A kind of enterohaemorrhagic Escherichia coli o157:h7 latex agglutination detection kit and its application

A detection kit, Escherichia coli technology, applied in the field of poultry immunology, can solve the problems of difficulty in product development, low stability and sensitivity, and achieve the effects of short detection time, strong specificity and simple operation

Active Publication Date: 2016-08-24
广东正大康生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used ways to sensitize latex are mainly physical adsorption method and chemical cross-linking method. The stability and sensitivity of physical adsorption method are low, and it is difficult to meet the needs of product development.

Method used

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  • A kind of enterohaemorrhagic Escherichia coli o157:h7 latex agglutination detection kit and its application
  • A kind of enterohaemorrhagic Escherichia coli o157:h7 latex agglutination detection kit and its application
  • A kind of enterohaemorrhagic Escherichia coli o157:h7 latex agglutination detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Preparation of anti-enterohaemorrhagic Escherichia coli O157: H7intimin protein monoclonal antibody sensitization latex reagent

[0019] The preparation method of the anti-Intimin protein monoclonal antibody is as follows: from the 5 strains of hybridoma cells that have been established, one hybridoma cell with high antibody titer and strong specificity is selected for expansion and culture, and at the same time, it is induced in BALB / c mice. Mouse ascites produced monoclonal antibodies; 20 8-week-old BALB / c mice were injected intraperitoneally with incomplete Freund's adjuvant 0.5mL / mouse, and 7 days later, the expanded cultured hybridoma cells were injected intraperitoneally with 5×10 6 ~5×10 7 / mouse, feeding observation, when the abdomen of the mouse protrudes and the movement is sluggish, the ascites of the mice is extracted one by one, centrifuged at 2000r / min for 8min, the fat is discarded to collect the upper layer to clarify the ascites, and the as...

Embodiment 2

[0042] The using method of embodiment 2 enterohaemorrhagic Escherichia coli O157:H7 latex agglutination detection kit

[0043] 1. Sample processing: put the collected sample to be tested in the sample processing solution and incubate at 37°C for 4-6 hours.

[0044] 2. Detection of tested samples: 1) Add 10 ul of positive control sample, 10 ul of negative control sample and 10 ul of sample to be tested dropwise on a clean glass slide or glass plate. Then add 10 ul of the latex detection reagent dropwise to the positive control sample, negative control sample, and sample to be tested, stir evenly, and observe the result under a blue background after shaking for 1 minute. 2) Judgment of results: The test can only be established if the following results appear within 1 minute of the control test: latex agglutination of more than +++ occurs between the positive control sample and the latex detection reagent, and no agglutination between the negative control sample and the latex det...

Embodiment 3

[0046] Example 3 Preliminary Application of Enterohaemorrhagic Escherichia coli O157:H7 Latex Agglutination Detection Kit

[0047] 1. Sensitivity test: Pick a single colony of enterohemorrhagic Escherichia coli after cultured at 37°C for 18-24 hours on a Sorbitol MacConkey plate, culture it in 5 mL TSB liquid medium for 18-24 hours, and count the bacteria by plate counting method. And the bacterial solution was diluted in different gradients, and the OD of the bacterial solution was detected by a spectrophotometer 600 value, and then detected with a good latex reagent for sensitization (test results are shown in Table 7). Test results: the bacteria solution was diluted to 1:256 times (bacteria quantity 2.3×10 7 CFU / ml), there can still be obvious agglutination, and the test results show that the sensitivity is better.

[0048] Table 7 Sensitivity Test

[0049]

[0050] 2. Specificity test: Carry out latex agglutination detection with pathogens such as Salmonella, other s...

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Abstract

The invention relates to an enterohemorrhagic E.coli O157:H7 latex agglutination detection kit and an application of the detection kit in detection of enterohemorrhagic E.coli O157:H7. The kit comprises: a) a latex detection reagent; b) a sample treating fluid; c) a positive control sample; and d) a negative control sample. The latex detection reagent is a latex reagent which can be subjected to a specific reaction with enterohemorrhagic E.coli O157:H7, and is obtained from Intimin monoclonal antibody sensitized carboxylated polystyrene latex. The kit provided by the invention has the following advantages: 1, the kit is simple to operate, is convenient and fast, has low detection cost and is suitable for rapid on-site detection; 2, coupling rate of antibody and sensitivity and stability of the latex detection reagent are improved, and requirements of product research and mass production are met; and 3, the kit can be used to directly detect enterohemorrhagic E.coli O157:H7 in a sample and has characteristics of strong specificity, high sensitivity and short detection time.

Description

technical field [0001] The invention relates to a latex agglutination detection kit for enterohaemorrhagic Escherichia coli O157:H7 and its application in the detection of enterohaemorrhagic Escherichia coli O157:H7, belonging to the technical field of poultry immunology. Background technique [0002] Enterohemorrhagic Escherichia coli O157:H7 is an important food-borne pathogen and one of the main causes of food safety and public health emergencies that are increasing in the world today. The bacteria can infect poultry and other animals, and people get sick mainly through contaminated food. In 1982, O157:H7 was first discovered in the United States, and since then it has been distributed or endemic around the world, and has become a global public health and food safety problem. Therefore, the development of rapid, sensitive and specific detection methods and reagents is an important means to ensure drinking water and food safety and prevent E. coli O157:H7 infection. At p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 罗玲艾地云罗青平邵华斌张腾飞王红琳汪宏才温国元张蓉蓉
Owner 广东正大康生物科技股份有限公司
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