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Bovine tuberculosis antibody identifying and detecting test strip prepared by applying Rv3872 novel fusion protein

A detection test strip, rv3872-cfp10-esat6 technology, applied in the field of animal bacteriology and zoonotic diseases, can solve the problems of inability to distinguish BCG and non-tuberculous mycobacterium infection, lack of differential diagnosis function, etc., and achieve short detection time , high sensitivity and convenient storage

Inactive Publication Date: 2010-12-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The previous work of the applicant is the authorized patent (Patent No. ZL 2006101665510; Invention name: immunocolloidal gold test strip for detecting bovine tuberculosis antibody, date of authorization announcement: June 3, 2009) is also a bovine tuberculosis antibody detection method, but This method does not have the function of differential diagnosis, and cannot distinguish between BCG and non-tuberculous mycobacterium infection, while the present invention is an immune colloidal gold test strip designed for the specific early secretion protein of virulent Mycobacterium bovis, except that it has been In addition to the patented sensitivity and specificity, it also has new functions for differential diagnosis

Method used

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  • Bovine tuberculosis antibody identifying and detecting test strip prepared by applying Rv3872 novel fusion protein
  • Bovine tuberculosis antibody identifying and detecting test strip prepared by applying Rv3872 novel fusion protein
  • Bovine tuberculosis antibody identifying and detecting test strip prepared by applying Rv3872 novel fusion protein

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Experimental program
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Embodiment 1

[0048] Embodiment 1 (preparation embodiment)

[0049] (1) Cloning of Mycobacterium bovis-specific secretory protein RCE gene and its expression and purification method in recombinant Escherichia coli refer to (patent application number: 200910060913.1; publication number CN101538578, announcement date: September 23, 2009) method.

[0050] (2) Purify the IgG antibody of RCE protein

[0051] Centrifuge the serum containing RCE antibody at 4000r / min for 10 minutes and take the supernatant for purification: take 10ml of the supernatant and centrifuge at 12000r / min for 10 minutes at 4°C, discard impurities, and then add 40ml of 0.06M acetate buffer at pH4.5 solution, and slowly add 330 μl octanoic acid at room temperature (25°C), stirring while adding dropwise. Slowly add saturated ammonium sulfate to a final concentration of 45%, dissolve with 0.01M PBS (pH7.4) and dialyze overnight. SDS-PAGE electrophoresis analysis (as attached Figure 11 shown) shows two bands, one for the ...

Embodiment 2

[0052] Embodiment 2 (preparation embodiment)

[0053] Assembling and preparation method of diagnostic test strip for Mycobacterium bovis specific secretory protein RCE antibody

[0054] 1. RCE test strips include:

[0055] The sample pad (2), binding pad (3), nitrocellulose membrane (4), water-absorbent pad (7) and PVC backing (1) are commercially available, and its specific structure is: on the PVC backing (1) press A sample pad (2), a binding pad (3), a nitrocellulose membrane (4), and a water-absorbing pad (7) are adhered in sequence; the binding pad (3) is coated with MPBrce protein-colloidal gold markers ; The nitrocellulose membrane (4) is respectively coated with a detection line (5) of purified RCE protein and a quality control line (6) composed of purified RCE protein IgG.

[0056] 2 Preparation of colloidal gold:

[0057] Dilute 1% chloroauric acid to 0.01% with ultrapure water, stir and boil on a magnetic heating stirrer, add 2.0ml 1% trisodium citrate for every 10...

Embodiment 3

[0068] Embodiment 3 (application embodiment)

[0069] How to use the immune colloidal gold test strip for bovine tuberculosis antibody

[0070] 1. Pretreatment of serum samples

[0071] Serum samples were centrifuged at 4000rpm / min for 10min to remove blood cells, stored at -20°C for a long time, and stored at 4°C for a short term.

[0072] 2. Detection steps

[0073] Use a pipette to draw 40 μl of the serum to be tested and mix it with 40 μl of serum diluent, then slowly drop it on the sample pad of the colloidal gold test strip, and observe the result after 20 minutes.

[0074] 3. Result judgment

[0075] Such as image 3 As shown, if the color of the detection line of the test strip of the sample to be tested is obviously darker than that of the test strip of the positive standard product, and a red band appears on the quality control line, it is judged as a positive sample, that is, there is bovine tuberculosis in the sample to be tested. Mycobacterium-specific secret...

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Abstract

The invention belongs to the technical field of animal infectious disease gene engineering and discloses a bovine tuberculosis antibody detecting immune colloidal gold test strip prepared by utilizing RV3872, ESAT6 and CFP10 fusion protein, and a preparation method and application. A colloidal gold immunochromatographic test strip is established by using the fusion protein as a colloidal gold labeled antigen and a capture antigen in a detection region of a nitrocellulose membrane. The detection of the bovine tuberculosis antibody by using test strip has prominent advantages of strong specificity and high sensitivity, and simultaneously bacillus calmette-guerin immunity and nontuberculosis mycobacteria infection can be identified and detected. The test strip comprises recombinant Escherichia coli BL21 / pET28a-MPBrce, and the strain expresses mycobacterium bovis RCE proteins and is preserved in the China Center for Type Culture Collection with the collection number of CCTCC No:M208244.

Description

technical field [0001] The invention relates to the technical fields of animal bacteriology and animal infectious disease. Specifically, it is an immune colloidal gold test strip for rapid and differential detection of bovine tuberculosis antibody by means of an immunochromatographic reaction of colloidal gold labeling and color development, and a preparation method thereof. Background technique [0002] Bovine tuberculosis is a chronic wasting zoonotic disease caused by Mycobacterium bovis, which is one of the diseases that must be notified by the International Organization for Animal Health (OIE). Part of the statistical results in 2001 and 2002 showed that the positive rate of cow tuberculosis in some provinces and cities in my country was as high as 10%. The prevention and control measure of bovine tuberculosis is "quarantine-culling", that is, the allergy of bovine purified tuberculin (PPD) is used for quarantine, and the positive cattle detected are culled. The basic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/544G01N33/532C12N15/62C12N15/70C12N1/21C07K19/00C12R1/19
Inventor 郭爱珍于清龙刘冬光涂玲玲陈颖钰廖娟红凌洁玉陈焕春
Owner HUAZHONG AGRI UNIV
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