Early detection method for arch insect infection

A technology for early detection and toxoplasma gondii, applied in measuring devices, biological tests, material inspection products, etc., can solve problems such as severe fetal damage

Inactive Publication Date: 2008-12-03
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The degree of fetal damage is negatively correlated with gestational age. The earlier the infection occurs, the more severe the fetal damage will be. At present, there is no effective method for early diagnosis of toxoplasmosis

Method used

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  • Early detection method for arch insect infection
  • Early detection method for arch insect infection
  • Early detection method for arch insect infection

Examples

Experimental program
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Embodiment 1

[0028] The preparation method of the fusion protein NTPase-II of Toxoplasma gondii in this detection method comprises the following steps:

[0029] (1) Preparation of Toxoplasma gondii genomic DNA;

[0030] (2) Amplification of the Toxoplasma gondii NTPase-II gene;

[0031] (3) Toxoplasma gondii NTPase-II gene insertion cloning plasmid;

[0032] (4) Toxoplasma gondii NTPase-II gene insertion expression plasmid;

[0033] (5) transferring the recombinant obtained in (4) into a host cell;

[0034] (6) Induced expression of fusion protein NTPase-II;

[0035] (7) Separating and purifying the product of (5) to obtain NTPase-II expression protein.

[0036] Concrete preparation process is as follows:

[0037] (1) Preparation of Toxoplasma gondii genomic DNA:

[0038] The tachyzoites of Toxoplasma gondii RH strain were continuously passaged in mice, and the ascites was aseptically extracted 3 days after the infection, the parasites were washed with normal saline for 3 times, the ...

Embodiment 2

[0076] Dilute the purified NTPase-II with carbonic acid buffer to make a coating solution of 5 μg / ml, coat 100 μl / well on a 96-well ELISA plate, and coat overnight at 4°C; wash the plate 3-5 times with PBST, usually 3 times. Yes, 2-3min / time, pat dry; add 5% skimmed milk powder, seal at 37°C for 2h; PBST wash plate 3-5 times, usually 5 times, 2-3min / time, pat dry; add dilution of 1:200 serum of the above-mentioned tested mice, 100 μl / well, incubate at 37°C for 1.5 h; wash the plate with PBST 5-7 times, preferably 7 times, 2-3 min each time, and pat dry; the dilution ratio is 1: 4 000 goat anti-mouse enzyme-labeled secondary antibody IgG, 100μl / well, incubate at 37°C for 1h, wash the plate with PBST 5-7 times, preferably 7 times, 2-3min / time, pat dry; add substrate chromogenic solution Solution A and B, develop color at 37°C in the dark for 10-15 minutes, add stop solution 2mol / L concentrated H 2 SO 4 Terminate the reaction, 50 μl / well; use a microplate reader to detect the a...

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Abstract

The invention relates to a method for detecting toxoplasma infection early. The nucleoside triphosphate hydrolase-II (NTPase-II) of toxoplasma is an indispensable key enzyme in the process that purine is remedied and synthesized by the toxoplasma by utilizing host cells, play an important part in parasitism and propagation of the toxoplasma, and is a major antigen for causing a body to generate immune reaction in the acute infection stage of the toxoplasma. The gene of the toxoplasma nucleoside triphosphate hydrolase-II is expanded by utilizing a polymerase chain reaction through the invention, expression plasmids are constructed and recombined in a expression vector, the recombined expression plasmids are transferred to procaryotic cells for expression, recombined fusion protein NTPase-II is obtained, the recombined fusion protein NTPase-II is used as an indirect ELISA method to establish a coating antigen, and is used as the method for detecting the toxoplasma infection early.

Description

technical field [0001] The invention relates to a detection method of obligate intracellular parasitic opportunistic pathogenic protozoa, in particular to a method for early detection of toxoplasma gondii infection. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic opportunistic pathogenic protozoan, which parasitizes and reproduces in the nucleated cells of humans and animals, causing toxoplasmosis that is zoonotic. Toxoplasmosis is distributed worldwide, and the average infection rate of human Toxoplasma is 33%. Healthy adults generally do not show obvious clinical symptoms after infection, but they can persist in the body for many years. When the body's immunity is weakened or severely infected, this latent infection can show significant symptoms of toxoplasmosis, such as toxoplasma eye disease, toxoplasma hepatitis, toxoplasma lymphadenitis, etc. Toxoplasma gondii infection often presents as a fatal lesion in imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/53
Inventor 谭峰潘长旺沙丹李丽宁
Owner WENZHOU MEDICAL UNIV
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