Kit for rapid detection of Vibrio parahaemolyticus tdh toxin in food and its application

A hemolytic vibrio and kit technology, applied in biological testing, material inspection products, measuring devices, etc., can solve the problems of short detection time, difficulty in distinguishing whether it is pathogenic, long detection time of vibrio parahaemolyticus, etc.

Active Publication Date: 2016-08-17
JIANGSU VOCATION & TECHNICAL COLLEGE OF FINANCE & ECONOMICS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Technical problem: A technical problem to be solved by the invention is to overcome the shortcomings of the existing detection of Vibrio parahaemolyticus in food, such as long detection time, high detection cost, and difficulty in distinguishing whether it is pathogenic or not, and provides a kind of vibrio parahaemolyticus in food DAS-ELISA Rapid Detection Kit for Vibrio pathogenic factor TDH toxin, which has high detection accuracy and short detection time, and can realize the standardized detection of ELISA detection method

Method used

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  • Kit for rapid detection of Vibrio parahaemolyticus tdh toxin in food and its application
  • Kit for rapid detection of Vibrio parahaemolyticus tdh toxin in food and its application
  • Kit for rapid detection of Vibrio parahaemolyticus tdh toxin in food and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0054] The TDH toxin extraction and purification of Vibrio parahaemolyticus comprises the following steps:

[0055] (1) Inoculate TDH toxin-producing Vibrio parahaemolyticus in 3% sodium chloride alkaline peptone water at 37°C for 24 hours on a shaking table at 150r / min, then centrifuge at 6000r / min to remove the bacteria, and press 35.1g / 100mL Add the amount of supernatant (NH 4 ) 2 SO 4 , after fully dissolving, let stand at 4°C for salting out overnight to fully precipitate TDH toxin, centrifuge at 10,000r / min, 4°C for 10min, discard the supernatant, and dissolve the precipitate in a small amount of 0.01mol / L PBS solution with pH=7.0 , and dialyzed overnight with this PBS solution to obtain the crude product of TDH toxin;

[0056] (2) DEAE-cellulose ion exchange column chromatography: use the crude toxin solution on DEAE-cellulose DE-25 for column chromatography, the method is as follows: slowly drop the crude TDH toxin in the above PBS solution at 4°C Add it to the chr...

Embodiment 2

[0069] The application of DAS-ELISA rapid detection kit, the method is as follows:

[0070] (1) Sample pretreatment, weigh 25g large (small) yellow croaker sample by aseptic operation, add 225mL 0.01mol / L, pH 7.4 phosphate buffer, homogenize in a homogenizing cup to make sample homogenate (1:10). Filter the sample homogenate with silk gauze, centrifuge at 8000r / min for 10min, and take the supernatant for DAS-ELISA detection.

[0071] (2) Take out the ELISA plate coated with the capture antibody, and add 100 μL of the sample extract to be tested and TDH standard dilution (for drawing a standard curve, the concentrations are 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL), at the same time, 100 μL rabbit anti-Vibrio parahaemolyticus negative serum was added as a negative control, 100 μL of PBST was added as a blank control, the plate was sealed with film, and incubated at 37°C for 60 minutes.

[0072] (3) Wash the plate manually: Discard the liquid in the wells, fill e...

Embodiment 3

[0083] The application of DAS-ELISA rapid detection kit, the method is as follows:

[0084] (1) Pre-treatment of the sample, weighing 25g of crustacean aquatic product samples such as prawns and river crabs by aseptic operation, adding 225mL of 0.01mol / L, pH7.4 phosphate buffer, homogenizing in a homogenizing cup to prepare Sample homogenization (1:10). Filter the sample homogenate with silk gauze, centrifuge at 10000r / min for 8min, and take the supernatant for DAS-ELISA detection.

[0085] (2) Take out the ELISA plate coated with the capture antibody, and add 100 μL of the sample extract to be tested and TDH standard dilution solution (for drawing a standard curve, the concentrations are 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL), while adding 100 μL of rabbit anti-Vibrio parahaemolyticus negative serum as a negative control, adding 100 μL of PBST as a blank control, sealing the plate with film, and incubating at 37°C for 80 minutes.

[0086] (3) Wash the plate...

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Abstract

The invention provides a kit for rapidly detecting vibrio parahaemolyticus TDH toxin in food and application thereof. The kit comprises an elisa plate of a captured antibody, TDH standard diluent, rabbit anti-vibrio parahaemolyticus negative serum, rabbit anti-vibrio parahaemolyticus polyclone antibody serum, a phosphate buffer solution of goat rabbit anti IgG-HRP, a phosphate buffer solution containing tween-20, a substrate solution and a 2 mol / L stop solution of H2SO4. According to the kit for rapidly detecting the vibrio parahaemolyticus TDH toxin in the food and the application thereof, the kit for detecting the vibrio parahaemolyticus TDH toxin in the food serves as a study object, an anti-TDH polyclone antibody is prepared through the prepared TDH toxin protein immune BALB / C mice, meanwhile, a thalli of pathogenic vibrio parahaemolyticus serves as an immunogen to immune New Zealand white rabbits to prepare the rabbit anti-vibrio parahaemolyticus polyclone antibody, the anti-TDH polyclone antibody serves as a capture antibody, an antibiotic body polyclone antibody serves as a detection antibody, the double-antibody sandwich ELISA detection method is built, the detection method is used for detecting the vibrio parahaemolyticus TDH toxin in the food, and the kit for rapidly detecting the vibrio parahaemolyticus TDH toxin ELISA in the food is developed.

Description

technical field [0001] The present invention relates to an ELISA kit, in particular to a double-antibody sandwich ELISA detection kit for rapidly detecting TDH toxin of Vibrio parahaemolyticus in food, and also relates to the rapid detection of TDH toxin of Vibrio parahaemolyticus in food by the kit The application in the invention belongs to the technical field of food safety detection. Background technique [0002] Vibrio parahaemolyticus (V.p) is a common foodborne pathogenic microorganism in food. It mainly exists in aquatic products, especially seafood. It is an important pathogen that causes food poisoning and summer diarrhea in coastal areas of my country every year. The hemolytic toxin produced by V.p is the main cause of the pathogenicity of the bacteria, including thermostable direct hemolysin (TDH for short), thermostable direct hemolysin-related hemolysin (TDH-related hemolysin, TRH) and thermolabile hemolysin (Thermolabile Hemolysin, referred to as TLH), epidem...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/543G01N33/68G01N2333/28
Inventor 窦勇胡佩红顾鹏程董静姚妙爱吴存兵
Owner JIANGSU VOCATION & TECHNICAL COLLEGE OF FINANCE & ECONOMICS
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