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Edwardsiella tarda rapid detection test paper as well as rapid detection method and application

A technology for detecting test paper and test strips, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complicated operation procedures, difficult promotion, time-consuming and cumbersome, etc., and achieve the effect of simple operation and accurate detection

Active Publication Date: 2011-04-27
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional bacteriological, physiological and biochemical identification methods are time-consuming and cumbersome. Although general immunoenzyme tests, immunofluorescence methods, and ELISA can be used for the detection of Edwardsiella tarda, the operating procedures are complicated and the detection is relatively time-consuming and laborious.
PCR technology is simple, fast, and highly sensitive, but it requires high equipment and experimental skills, and it is difficult to promote it on a large scale in aquaculture grassroots units

Method used

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  • Edwardsiella tarda rapid detection test paper as well as rapid detection method and application
  • Edwardsiella tarda rapid detection test paper as well as rapid detection method and application
  • Edwardsiella tarda rapid detection test paper as well as rapid detection method and application

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Embodiment 1

[0034] Embodiment 1: Preparation of polyclonal antibody:

[0035] 1) Cultivate Edwardsiella tarda EIB202 (CCTCC-M 208068) to the late logarithmic phase with TSB medium, take the bacterial liquid at 4°C, 4000r·min -1 Centrifuge for 5min, use 0.05mol·L -1 After washing three times with PBS buffer (pH 7.4), resuspend in PBS buffer, add 0.4% formaldehyde to inactivate for 48h;

[0036] 2) After fully emulsifying the inactivated bacterial solution with an equal amount of complete Freund's adjuvant, inject multi-point intradermally into adult New Zealand female rabbits on the back, the antigen dose is 2×10 7 CFU / piece;

[0037] 3) After 2 weeks, fully emulsify the inactivated bacterial solution with the same amount of incomplete Freund's adjuvant, inject multi-point intradermally on the back for immunization, and then boost immunization once every 2 weeks, and prepare serum after 3 times of boosting immunization, and use Indirect enzyme-linked immunoassay (ELISA) was used to dete...

Embodiment 2

[0040] Embodiment 2: the preparation of colloidal gold and the rapid detection test strip assembly of Edwardsiella tarda:

[0041] 1. Preparation of colloidal gold solution by trisodium citrate reduction method

[0042] 1) Take a 250ml triangular flask, add 99ml ultrapure water and 1ml 1% chloroauric acid, heat to boil;

[0043] 2) Take 1.8mL of 1% sodium citrate and add it to the above solution, mix quickly and evenly, keep boiling for 10min, and restore the volume of distilled water to 100mL after natural cooling;

[0044] 3) The colloidal gold solution is stored at 4°C in a siliconized reagent bottle for later use.

[0045] 2. Preparation of gold-labeled antibody

[0046] 1) Dilute the Edwardsiella tarda polyclonal antibody with PBS buffer (pH 7.4), mix it with colloidal gold solution (pH 8.0), and stir it electromagnetically for 1 hour;

[0047] 2) Add PBSTB blocking solution (PBS buffer containing 0.05% Tween 20 and 5% BSA) to block for 1 h;

[0048] 3) Centrifuge at 16...

Embodiment 3

[0055] Embodiment 3: Edwardsiella tarda rapid detection test strip specificity detection

[0056] 1) Common bacteria in water bodies and common fish pathogens Escherichia coli, Staphylococcus aureus, Vibrio anguillarum, Vibrio harveyi, Edwardsiella tarda ATCC15947, ATCC23692 from ATCC and Edwardsiella catfish ATCCA33202 were respectively placed in the optimal culture conditions Cultivate to logarithmic phase.

[0057] 2) Resuspend the cells with PBS buffer solution and dilute to 10 5 CFU / ml.

[0058] 3) Take 100 μl each to test with Edwardsiella tarda rapid detection test strips to determine the specificity of the test strips. see results Image 6 . It can be seen from the figure that all Edwardsiella tarda were positive, and Escherichia coli, Staphylococcus aureus, Vibrio harveyi and Vibrio anguillarum were negative. It can be seen that the test strip has good specificity for Edwardsiella tarda. It is negative for cross-reactivity to common bacteria in water bodies.

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Abstract

The invention relates to an edwardsiella tarda rapid detection test paper which is a colloidal gold immunochromatography detection test paper. An edwardsiella tarda colloidal gold labeled antibody is adsorbed on a combined pad of the colloidal gold immunochromatography detection test paper, a detection line and a quality control line are arranged on an analyzing film of the colloidal gold immunochromatography detection test paper, an edwardsiella tarda antibody coats the detection line, and a second antibody coats the quality control line wherein the immunogen of the second antibody is the same as that of edwardsiella tarda antibody. The invention also provides a method for rapidly detecting edwardsiella tarda and application of the edwardsiella tarda rapid detection test paper in detecting the edwardsiella tarda. The edwardsiella tarda rapid detection test paper can conveniently, rapidly, sensitively and accurately detect the edwardsiella tarda to control the spread of edwardsiella tarda diseases in the early days, has an important significance in controlling the edwardsiella tarda diseases in aquaculture and is suitable for large-scale generalization and application.

Description

technical field [0001] The invention relates to the technical field of detection test strips, in particular to the technical field of detection test strips for Edwardsiella tarda, in particular to a rapid detection test strip for Edwardsiella tarda, a rapid detection method and its application. Background technique [0002] Edwardsiella tarda (Edwardsiella tarda) is currently a Gram-negative pathogen that is extremely harmful in the aquaculture industry, and can infect a variety of fish including marine fish and freshwater fish. It has caused different degrees of fish disasters in many countries and regions in the world, and has caused great harm to my country's aquaculture industry in recent years. [0003] Establishing a rapid, simple and accurate detection method for Edwardsiella tarda is of great significance to the control of Edwardsiella tarda disease in aquaculture. The current traditional detection method is to enrich the pathogenic microorganisms, and determine the...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/569G01N33/544
Inventor 肖婧凡张元兴刘和松王启要刘琴
Owner EAST CHINA UNIV OF SCI & TECH
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