Enzyme-linked immunoassay method for detecting sheep toxoplasma gondii igg antibody

An enzyme-linked immunosorbent assay, Toxoplasma gondii technology, applied in immunoassays, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of inappropriate large-scale inspection, low detection rate, time-consuming and laborious, etc., and achieve high sensitivity. , the effect of good specificity and easy automation

Inactive Publication Date: 2019-02-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological detection is to detect oocysts or trophozoites from isolated disease materials. This method is accurate and reliable, but it is time-consuming and laborious, with a low detection rate and is not suitable for large-scale inspections

Method used

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  • Enzyme-linked immunoassay method for detecting sheep toxoplasma gondii igg antibody
  • Enzyme-linked immunoassay method for detecting sheep toxoplasma gondii igg antibody
  • Enzyme-linked immunoassay method for detecting sheep toxoplasma gondii igg antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of dense granule protein GRA1

[0029] After extracting the RNA of Toxoplasma gondii, use the Takara reverse transcription kit to obtain cDNA, use the clonmanager molecular cloning software to design relevant primers according to the required target fragment, and amplify the target fragment by PCR method, and use the method of homologous recombination to synthesize GRA1-CDs The fragment was ligated into the pE-SUMO vector, and after being verified by enzyme digestion and sequencing, the constructed pE-SUMO-GRA1 plasmid was transferred to Escherichia coli BL21(DE3) for expression.

[0030] 1. Obtaining the target fragment of GRA1

[0031] (1) Extraction of Toxoplasma gondii RNA

[0032] Centrifuge the collected parasites, discard the supernatant, add 1ml Trizol to resuspend the pellet, and repeatedly blow and mix, then transfer the sample to a 1.5ml RNase-free centrifuge tube, shake and vortex vigorously for 3 minutes to fully lyse the parasites, ...

Embodiment 2

[0064] Embodiment 2: establish fast and accurate sheep toxoplasmosis on-the-spot diagnosis method (GRA1-iELISA)

[0065] 1. (1) Determination of antigen coating concentration and serum dilution multiple: Dilute GRA1 protein in gradients of 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, and 0.3125 μg / mL, respectively, and coat ELISA plate, each concentration is coated with a column of 5 wells. Dilute negative and positive control sera with warming solution at 1:25, 1:50, 1:100, 1:200, and 1:400, respectively, and add each dilution to 5 wells in a row. Operate according to the routine steps of ELISA, and measure OD with a microplate reader 630 value, compare the size of the P / N value corresponding to each antigen, and select the antigen coating concentration and serum dilution factor corresponding to the maximum P / N value of the optimal antigen as the optimal condition.

[0066] (2) Optimization of the optimal blocking concentration and optimal blocking time: optimize the blocki...

Embodiment 3

[0088] Embodiment 3: Sensitivity test and specificity test of sheep Toxoplasma gondii indirect ELISA detection method

[0089] According to the method of Example 2, 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 dilutions were respectively performed on 5 parts of positive sera for detection, and set positive, negative, Blank control. The results are shown in Table 2. The results show that the sensitivity of the GRA1-iELISA diagnostic method established in the present invention is greater than 1:100. The established method was used to detect the positive sera of goat aplasma, goat Babesia mosoni, goat Theileria reuteri and goat positive sera of Haemonchus contortus. The results are shown in Table 3. The results showed that the specificity of this method was good, and there was no cross-reaction with Babesia mosoni, Theileria reuvei and Haemophilus contortus.

[0090] Table 2 Sensitivity test results

[0091] Dilution factor

1:25

1:50

1:100

1:200

1:40...

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Abstract

The invention discloses a toxoplasma gondii dense granular protein GRA1. A nucleotide sequence for encoding the protein is shown as SEQ ID NO:1. The invention further discloses an application of the dense granular protein GRA1 to the preparation of an enzyme-linked immunoassay detecting kit for treating toxoplasmosis and an enzyme-linked immunoassay method for detecting a sheep toxoplasma gondii IgG antibody. The method has the advantages such as high sensitivity, strong specificity, good accuracy as well as high speed, simplicity and convenience when being used for detecting the toxoplasmosis.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent method (ELISA) for detecting goat toxoplasma IgG antibody, and also relates to a toxoplasma gondii dense granule protein GRA1 which can specifically react with goat toxoplasma gondii IgG antibody. technical background [0002] Toxoplasma gondii is a zoonotic protozoan widely distributed in the world, which can cause diseases in humans and various animals. It is estimated that about 1 / 3 of the world's people are infected with Toxoplasma gondii. Among animals, pigs have the highest infection rate, generally above 20%, and some farms even reach 100%. Sheep are the most sensitive animals to Toxoplasma gondii except mice. After being infected with Toxoplasma gondii, sheep are prone to miscarriage, stillbirth, weak fetus, etc. In some countries, up to 20% of sheep abortions are caused by Toxoplasma gondii infection. industry has caused great losses. In China, the average infection rate of flocks is ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/45C12N15/30G01N33/569G01N33/543
CPCC07K14/45G01N33/54306G01N33/56905G01N2333/45G01N2469/20
Inventor 申邦李迎迎赵俊龙周艳琴方瑞贺兰王敏侯伦李明俊
Owner HUAZHONG AGRI UNIV
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