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Applications of echinococcus granulosus dihydrofolate reductase

A technology of Echinococcus granulosus and dihydrofolic acid, which is applied in the biological field, can solve the problems of inaccurate diagnosis of Echinococcus granulosus and high probability of cross-reaction, and achieves the effects of good diagnostic effect, high specificity and sensitivity

Active Publication Date: 2016-12-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on recombinant diagnostic antigens of echinococcosis in animals. Liu Hongxia et al. used Echinococcus granulosus CE18 recombinant protein to detect echinococcosis granulosus in sheep and found that other tapeworm-positive sera such as sheep brain There is a cross-reaction between the serum of polycephalic cysticercosis and the serum of sheep fine-necked cysticercosis, especially the high probability of cross-reaction with the serum of sheep fine-necked cysticercosis, which cannot accurately diagnose sheep echinococcosis

Method used

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  • Applications of echinococcus granulosus dihydrofolate reductase
  • Applications of echinococcus granulosus dihydrofolate reductase
  • Applications of echinococcus granulosus dihydrofolate reductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Extraction of total RNA from Echinococcus granulosus

[0044] Echinococcus granulosus cysts were obtained from naturally infected sheep livers. The samples were collected from Xining, Qinghai or Ganzi, Sichuan, and stored in liquid nitrogen.

[0045] Echinococcus granulosus preserved in liquid nitrogen was taken out, ground with a mortar, and then total RNA was extracted according to the instructions of Tiangen's animal tissue RNA extraction kit.

[0046] (1) Add 300 μL of lysate for every 10-20 mg of protocephalus, and grind with a grinding rod;

[0047] (2) Add Proteinase K (10 μL) and RNase-Free ddH2O (590 μL) to the homogenate, mix well and react for 15 minutes (56°C).

[0048] (3) Centrifuge for 5min (12,000rpm), transfer the supernatant to a clean tube; add 0.5 times the volume of supernatant to the tube with absolute ethanol, mix well and transfer to the adsorption column, centrifuge for 1min (12,000rpm) Discard the waste liquid.

[0049] (4) Add pr...

Embodiment 2

[0055] Example 2: Synthesis of first-strand cDNA

[0056] Using the extracted total RNA of Echinococcus granulosus as a template and Oligo dT(18) as a reverse transcription primer, operate according to the instructions of the reverse transcription kit from Thermo Company:

[0057] (1) Prepare the reaction mixture on ice

[0058] Oligo dT 18 1μL

[0059] Template RNA 1 μL

[0060] DEPC ddH 2 O 1 μL

[0061] (2) After incubation at 70°C (5min), transfer to ice to cool

[0062] (3) Add the following reactants in order on ice:

[0063] 5 reaction buffer 4μL

[0064] RNase inhibitor 1 μL

[0065] 10dNTP mix 2 μL

[0066] (4) Add RevertAid after incubation at 37°C (5min) TM Reverse transcriptase 1 μL.

[0067] (5) PCR program: 42°C, 1h; 70°C, 10min.

[0068] (6) Store the obtained cDNA at -80°C.

Embodiment 3

[0069] Embodiment 3: Amplification of Eg-DHFR gene

[0070] According to the gene sequence of Eg-DHFR (EgrG_000572400) published in GeneDB (http: / / www.genedb.org / Homepage / Egranulosus), primers were designed with Primer Premier 5.0 software:

[0071] Upstream: 5'-CGC GGATCC ATGGGGCTGAAGCGTCT-3' is underlined as BamH I

[0072] Downstream: 5'-CCG CTCGAG ATGATCATTAAGGGGATGCG-3' Underline Xho I

[0073] Amplification system (25 μL): DNA template 1 μL each, upstream and downstream primers 1 μL each, PCR Mixture 12.5 μL, sterilized double distilled water 9.5 μL.

[0074] Amplification conditions: pre-denaturation: 95°C for 5min; 38 cycles (denaturation: 95°C, 40s; annealing: 54°C, 45s; extension: 72°C, 45s); final extension: 72°C, 10min.

[0075] A band of about 576 bp was amplified using the cDNA of Echinococcus granulosus Protoscoleum as a template ( figure 1 ).

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Abstract

The invention relates to the technical field of biology, and particularly discloses applications of echinococcus granulosus dihydrofolate reductase to preparing an immunizing antigen and / or a kit which are / is used for detecting echinococciasis granulous. The echinococcus granulosus dihydrofolate reductase serves as the immunizing antigen and can be identified by sheep positive blood serums naturally infected by the echinococciasis granulous; when the echinococcus granulosus dihydrofolate reductase is applied to indirect ELISA detection, the high specificity and the high sensitivity are achieved, and the clinical-detection coincidence rate is up to 97.9%. A detection method built with the echinococcus granulosus dihydrofolate reductase serving as the immunizing antigen has the good diagnosing effect, and can be used for preliminary screening of the sheep echinococciasis granulous in an affected area.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the application of Echinococcus granulosus dihydrofolate reductase. Background technique [0002] Echinococcus granulosus, also known as cystic echinococcosis, is a zoonotic disease caused by the middle-stage larvae of Echinococcus granulosus, which mainly parasitizes various mammals such as humans and domestic animals. inside the animal. More than 90% of Echinococcus granulosus cysts grow in the host's liver, lung or both. Echinococcus granulosus is distributed worldwide, causing a series of economic and public health problems. In some endemic areas, the infection rate can be as high as 5-10%, and the mortality rate can be as high as 2-4%. It is estimated that human cystic echinococcosis loses at least 1-3.6 million disability-adjusted life-years (DALYs) annually globally, while animal cystic echinococcosis causes economic losses of at least $2 billion per year. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N33/573G01N2333/43543
Inventor 杨光友宋星桔
Owner SICHUAN AGRI UNIV
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