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Application of EG-TPx and kit for diagnosing echinococcosis granulosa of livestock

A technology for Echinococcus granulosus and Echinococcus granulosus, which is applied in the field of kits for diagnosing Echinococcus granulosus in livestock, can solve the problems of low sensitivity and specificity, and achieves high specificity and sensitivity, Good diagnostic effect, good immunogenicity effect

Inactive Publication Date: 2018-12-11
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on recombinant diagnostic antigens for this disease, and there are problems such as low sensitivity and specificity. Therefore, screening an effective, sensitive and specific recombinant diagnostic antigen and establishing accurate diagnostic methods are the focus of breakthroughs in the future. One of the research directions

Method used

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  • Application of EG-TPx and kit for diagnosing echinococcosis granulosa of livestock
  • Application of EG-TPx and kit for diagnosing echinococcosis granulosa of livestock
  • Application of EG-TPx and kit for diagnosing echinococcosis granulosa of livestock

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Preparation of recombinant antigen

[0027] 1. Extraction of total RNA from Echinococcus granulosus

[0028] 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.

[0029] 2. Synthesis of first-strand cDNA

[0030] The extracted Echinococcus granulosus total RNA was used as a template, Oligo dT(18) was used as a reverse transcription primer, and the operation was performed according to the instructions of the reverse transcription kit from Thermo Company.

[0031] 3. Amplification of Eg-TPx gene

[0032] According to the published gene sequence of Eg-TPx (EgrG_000791700), primers were designed using Primer Premier 5.0 software:

[0033] TPx-F: 5'-CGC GGATCC ATGGCTGCTGTTGTTGG-3' (SEQ ID NO: 1), the underline is BamHI;

[0034] TPx-R: 5'-CCG GAATTC TCACGAGCTCATGAACGA-3' (SEQ ID NO: 2), EcoRI is underlined.

[0...

Embodiment 2

[0090] The establishment of embodiment 2 indirect ELISA method

[0091] 1. Indirect ELISA operation steps

[0092] (1) Dilute the recombinant antigen rEg-TPx in proportion with the antigen coating solution, add 100 μL per well to a 96 microplate plate for coating;

[0093] (2) Pour off the coating solution, pat dry the liquid in the well, wash with PBST, 5min each time, repeat four times;

[0094] (3) Add blocking solution to seal;

[0095] (4) After washing, the serum was diluted in proportion with PBS, and 100 μL of each well was added to the enzyme-labeled wells for incubation, and the liquid was poured off;

[0096] (5) After washing, add 100 μL diluted HRP-labeled goat or sheep anti-rabbit secondary antibody to each well and incubate;

[0097] (6) Add the soluble one-component substrate TMB to the well under the condition of avoiding light to carry out the color reaction;

[0098] (7) Add 100 μL 2M H 2 SO 4 Terminate the reaction, measure its OD value when the UV ab...

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Abstract

The invention discloses application of EG-TPx and a kit for diagnosing echinococcosis granulosa of livestock. The EG-TPx can serve as an immunizing antigen and can be recognized by positive sheep serum with natural infection of echinococcosis granulosa. When applied to indirect ELISA (Enzyme-Linked Immuno Sorbent Assay), the EG-TPx has excellent immunogenicity and high specificity and sensitivity,the sensitivity is 92.6%, the specificity is 99.0%, and the cross reaction with other taeniasis positive serum is low. The ELISA detection method established by an immunizing antigen prepared from echinococcus granulosus thioredoxin peroxidase has an excellent diagnosis effect, and can be applied to preliminary screening of echinococcosis granulosa of cattle and sheep in epidemic areas.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to the application of Echinococcus granulosus thioredoxin peroxidase (Eg-TPx) and a kit for diagnosing echinococcosis granulosus in domestic animals. Background technique [0002] Echinococcus granulosus is a zoonotic disease caused by the middle-stage larvae of Echinococcus granulosus parasitizing the liver and lungs of various mammals. The disease, also known as hydatid disease, is distributed worldwide and seriously threatens the development of public health and animal husbandry. Currently, the disease infects about 3 million people and costs an estimated $760 million annually. Additionally, the disease has caused economic losses of at least $3 billion in the livestock industry, including problems such as reduced animal weight, reduced milk production and reduced fertility. Therefore, the disease has been listed by the World Health Organization as a Neglected Tr...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/535G01N33/536C12N9/08C12N15/70
CPCC12N9/0065C12Y111/01015G01N33/535G01N33/536G01N33/573G01N2333/908
Inventor 杨光友梁雨琴
Owner SICHUAN AGRI UNIV
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