Diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay

A diagnostic kit and spot immunization technology are applied in the field of immunodetection to achieve the effect of shortening diagnosis time, realizing diagnosis and on-site seroepidemiological investigation, and being easy to interpret.

Inactive Publication Date: 2010-07-21
ZHENGZHOU UNIV
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Problems solved by technology

[0005] Dot-immuogold filtration assay (DIGFA) based on colloidal gold labeling technology is a new method established in recent years for the detection of various viruses, bacteria, parasitic diseases and autoimmune diseases. It is an indirect method or a sandwich method (indirect method for antibody detection: specific antigen immobilized on the membrane + corresponding antibody in the specimen + gold-labeled antibody or aureus A (SPA) for color development; sandwich method fo

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  • Diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay
  • Diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay
  • Diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay

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[0024] Trichinellosis dot immunogold filtration method diagnostic kit for trichinellosis of the present invention, it comprises following preparation steps:

[0025] Step 1: Preparation of Ts21 recombinant protein

[0026] 1.1 Construction of Ts21 prokaryotic expression plasmid: According to the gene sequence of Trichinellaspiralis Hypothetical ORF17.20 in the GenBank nucleic acid database, primers were designed using the bioinformatics software Primer5.0, the Ts-21 gene of Trichinella spiralis was obtained by RT-PCR, and the expression vector pAML- c2X-Ts21, transform Escherichia coli TB1, and obtain the positive clone pAML-c2X-Ts21 / TB1;

[0027] 1.2 Induced expression of Ts21 recombinant protein: (1) Pick a single colony of pAML-c2X-Ts21 / TB1 into 5 mL LB medium containing 100 μg / mL ampicillin, and culture overnight at 37°C; (2) Expand culture at a ratio of 1:50 , to OD 600 ≌0.6, add the inducer isopropyl-β-D-thiogalactoside to a final concentration of 0.25mmol / L, induce cu...

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Abstract

The invention discloses a diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay. The invention has the following advantages: gold labeled SPA is taken as an antibody labeling tag, isopropyl beta-D-thiogalactoside is used for inducing the pMAL-c2X-Ts21/TB1 recombinant strain, expressing and purifying trichina Ts21 gene-encoded protein (Ts21 antigenic gene, ORF17.20, the access number at GenBank is U88239), after being purified on a nitrocellulose membrane (NCM), the Ts21 recombinant protein coats the detection point T, and human IgG coats the quality control point C, thus constructing Ts21 recombinant protein-dot-immunogold filtration assay (Ts21-DIGFA) and assembling the rapid diagnostic reagent kit for trichina with Ts21 recombinant protein by employing dot-immunogold filtration assay. The kit is simple to operate and has high sensitivity and specificity and good stability and repeatability; the detection results can be observed and judged within 1min immediately after adding samples, the diagnosis results are accurate and clear and misjudgment can not happen; and the kit is suitable for rapid diagnosis and serum epidemiological survey at the scene and is easy to popularize and apply extensively.

Description

technical field [0001] The invention relates to immune detection technology, in particular to a dot immunogold filtration method diagnostic kit for detecting anti-trichinella antibody in serum with trichinella spiralis Ts21 recombinant antigen. Background technique [0002] Trichinellosis is a serious zoonotic disease, which is mainly infected by eating raw or half-cooked pork or other animal meat containing Trichinella larva cysts. This disease not only poses a serious threat to human health, but also The pig industry and the meat import and export trade cause huge economic losses. [0003] At present, the clinical diagnosis of trichinellosis mainly depends on muscle biopsy, but it is often difficult to detect mild and early infections. Even in advanced patients, the positive rate of biopsy is only about 50% due to limited material collection, and it is not easy to be accepted by patients; A lot of research has been done on the serological diagnosis methods of insect disea...

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Application Information

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IPC IPC(8): G01N33/532C12N15/12C12N15/70C07K14/435
Inventor 崔晶王中全侯小君姜鹏王烨王来王睿逯丽秦银霞
Owner ZHENGZHOU UNIV
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