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Kit used for rapidly detecting Escherichia coli O157:H7 in sample, and detection method thereof

A technology for Escherichia coli and O157, which is applied in the field of immunological detection, can solve the problems of time-consuming, low detection sensitivity, complex and cumbersome inspection procedures, etc., and achieve rapid and accurate detection results

Inactive Publication Date: 2012-05-02
王利兵 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional methods for detecting bacteria include biochemical culture, isolation and identification, the inspection procedures are complex and cumbersome, and it takes about 4-7 days to report the inspection results, which takes too long and the detection sensitivity is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Preparation of immunogen and positive standards

[0052] Escherichia coli O157∶H7 (ATCC 35150) was inoculated on a sorbitol MacConkey (SMAC) plate, cultured at 37°C for 24 hours, and a single colony was picked up in the modified EC novobiocin enrichment broth, 37°C, 200r / min shaking Incubate for 17 hours, count, and inactivate by heating in a boiling water bath overnight. Adjust the concentration of Escherichia coli O157:H7 (ATCC 35150) to 5×10 with normal saline. 9 cfu / mL, as the immunogen; adjust the concentration to 10 with PBS buffer 7 cfu / mL is the positive control standard, and PBS buffer is the negative control standard.

Embodiment 2

[0053] Example 2. Preparation of coated antiserum

[0054] 1) Experimental animals: Choose 3 healthy New Zealand white rabbits of 2 months old and weighing 1.5-2 kg as experimental animals.

[0055] 2) Emulsification: Emulsify 0.5 mL of immunogen and the same amount of complete or incomplete Freund's adjuvant by mixing and stirring until a drop of emulsion is dropped into the water and floats on the water without dispersing.

[0056] 3) Immunization method: In the first immunization, the emulsified reagent is injected into the rabbit's back at multiple points, 1 mL / head. The immunization after the initial immunization is called booster. The booster is emulsified with Freund's incomplete adjuvant. The booster is intramuscular injection. The booster is once every two weeks, and the dose is the same as the first immunization.

[0057] 4) Blood sampling: blood was collected from the ear vein after 3 booster immunizations, and the antiserum titer was determined by indirect non-competitive...

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Abstract

The invention relates to a kit used for rapidly detecting Escherichia coli O157:H7 in a sample, and a detection method thereof. The invention belongs to the technical field of immunological detection. According to the invention, a heated and deactivated Escherichia coli O157:H7 immunogen is used for immunizing a healthy New Zealand rabbit, such that a polyclonal antibody is obtained, and the polyclonal antibody is adopted as a coating antibody; a BALB / C mouse is immunized, and cell fusion is carried out, such that a monoclonal antibody is obtained, and the monoclonal antibody is adopted as a secondary antibody; and a double-antibody sandwich ELISA kit of Escherichia coli O157:H7 in foodstuffs (meat) is established. With the kit, a rapid and highly efficient detection means is provided for the detection of the residue of Escherichia coli O157:H7 in foodstuffs. The kit is advantaged in relatively low cost, relatively good stability, and relatively good repeatability. According to the invention, a detection limit is 105cfu / mL. The kit and the method are suitable for large-batch detections of samples.

Description

Technical field [0001] A kit and a detection method for rapidly detecting Escherichia coli O157:H7 in a sample belong to the technical field of immunological detection. Background technique [0002] Escherichia coli O157∶H7 is an important zoonotic pathogenic bacteria, which is widespread in nature, often causing severe diarrhea and sepsis, severe hemolytic uremic syndrome (HUS), thrombosis Thrombocytopenic purpura (TTP), in which the mortality rate of patients with HUS and TTP is as high as about 80%, which is very serious. [0003] A large number of studies have shown that: cattle, pigs, poultry and other animals can be the natural host of E. coli. The bacteria can exist in the animal body for a long time and continue to excrete bacteria, polluting the surrounding environment and the carcass of animals, resulting in both human and animal origin. There is a high rate of bacteria, and there are many opportunities for food to be contaminated. How to control O157:H7 infection is cu...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 王利兵胥传来勇倩倩
Owner 王利兵
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