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Method for detecting living bacteria body of listeria monocytogenes in sample

A technology for Listeria monocytogenes and sample detection, which is applied in the field of biomedicine and can solve the problem of inability to distinguish between live and dead bacteria of Listeria monocytogenes.

Active Publication Date: 2015-05-27
BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the method for detecting pathogenic bacteria can only detect the total number of pathogenic bacteria in the sample, but cannot distinguish the number of living and dead bacteria of Listeria monocytogenes in the sample

Method used

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  • Method for detecting living bacteria body of listeria monocytogenes in sample

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Experimental program
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Embodiment approach

[0016] Wherein, as a particularly preferred embodiment of the present invention, the antibody capable of specifically binding to live Listeria monocytogenes is the California Bioscience monoclonal antibody numbered MC0007, which can be purchased from California Bioscience Available commercially under item number MC0007.

[0017] Wherein, the amount of the antibody may be 0.05-2 mg relative to each mg of magnetic balls.

[0018] Wherein, relative to the sample to be tested in liquid form per milliliter, the amount of the immunomagnetic balls may be 10-25 μg in terms of the weight of the magnetic balls.

[0019] Wherein, in step (3), the liquid used for elution may be a phosphate buffer containing 0.144-0.162 mol / L disodium hydrogen phosphate and 0.038-0.056 mol / L sodium dihydrogen phosphate.

[0020] Wherein, as a particularly preferred embodiment of the present invention, for the live cells of Listeria monocytogenes, the primers used for PCR amplification include the forward ...

preparation Embodiment 1

[0025] In Example 1 of this preparation, Antibody 1 was used to prepare immunomagnetic beads used in the method of the present invention.

[0026] Surface-modified superparamagnetic Fe with carboxyl groups activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) 3 o 4 Polystyrene composite microspheres (purchased from Shanghai Aorun Micro-Nano New Material Technology Co., Ltd., PM3-020 polymer magnetic balls, with a concentration of 10 mg / mL, a particle size of 180 nm, and carboxyl functional groups on the surface) were used as magnetic balls , take 2 mg of magnetic spheres and disperse them in the phosphate buffer solution, the concentration of the magnetic spheres in the dispersion is 2 mg / mL. Mix 0.2 mg of specific antibody (dissolved in 1 mL of phosphate buffer) with 2 mg of magnetic balls, keep in a shaker (200 r / min) for 3 hours at room temperature, and then wash by magnetic separation to remove the uncoupled magnetic balls. Surf...

Embodiment 1

[0029] Cultivate Listeria monocytogenes (purchased from ATCC, product number 35152) in LB medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L) to logarithm period, adjust the cell concentration to 10 with LB medium 3 pc / L, as a positive sample containing viable Listeria monocytogenes.

[0030] The above positive samples were inactivated by high pressure (121°C, 15min) to obtain viable cells that did not contain Listeria monocytogenes but contained 10 3 Negative samples of / L dead bacteria.

[0031] Mix the positive sample and the negative sample in equal volumes to obtain 5×10 2 each / L of dead bacteria and 5×10 2 A mixed sample of viable cells / L.

[0032] Mix 1mL of positive samples, negative samples and mixed samples with immunomagnetic balls 1 (15 mg based on the weight of magnetic balls) to obtain the mixed materials, incubate at 29°C for 30 min, and then fix the immunomagnetic beads in a sorting magnetic field. Magnetic spheres, to elute the substances t...

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Abstract

The invention discloses a method for detecting living bacteria bodies of listeria monocytogenes in a sample. The method includes following steps: (1) coupling an antibody capable of being specifically combined with the living bacteria bodies of listeria monocytogenes onto magnetic balls to obtain immune magnetic balls, wherein the antibody capable of being specifically combined with the living bacteria bodies of listeria monocytogenes cannot be combined with dead bacteria bodies; (2) mixing the immune magnetic balls with a liquid sample to obtain a mixture, wherein the mixing condition is described as that when the living bacteria bodies of listeria monocytogenes exists in the sample, the immune magnetic balls can specifically adsorb the living bacteria bodies of listeria monocytogenes; (3) placing the mixture in a separation magnetic field for immobilizing the immune magnetic balls and eluting substances being not adsorbed on the immune magnetic balls to obtain eluted immune magnetic balls; (4) performing DNA extraction to substances adsorbed onto the immune magnetic balls to obtain a template sample; (5) performing PCR amplification to the template sample with a specific primer aiming to the living bacteria bodies of listeria monocytogenes, and determining the content of the living bacteria bodies of listeria monocytogenes in the sample according to the content of targeted fragments in a PCR amplification product.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for detecting live cells of Listeria monocytogenes in a sample. Background technique [0002] Pathogenic bacteria (Pathogenic bacteria) Microorganisms that can cause disease are called pathogenic microorganisms or pathogenic bacteria. Pathogenic microorganisms include bacteria, viruses, spirochetes, rickettsia, chlamydia, mycoplasma, fungi, and actinomycetes. Generally speaking, pathogenic bacteria refer to bacteria in pathogenic microorganisms. The pathogenicity of bacteria is related to its virulence, invasion quantity and invasion portal. While the vast majority of bacteria are harmless or even beneficial, a significant number can cause disease. Opportunistic pathogens only cause disease under certain conditions, such as when there is a wound that allows bacteria to enter the bloodstream, or when immunity is weakened. For example, staphylococcus aureus and streptococcus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6804C12Q2563/143C12Q2563/149
Inventor 杜美红孙永军刘清珺杨寅陈婷陈舜琮
Owner BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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