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Escherichia coli O157:H7 enrichment and rapid detection method

A technology of O157 and Escherichia coli, applied in the field of microbial detection, can solve the problems of sensitivity limitation, influence results and sensitivity, etc., to achieve the effect of high coupling rate, small coefficient of variation, reducing workload and probability of contamination by miscellaneous bacteria

Active Publication Date: 2013-12-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some samples with extremely low antigen or antibody content, the color of the marker is so light that it is almost difficult to judge the result with the naked eye. The instrument can only detect the color of the marker on the surface of the solid phase carrier, but it is difficult to detect the marker inside the solid phase carrier, so the detection sensitivity is greatly limited
The biological samples detected at the same time may contain color substances, which will seriously affect the results and sensitivity of the detection

Method used

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  • Escherichia coli O157:H7 enrichment and rapid detection method
  • Escherichia coli O157:H7 enrichment and rapid detection method
  • Escherichia coli O157:H7 enrichment and rapid detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Detection of Escherichia coli O157:H7 in milk using nano-gold magnetic particles

[0028] 1. Preparation of gold magnetic particles coupled with monoclonal antibody:

[0029] 1.1 Treatment of nano-gold magnetic particles: Take 200-400 μL of coupling buffer in a 2 mL centrifuge tube, mix with 0.5-1.0 mg of 50 nm nano-gold magnetic particles, magnetically separate for 3-5 minutes, and discard the supernatant.

[0030] 1.2 Coupling reaction: Take 200-300 μg of the prepared anti-Escherichia coli O157:H7 monoclonal antibody, mix with 0.5-1.0 mg of 50 nm nano-gold magnetic particles, and place in 1 mL of coupling buffer. At a temperature of 37°C, place on a rotator with a rotation speed of 10-15rpm, couple for 30-60min, magnetically separate for 3-5min and discard the supernatant. Wash 3 times with 1 mL of wash buffer.

[0031] 1.3 Blocking: After washing, mix 1 mL of blocking agent with magnetic beads to block for 1 h.

[0032] 2. Capturing Escherichia coli O...

Embodiment 2

[0040] Example 2: Detection of Escherichia coli O157:H7 in beef using nano-gold magnetic particles

[0041] 1. Preparation of gold magnetic particles coupled with monoclonal antibody:

[0042] 1.1 Treatment of nano-gold magnetic particles: Take 200-400 μL of coupling buffer in a 2 mL centrifuge tube, mix with 0.5-1.0 mg of 50 nm nano-gold magnetic particles, magnetically separate for 3-5 minutes, and discard the supernatant.

[0043] 1.2 Coupling reaction: Take 200-300 μg of the prepared anti-Escherichia coli O157:H7 monoclonal antibody, mix with 0.5-1.0 mg of 50 nm nano-gold magnetic particles, and place in 1 mL of coupling buffer. At a temperature of 37°C, place on a rotator with a rotation speed of 10-15rpm, couple for 30-60min, magnetically separate for 3-5min and discard the supernatant. Wash 3 times with wash buffer.

[0044] 1.3 Blocking: After washing, mix 1 mL of blocking agent with magnetic beads to block for 1 h.

[0045] 2. Capturing Escherichia coli O157:H7 i...

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Abstract

The invention applies difunctional nanogold magnetic particles and provides an escherichia coli O157:H7 rapid detection method integrating an immunomagnetic bead capture technique and an immunochromatography technique. The immune magnetic separation and immunochromatography are organically integrated, the step of eluting the escherichia coli O157:H7 from an immunomagnetic bead is eliminated, as a result, the capture efficiency is improved; the step of spraying colloidal gold on a binding pad is eliminated, so that the immunological reaction is much evener, and the variable coefficient is small during quantitative determination; the workload and the living contaminant rate are reduced. The basic train of thought of the detection method is exploring the effective combination method of the nanogold magnetic particles and an antibody, optimizing the condition of enriching the escherichia coli O157:H7 on the nanogold magnetic particles and the antibody, and conducting rapid quantitative detection by taking the immunochromatography technique as a carrier and a double-antibody sandwich as a detection principle.

Description

technical field [0001] The invention relates to the field of microorganism detection, and specifically adopts a bifunctional nano-gold magnetic particle integrated immune magnetic bead capture technology and immune chromatography to rapidly detect Escherichia coli O157:H7. technical background [0002] Foodborne diseases caused by microorganisms are the number one food safety problem in the world and also in my country. Among food-borne pathogens, Escherichia coli O157:H7 is the most dangerous one. Its infectious dose is very low, and a minimum of 10 viable bacteria may cause infection. After E. coli O157:H7 infection, abdominal cramps and non-hemorrhagic diarrhea generally appear first, and more than 70% of patients can develop hemorrhagic diarrhea, 30% to 60% of cases have vomiting, and 30% of cases have low-grade fever symptom. 3%-5% of patients can develop hemolytic uremic syndrome leading to acute renal failure and death. It can contaminate pork, poultry meat, beef, ...

Claims

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Application Information

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IPC IPC(8): G01N33/569
Inventor 赖卫华山珊
Owner NANCHANG UNIV
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