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Method for detecting live enterohemorrhagic escherichia coli O157: H7

A technology of Escherichia coli and O157, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Viable bacteria and other problems, to achieve the effect of shortening the detection period, short detection period, and eliminating interference

Inactive Publication Date: 2013-07-31
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention aims at the problem that the existing real-time fluorescent PCR cannot effectively distinguish between dead bacteria and live bacteria, resulting in excessive detection of pathogenic bacteria, and provides a method for qualitatively detecting live bacteria O157:H7 of enterohaemorrhagic E. The problem that existing technologies cannot quickly identify live enterohaemorrhagic Escherichia coli O157:H7 bacteria in the environment

Method used

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  • Method for detecting live enterohemorrhagic escherichia coli O157: H7
  • Method for detecting live enterohemorrhagic escherichia coli O157: H7
  • Method for detecting live enterohemorrhagic escherichia coli O157: H7

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Embodiment 1

[0024] A method for detecting enterohemorrhagic escherichia coli O157:H7 viable bacteria, carried out according to the following steps:

[0025] (1) Pretreatment of the sample to be tested: use an inoculation loop to dip the bacterial solution of the sample to be tested stored in a glycerol tube, streak on the LB solid medium, and incubate in a constant temperature incubator at 37°C for 24 hours. Pick a single colony and inoculate it in 50 mL of liquid LB medium, and cultivate it on a shaker at 37°C and 180 rpm until the logarithmic growth phase (OD600≈1.0). Adjust the OD600 value to 1.0 with sterile LB medium, in which the bacterial concentration is 10 9 CFU / mL. Draw 1mL of the bacterial culture solution in the logarithmic phase into a 1.5mL centrifuge tube, treat it in a boiling water bath for 50s, and immediately place it on ice to cool it, which is the heat-killed Escherichia coli. Through this treatment, the cell wall and cell membrane are damaged to varying degrees to ...

Embodiment 2

[0036] A method for detecting enterohemorrhagic escherichia coli O157:H7 viable bacteria, carried out according to the following steps:

[0037] (1) Pretreatment of the sample to be tested: use an inoculation loop to dip the bacterial solution preserved in the glycerol tube, streak on the LB solid medium, and incubate in a constant temperature incubator at 37°C for 24 hours. Pick a single colony and inoculate it in 50 mL of liquid LB medium, and cultivate it on a shaker at 37°C and 180 rpm until the logarithmic growth phase (OD600≈1.0). Adjust the OD600 value to 1.0 with sterile LB medium, in which the bacterial concentration is 10 9 CFU / mL. Draw 1mL of the bacterial culture solution in the logarithmic phase into a 1.5mL centrifuge tube, treat it in a boiling water bath for 50s, and immediately place it on ice to cool it, which is the heat-killed Escherichia coli. Through this treatment, the cell wall and cell membrane are damaged to varying degrees to achieve the purpose of...

Embodiment 3

[0047] (1) Pretreatment of the sample to be tested: use an inoculation loop to dip the bacterial solution preserved in the glycerol tube, streak on the LB solid medium, and incubate in a constant temperature incubator at 37°C for 24 hours. Pick a single colony and inoculate it in 50 mL of liquid LB medium, and cultivate it on a shaker at 37°C and 180 rpm until the logarithmic growth phase (OD600≈1.0). Adjust the OD600 value to 1.0 with sterile LB medium, in which the bacterial concentration is 10 9 CFU / mL. Draw 1mL of the bacterial culture solution in the logarithmic phase into a 1.5mL centrifuge tube, treat it in a boiling water bath for 50s, and immediately place it on ice to cool it, which is the heat-killed Escherichia coli. Through this treatment, the cell wall and cell membrane are damaged to varying degrees to achieve the purpose of damaging the bacteria.

[0048] (2) For a concentration of 10 9 CFU / mL live bacteria or heat-treated dead bacteria samples were centrifu...

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Abstract

The invention discloses a method for detecting live enterohemorrhagic escherichia coli O157: H7. The method solves the problem that the existing real-time fluorescence PCR technology cannot effectively distinguish live or dead enterohemorrhagic escherichia coli thereby leading to overestimation of a pathogenic bacterium risk. The method comprises the following steps of pre-treating a liquid sample needing to be detected, adding propidium monoazide (PMA) into the pre-treated liquid sample, controlling a PMA final concentration to 15-20 micrograms per milliliter, carrying out incubation in the dark at a normal temperature, carrying out halogen tungsten lamp radiation treatment, extracting a genome DNA of the treated sample, utilizing the extracted sample genome DNA as a template, carrying out real-time fluorescence PCR quantitative detection of object enterohemorrhagic escherichia coli in the sample, wherein primers and fluorescent label probes used by the real-time fluorescence PCR quantitative detection are shown in the formula of SEQ ID NO.1. The method has short operation time of about 1.5h and can be widely used in the field of food-borne pathogenic bacterium detection.

Description

technical field [0001] The invention relates to a method for detecting Escherichia coli, in particular to a method for detecting live enterohaemorrhagic Escherichia coli O157:H7. Background technique [0002] Enterohaemorrhagic Escherichia coli O157:H7 (E.coli O157:H7) is the most important serotype of hemorrhagic Escherichia coli related to public health. It has strong environmental resistance, acid resistance and low temperature resistance, and is sensitive to heat. They can survive in water for weeks or even months. Enterohaemorrhagic Escherichia coli infection is a zoonotic disease. The disease can be transmitted by patients, carriers, livestock and poultry with enterohemorrhagic Escherichia coli infection in the body. Enterohaemorrhagic Escherichia coli O157:H7 has strong pathogenicity and resistance to gastric acid, and is destructive to cells. The crowd is generally susceptible, but mainly the elderly and children, and the symptoms of the elderly and children are o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/19
Inventor 肖性龙余以刚李聪聪吴晖李晓凤
Owner SOUTH CHINA UNIV OF TECH
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