optimal labeling pH value

[0076] With 0.1mol / L K 2 CO 3 solution or 0.1mol / L HCl solution to adjust the pH value of the colloidal gold solution to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, take 10 1.5mL centrifuge tubes, add 1mL of different pH Add 50 μL of 1mg / mL rR7 protein to each tube, shake and mix for 5 minutes, then let it stand at room temperature for 15 minutes, then add 100 μL of 10% NaCl solution to each tube, shake and mix for 5 minutes, then let it stand for 2 hours at room temperature, observe Colloidal gold color changes, record the lowest pH that remains red.

[007...

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Abstract

The invention belongs to the technical field of biology, and discloses a colloidal gold test strip for detecting a Leucocytozoon caulleryi antibody. The colloidal gold test strip comprises a bottom plate, and a sample pad, a gold-labeled pad, a reaction pad and a water absorption pad which are arranged on the bottom plate, wherein the gold-labeled pad is a glass fiber membrane coated with a colloidal gold labeled recombinant R7 protein antigen, and the reaction pad is a nitrocellulose membrane coated with a detection line of the recombinant R7 protein antigen and a quality control line of a rabbit anti-R7 protein polyclonal antibody. When the colloidal gold test strip is used for detecting the Leucocytozoon caulleryi antibody, a result can be judged by naked eyes without any instrument andequipment. The colloidal gold test strip can be used for detecting on a sample collection site and is particularly suitable for field or on-site application. The number of detection steps is small, aresult is obtained within 10-15min, and the colloidal gold test strip is particularly suitable for large-scale detection and epidemiological investigation.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a colloidal gold test strip for detecting the antibody of Leucocystria carinii and a preparation method thereof. Background technique [0002] Leukocytozoon cariniiosis, also known as "white crown disease", is a hemosporidiosis caused by Leukocytosis carinii parasitizing in the red blood cells, white blood cells and visceral tissue cells of chickens. Leukocytosis cariniiosis mostly occurs in chicks aged 3 to 6 weeks, with severe disease and high mortality; the infection rate of young chickens is higher than that of young chickens, but the mortality rate is not high; while the infection rate of adult chickens is the highest, but the mortality rate is very low , with mild symptoms. The main clinical symptoms are pale combs, emaciation, watery white or green feces, stunted growth of chickens, decline or even stop of egg production in adult chickens, which w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/58G01N33/52C07K14/44C12N15/30C12N15/70
CPCG01N33/56905G01N33/558G01N33/58G01N33/52C07K14/44C12N15/70G01N2333/44
Inventor 邝春曼谭志坚王新秋刘丽丹翁亚彪黄仪娟
Owner FOSHAN STANDARD BIO TECH
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