Listeria monocytogenes enrichment and rapid detection method

A technology for Listeria and detection methods, applied in measurement devices, instruments, scientific instruments, etc., can solve problems such as sensitivity limitations, affect results and sensitivity, achieve high coupling rate, small coefficient of variation, reduce workload and Effect of bacterial contamination probability

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

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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 car...

Method used

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  • Listeria monocytogenes enrichment and rapid detection method
  • Listeria monocytogenes enrichment and rapid detection method
  • Listeria monocytogenes enrichment and rapid detection method

Examples

Experimental program
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Embodiment 1

[0027] Example 1: Detection of Listeria monocytogenes 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-Listeria monocytogenes 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 rotational speed of 10-15 rpm, couple for 30-60 min, magnetically separate for 3-5 min 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 hour.

[0032] 2. Capturing Listeria monoc...

Embodiment 2

[0040] Example 2: Detection of Listeria monocytogenes 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-Listeria monocytogenes 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 rotational speed of 10-15 rpm, couple for 30-60 min, magnetically separate for 3-5 min 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 Listeria monocytogenes in...

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Abstract

The invention applies difunctional nanogold particles and provides a listeria monocytogenes 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 listeria monocytogenes from an immunomagnetic bead is eliminated, so 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; and 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 listeria monocytogenes on the nanogold magnetic particles and the antibody, and conducting rapid quantitative detection by using 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 Listeria monocytogenes. technical background [0002] Listeria monocytogenes (Listeria monocytogenes) Listeria monocytogenes , LM), is the most important human foodborne pathogen in the Listeria genus. Meat, eggs, seafood, dairy products, vegetables and other foods are the main sources of LM pollution. The mortality rate of people infected by LM can reach 30%, and the mortality rate among newborn infants and people with low immunity is as high as 70%. The bacterium can still grow and multiply in food stored in a refrigerator at 4°C, making it one of the main pathogenic bacteria in refrigerated food. According to the detection method stipulated in my country's national standard law, it takes 72-96 hours to complete the LM primary ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531
Inventor 赖卫华山珊
Owner NANCHANG UNIV
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