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Application of echinococcus granulosus glutaredoxin-1

A technology for Echinococcus granulosus and Echinococcus granulosus, which is applied in the application field of Echinococcus granulosus glutaredoxin-1, can solve the problems of unstable antigen source, complex composition and high cost, and achieve good results. Effects of diagnostic efficacy, high specificity, and sensitivity

Active Publication Date: 2016-11-09
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on the immunodiagnosis of echinococcosis granulosus mainly focuses on the antigen of crude cyst fluid, but its composition is complex, and it has defects such as difficulty in large-scale purification, unstable source of antigen, high cost, and low diagnostic specificity.
Lack of standard validated specific recombinant antigens is an unsolved problem in the immunodiagnosis of echinococcosis

Method used

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  • Application of echinococcus granulosus glutaredoxin-1
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  • Application of echinococcus granulosus glutaredoxin-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0044] The cysts of middle-stage larvae (Echinococcus granulosus) of Echinococcus granulosus were collected from naturally infected sheep livers, and 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 1mi...

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-Grx1 gene

[0070] According to the gene sequence of Eg-Grx1 (EgrG_000124800) published in GeneDB (http: / / www.genedb.org / Homepage / Egranulosus), primers were designed with Primer Premier 5.0 software:

[0071] Upstream: 5'-CC GGAATTC ATGTGGCGCTTTTTATC-3' is underlined as EcoR I

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

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

[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 351bp 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 concretely discloses application of echinococcus granulosa glutaredoxin-1 to preparation of an immunizing antigen and / or a kit for detecting echinococcosis granulose. When being used as an immunizing antigen, the echinococcus granulosa glutaredoxin-1 can be recognized by sheep positive blood serum naturally infecting the echinococcosis granulose. When being applied to indirect ELISA detection, the echinococcus granulosa glutaredoxin-1 has high specificity and sensitivity; the clinic detection coincidence rate is as high as 97.9 percent. The detection method built by using the echinococcus granulosa glutaredoxin-1 as the immunizing antigen has a good diagnosis effect, and can be used for the primary screening of the echinococcus granulosa of sheep in an infected area.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the application of Echinococcus granulosus glutaredoxin-1. Background technique [0002] Echinococcus granulosus (Echinococcus granulosus) belongs to the band family (Taeniidae) Echinococcus (Echinococcus), in which tape stage larvae parasitize in the liver, lung and other organs of animals and humans and cause echinococcosis, also known as Cystic echinococcosis is a zoonotic disease that mainly parasitizes in many mammals such as humans and domestic animals. 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-ye...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 杨光友宋星桔
Owner SICHUAN AGRI UNIV
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