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Method for rapidly detecting live enterohemorrhagic Escherichia coli O157:H7

A technology for Escherichia coli and enterohemorrhagic bacteria, applied in the field of rapid detection of live E. The effect of reducing dosage

Inactive Publication Date: 2018-10-30
曾小敏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot distinguish between live and dead microorganisms present in the sample

Method used

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Effect test

Embodiment 1

[0021] A rapid detection method for live enterohaemorrhagic Escherichia coli O157:H7, comprising enrichment culture, extraction of bacterial strain genomic DNA, design and synthesis of primers and probes, PCR reaction amplification, and result judgment. The specific steps are as follows:

[0022] 1) Bacterial enrichment culture: Inoculate E. coli O157:H7 into the improved E.C novobiocin enrichment broth m(EC)n, and enrich the bacteria at 38°C for 20 hours to obtain a bacterial suspension for use in this step. The bacterial effect is remarkable, making E. coli O157:H7 reach the logarithmic growth phase. At this time, the bacterial structure is stable, the metabolism and physiological characteristics are relatively consistent and good, the defense ability is the strongest, and the effect of ethidium bromide azide is the most difficult;

[0023]2) Extraction of the genomic DNA of the strain: take 1ml of the cultured bacterial suspension, add ethidium bromide azide solution (EMA) a...

Embodiment 2

[0029] A rapid detection method for live enterohaemorrhagic Escherichia coli O157:H7, comprising enrichment culture, extraction of bacterial strain genomic DNA, design and synthesis of primers and probes, PCR reaction amplification, and result judgment. The specific steps are as follows:

[0030] 1) Bacterial enrichment culture: inoculate E. coli O157:H7 into the improved E.C novobiocin enrichment broth m(EC)n, and enrich the bacteria at 45°C for 15 hours to obtain a bacterial suspension for later use;

[0031] 2) Extraction of the genomic DNA of the strain: take 1ml of the cultured bacterial suspension, add ethidium bromide azide solution (EMA) at a concentration of 0.05mg / mL under dark conditions, so that the final concentration of EMA reaches 2.0mg / L, shake slightly to mix Let it stand in the dark for 16 minutes, then take out the bacterial solution, keep the lamp tube 14cm away, open the cover and put it on ice, and use a 700W halogen tungsten lamp for 5 minutes to photolyz...

Embodiment 3

[0037] A rapid detection method for live enterohaemorrhagic Escherichia coli O157:H7, comprising enrichment culture, extraction of bacterial strain genomic DNA, design and synthesis of primers and probes, PCR reaction amplification, and result judgment. The specific steps are as follows:

[0038] 1) Bacterial enrichment culture: inoculate E. coli O157:H7 into the improved E.C novobiocin enrichment broth m(EC)n, and enrich the bacteria at 41°C for 18 hours to obtain a bacterial suspension for later use;

[0039] 2) Extraction of the genomic DNA of the strain: take 1ml of the cultured bacterial suspension, add ethidium bromide azide solution (EMA) at a concentration of 0.05mg / mL under dark conditions, so that the final concentration of EMA reaches 3.0mg / L, shake slightly to mix Let it stand in the dark for 15 minutes, then take out the bacterial solution, keep the lamp tube 15cm away, open the cover and put it on ice, and use a 650W halogen lamp for 8 minutes to photolyze ethidiu...

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Abstract

The invention discloses a method for rapidly detecting live enterohemorrhagic Escherichia coli O157:H7. The detection method comprises the following steps: culturing Escherichia coli O157:H7 to obtaina bacterium suspension; adding an ethidium bromide monoazide solution into the bacterium suspension under a dark condition, standing in the dark, uncovering to place on ice, exposing, centrifuging, removing the supernatant, re-suspending with ultrapure water, centrifuging in a metal bath, and taking the supernatant as extracted bacterial genome DNA; designing and synthesizing primers and probes;diluting the DNA with sterile ultrapure water, and performing dual fluorescent PCR (Polymerase Chain Reaction) amplification; analyzing by selecting a 'maximum second derivative' method, and judging the results by the value CP. The method has the beneficial effects that the detection method is excellent in specificity and repeatability, high in sensitivity and accuracy, capable of rapidly identifying dead and live bacteria of the enterohemorrhagic Escherichia coli O157:H7 and low in detection cost.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a rapid detection method for live enterohaemorrhagic Escherichia coli O157:H7. Background technique [0002] In recent years, with the improvement of people's living standards, my country's food hygiene has been greatly improved, but the epidemic outbreaks of food-borne pathogenic microorganisms are still frequently reported, and the contamination of pathogenic bacteria in food is the most important aspect of food safety. Hazardous factors are also the main cause of food poisoning. In recent years, food pathogenic bacteria such as Salmonella, Listeria, Escherichia coli O157, and Campylobacter have become the number one killer that endangers food safety. Among them, Escherichia coli O157:H7 is the most important serotype of enterohemorrhagic Escherichia coli (EHEC), as a zoonotic intestinal pathogen, Escherichia coli O157:H7 is mainly infected through contaminated anim...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/10C12R1/19
CPCC12Q1/686C12Q2563/107C12Q2537/143
Inventor 曾小敏
Owner 曾小敏
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