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Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1

A detection kit and matrix protein technology, applied in the biological field, can solve problems such as difficult application in sick animals, and achieve the effects of good repeatability, good specificity, and easy source

Inactive Publication Date: 2014-11-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Serological diagnosis of Toxoplasma gondii is feasible for acute infections in normal hosts but difficult for immunosuppressed sick animals

Method used

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  • Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1
  • Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1
  • Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Prokaryotic expression and purification of Toxoplasma gondii MAG1 protein

[0046] 1. Materials

[0047] 10× PCR Buffer, Taq Reagents required for PCR reactions such as DNA polymerase and dNTP, DNA Marker DL2000, and gel purification and recovery kit were purchased from Aisigen; Ni-FF affinity chromatography column was purchased from Beijing Weishi Bohui Chromatography Technology Co., Ltd.; restrictions endonuclease Kpn Ⅰ and EcoR Ⅰ, T4 DNA ligase, nucleic acid electrophoresis dye GoldView, purchased from Saibaisheng Biological Company; Biometra T-Gradient Thermoblock gradient PCR instrument, electrophoresis instrument, products of Beijing Liuyi Instrument Factory; gel imaging analysis system, Shanghai Peiqing Technology Co., Ltd.; constant temperature shaker, ultrasonic cracker, biochemical incubator, micro oscillator, pipette gun, pipette tip.

[0048] 2. Amplification and purification of MAG1 fragments

[0049]

[0050] Using the target gene ...

Embodiment 2

[0057] Example 2 Indirect ELISA detection technology specific detection results of Toxoplasma gondii MAG1 protein

[0058] 1. Materials

[0059] Biochemical incubator, micro shaker, wet box, pipette, NanoDrop ND-1000 UV spectrophotometer. Toxoplasma recombinant MAG1 protein, Toxoplasma canine positive serum, Toxoplasma canine negative serum and Escherichia coli, Salmonella, Listeria, canine parvovirus positive sera preserved in our laboratory. HRP enzyme-labeled SPA (dry powder purchased from Sangon Biotechnology Co., Ltd.), OPD, 30% H 2 o 2 solution, 0.2 M Na 2 HPO 4 solution, 0.1 M citric acid solution, skimmed milk (purchased from Guangming Dairy), Tween20, 2 M H 2 SO 4 solution, 0.01M PBS solution with pH 7.4.

[0060] 2. Dilution of recombinant protein

[0061] The MAG1 protein preserved in the laboratory was diluted to 15 μg / ml with 10 mM PBS at pH 9.6, and 100 μl per well was used for coating.

[0062] 3. Dilution of serum

[0063] Toxoplasma gondii canine p...

Embodiment 3

[0069] Example 3 Indirect ELISA Detection Technology Reproducibility Detection Results of Toxoplasma gondii MAG1 Protein

[0070] 1. Dilution of Positive Sera

[0071] Take 14 canine serum, dilute 1:50 with 10 mM PBS, pH 7.4 containing 5% skimmed milk powder, and use it as the primary antibody.

[0072] 2. Repeatability detection

[0073] Method is the same as embodiment one. Take 7 diluted sera, do 4 tests for 4 consecutive days, compare the differences in the test results, and calculate the coefficient of difference between the plates; take another 7 diluted sera, and do 3 replicates of each serum on the same plate, compare The difference between the results was calculated by calculating the coefficient of variation within the plate.

[0074] 3. Test results

[0075] The test results are shown in Table 3 and Table 4. The coefficient of variation between plates is less than 3%, and the coefficient of difference within the plate is less than 10%. This method has good rep...

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Abstract

The invention provides an indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1. The indirect ELISA detection kit consists of a 96-hole elisa plate, a substrate coating substance, a standard substance, a reference substance, a substrate solution, skim milk powder, a sample diluent, washing liquid, reaction liquid, color developing liquid and stopping liquid. The indirect ELISA detection kit can identify IgGs of a variety of mammals such as humans, rabbits, pigs, dogs, cats, monkeys, mice, etc., can detect infection of toxoplasma gondii relatively rapidly, is high in specificity and sensitivity, and especially in large-scale clinical detection, can greatly improve the detection efficiency; in a detection method, an expensive PCR detector and other equipment are not required, but only an UV spectrophotometer, a thermostat and other equipment are needed; the indirect ELISA detection kit is simple in operation, high in specificity, good in repeatability and clear and stable in result; a sample to be detected is very easy to obtain.

Description

technical field [0001] The invention belongs to biotechnology, and relates to an indirect ELISA detection kit based on toxoplasma gondii matrix protein 1 (MAG1) and its application, which can detect toxoplasma gondii infection in humans, pigs, cats, dogs, mice and the like. Background technique [0002] Toxoplasma gondii is a worldwide distribution of intracellular parasitic protozoa, which can infect various warm-blooded animals including humans, causing toxoplasmosis. The broad host population makes it one of the most successful parasites (Boothroyd, 2009). In humans, toxoplasmosis is mostly an insidious infection with lymphadenopathy, low-grade fever, malaise, sore throat, and lethargy. In immunosuppressed patients, more severe symptoms are present, including toxoplasma encephalitis, myocarditis, pneumonia, hepatitis, and systemic organ disorders (Utsuki et al., 2011). In pregnant women, congenital infections can lead to miscarriage, birth defects, blindness, etc. In a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/54393G01N33/56905G01N33/6893G01N2333/45
Inventor 杜爱芳卓洵辉张智赵现锋周前进
Owner ZHEJIANG UNIV
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