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Sandwich type immunochromatographic test paper for detection of Escherichia coli o157:h7

A technology of Escherichia coli and O157, applied in the field of bioengineering, can solve the problems of long detection time, low sensitivity, and complicated detection process, and achieve the effect of low production cost, high sensitivity, and high sensitivity

Active Publication Date: 2017-11-07
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the above technical problems in the prior art, the invention provides a sandwich type immunochromatographic test paper for detecting Escherichia coli O157:H7, the described sandwich type immune layer for detecting Escherichia coli O157:H7 The analysis of test paper should solve the technical problems of low sensitivity, long detection time and complicated detection process of the method for detecting Escherichia coli O157:H7 in food in the prior art

Method used

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  • Sandwich type immunochromatographic test paper for detection of Escherichia coli o157:h7
  • Sandwich type immunochromatographic test paper for detection of Escherichia coli o157:h7
  • Sandwich type immunochromatographic test paper for detection of Escherichia coli o157:h7

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Embodiment 1

[0042] The preparation process of the test paper of the present invention comprises the steps of: preparation of Escherichia coli O157:H7 monoclonal or polyclonal antibody, preparation of an adsorption fiber layer, preparation of a cellulose film layer, assembly of a test paper strip, and the like.

[0043] (1) Preparation of anti-Escherichia coli O157:H7 monoclonal antibody or polyclonal antibody

[0044] Monoclonal antibody preparation: at an inactivating concentration of 10 8 Escherichia coli O157:H7 cfu / mL whole bacteria immunized 6-8 week-old Balb / C mice 3-4 times, with an interval of 3-5 weeks between each immunization. After confirming that the antibody titer met the requirements, super-strong immunization was performed, and then 3 On ~4 days, blood was collected from the infraorbital sinus of the immunized mice, and the positive serum was separated; the mice were killed by dislocation of the neck, and the mice were soaked in 75% alcohol for 5-10 minutes to disinfect th...

Embodiment 2

[0064] see figure 2 , image 3 .

[0065] In the figure, 1 is the support layer, made of plastic sheet strips, 2 is the adsorption fiber layer, made of glass fiber cotton, FITC fluorescent monoclonal antibody is adsorbed on the fluorescent antibody fiber layer 3, and the cellulose membrane layer 4 is made of nitrocellulose Membrane, the water-absorbing material layer 5 at the handle end is made of water-absorbing filter paper, and the layers of absorbent fiber layer 2, fluorescent antibody fiber layer 3, cellulose film layer 4, and water-absorbing material layer 5 are pasted and fixed on the support layer 1 in sequence from right to left , The fibers at the junction between each other cross-penetrate each other. On the cellulose membrane layer 4, there is a stealth detection blot 6, which is made of Escherichia coli O157:H7 monoclonal antibody; the stealth control blot 7 is blotted on the cellulose membrane with a goat anti-mouse IgG antibody solution to make "︱", The two ...

Embodiment 3

[0075] The test strip structure is the same as in Example 1. The difference is that the fluorescent antibody fiber layer 3 is adsorbed with FITC-labeled anti-Escherichia coli O157:H7 polyclonal antibody, the adsorption fiber layer 2 is made of nylon membrane, the cellulose membrane layer 4 is made of pure cellulose membrane, and the invisible imprint is sheep Anti-rabbit IgG antibody. Both the invisible detection imprinted tape and the invisible contrast imprinted tape are "ten", and 8-2 is the blue protective film at the handle end covered on the water-absorbing material layer 5 .

[0076] Preparation and testing steps of samples to be tested:

[0077] Test bread: aseptically take 25g sample and add it to 225mLEC or NB broth containing FTIC solution, put the above solution into a homogenizer, homogenize continuously for 1-2min on a slap-type homogenizer, and incubate at 37°C several hours.

[0078] Operation method: Insert the test paper into the sample solution, take it o...

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Abstract

The invention relates to a sandwiched immunochromatographic test paper used for detecting Escherichia coli O157:H7. The sandwiched immunochromatographic test paper comprises a supporting layer, the upper side surface of the supporting layer is provided with adsorption layers, the adsorption layers sequentially comprise an adsorption fiber layer, a fluorescence antibody fiber layer, a cellulose film and a water absorbing material layer from a test end, the water absorbing material layer is arranged at a handle end, the cellulose film is provided with a detection line and a quality control line, the detection line is formed by an anti-Escherichia coli O157:H7 monoclonal antibody or polyclonal antibody, the quality control line is formed by a goat anti-mouse or rabbit anti-mouse IgG antibody, or a goat anti-rabbit IgG antibody, the fluorescence antibody fiber layer is adsorbed with a fluorescence antibody, and the fluorescence antibody is formed by an FITC labeled anti-Escherichia coli O157:H7 monoclonal antibody or polyclonal antibody. The invention also discloses a production method of the sandwiched immunochromatographic test paper. The test paper has the advantages of simple detection method, strong specificity, high sensitivity, visual property, high accuracy, and realization of lowest detection of 1CFU / mL of trace quantity pollution.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an immunochromatographic test paper, in particular to a sandwich-type immunochromatographic test paper for detecting Escherichia coli O157:H7. Background technique [0002] Escherichia coli O157:H7 type (Escherichia coli O157:H7) is an enterohemorrhagic Escherichia coli, which is one of the main food-borne pathogens. Enteric infectious diseases caused by Escherichia coli O157:H7 have become a global public health concern. Escherichia coli O157:H7 infection can cause serious gastrointestinal complications such as hemorrhagic colitis (HC), appendicitis and colon perforation, and severe thrombotic platelet purpura (TTP) and hemolytic urinary tract syndrome (HUS), etc. Systemic complications, 3-5% of patients died, about 12% of patients had serious sequelae. Moreover, the human infection dose is extremely low, and the ingestion of 10-100 live bacteria can cause the disease. In recent y...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56916G01N2333/265
Inventor 宋春美刘箐刘金鑫李建武
Owner UNIV OF SHANGHAI FOR SCI & TECH
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