ELISA detection method capable of distinguishing acute and chronic toxoplasma gondii infection and application thereof

A technology of Toxoplasma gondii and specificity, applied in the field of bioengineering, can solve the problems of low sensitivity and poor specificity of Toxoplasma gondii, and achieve the effect of improving sensitivity, strong specificity and high sensitivity

Pending Publication Date: 2021-10-15
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The present invention aims to solve the common technical problems of low sensitivity and poor specificity in current serological detection methods for Toxoplasma gondii, and provides two antigens for detecting acute and chronic Toxoplasma gondii infection, which can be detected accurately and quickly by using the antigens for ELISA detection Acute and chronic Toxoplasma gondii infection with high sensitivity and specificity

Method used

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  • ELISA detection method capable of distinguishing acute and chronic toxoplasma gondii infection and application thereof
  • ELISA detection method capable of distinguishing acute and chronic toxoplasma gondii infection and application thereof
  • ELISA detection method capable of distinguishing acute and chronic toxoplasma gondii infection and application thereof

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Embodiment 1

[0027] Example 1 Establishment of Toxoplasma gondii ELISA detection method

[0028] Toxoplasma gondii strain culture and nucleic acid extraction: The Toxoplasma gondii RH strain used in the test is an international standard type I strain, which was recovered by our laboratory and obtained through subculture of human foreskin fibroblasts (HFF). For the culture of Toxoplasma gondii tachyzoites, the parasites were taken out of liquid nitrogen and resuscitated and inoculated into HFF cells cultured in vitro, 37 ° C, pH 7.2-7.4 DMEM medium, containing 8% heat-inactivated fetal bovine serum, 5 %CO 2 Cultivate in an incubator and observe the worms with a microscope. For the purification of worms, the cells with worms were washed with pre-cooled PBS, suspended, passed through a 27-G needle three times, filtered through a 5 μm filter membrane (Millipore, USA), and then centrifuged at 1500rpm for 10 minutes to collect the worms, suspended in PBS, and blood cells Counting plate counts....

Embodiment 2

[0031] Example 2 Application of Toxoplasma gondii ELISA detection method

[0032] ELISA specific test: positive sera for coccidia, cryptosporidium and schistosomiasis were provided by the key open laboratory of animal parasitology of the Ministry of Agriculture of the Institute. Dilute all the worm positive serum as the sample to be tested, use the above SAG2 and SAG4 as antigens to detect by ELISA method, and observe its specificity. The results showed that the specificity analysis of ELISA on the positive sera of various parasites showed strong specific reactions only to the positive sera of Toxoplasma gondii, and no specific reactions to the sera of other parasites. ELISA sensitivity comparison test: Dilute the purified SAG2 or SAG2 and SAG4 mixed protein (SAG2+SAG4) to 5 μg / mL, add 100 μL to each well, and mix positive Toxoplasma-infected mouse serum and healthy mouse serum at a concentration ratio of 1: 200, 1:400, 1:800 and 1:1600 for doubling dilution, 100 μL / well, eac...

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Abstract

The invention provides a detection method capable of simultaneously detecting acute and chronic toxoplasma gondii infection and application thereof, provides an antigen for detecting toxoplasma gondii infection, and establishes a toxoplasma gondii ELISA detection technology by using the antigen. The ELISA detection technology is further established for detecting toxoplasma gondii infection, the toxoplasma gondii infection serum prepared in vivo is used for carrying out condition exploration of the detection method, and the detection method is compared with a single protein coating method for comparative analysis. Meanwhile, the established method is used for detecting a serum sample of a clinical animal. The result shows that the toxoplasma gondii ELISA detection technology with high sensitivity and strong specificity is successfully constructed, and the method can be used for clinical detection.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an ELISA detection method capable of distinguishing acute and chronic Toxoplasma gondii infection and its application. Background technique [0002] Toxoplasma gondii, belonging to the subphylum Apicomplexa, class Sporozoa, order Eucoccidia, family Isospora, and genus Toxoplasma, is an obligate intracellular protozoan parasite capable of infecting a wide range of hosts, including humans and Most warm-blooded animals. Approximately one-third of the world's population is infected with Toxoplasma gondii, the parasite is controlled by host immunity, and infection in most people is asymptomatic; however, the infection can lead to immunocompromise, which can cause complications and even death, with Toxoplasma gondii infection in pregnant women Worms can cause fetal abortion, fetal death, and fatal encephalitis in AIDS patients. Toxoplasma gondii spreads among livestock and pou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/56905G01N33/543G01N2333/45
Inventor 张厚双周金林曹杰周勇志林晓幸
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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