Quantitative detection method for escherichia coli O157:H7 live bacteria in food

A quantitative detection method, Escherichia coli technology, which is applied in the field of microbial detection, can solve the problems of inability to effectively inhibit PCR amplification, and achieve the effect of improving detection sensitivity and high sensitivity

Inactive Publication Date: 2014-01-15
WUXI ZODOLABS BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the PMA-PCR combined method also has certain limitations.
PMA cannot effectiv...

Method used

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  • Quantitative detection method for escherichia coli O157:H7 live bacteria in food
  • Quantitative detection method for escherichia coli O157:H7 live bacteria in food
  • Quantitative detection method for escherichia coli O157:H7 live bacteria in food

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The optimization of embodiment 1 deoxycholate concentration and processing time

[0026] Weigh a certain amount of deoxycholate (SD) and dissolve it in 0.1% (m(g) / v(mL)) peptone water to make SD with a concentration of 5% (m(g) / v(mL)). The mother solution was stored at 4°C in the dark.

[0027] Propidium azide bromide (PMA) was dissolved in dimethyl sulfoxide (DMSO), prepared into a 0.5 mg / mL PMA solution, and stored at -20°C in the dark.

[0028] Pick a single colony of Escherichia coli O157:H7 (British Type Culture Collection) in 5mL of LB medium, culture overnight at 37°C, take 1mL of the bacterial solution and dilute two gradients serially tenfold with PBS to obtain about 10 6 CFU / mL. Centrifuge at 12000rpm for 10min, and resuspend with an equal volume of 0.1% (m(g) / v(mL)) peptone water. The resuspended bacteria solution was placed at -70°C for 10 minutes and then bathed in water at 9°C for 30 minutes to obtain a mixture of injured bacteria, live bacteria and dea...

Embodiment 2

[0030] Pick a single colony of Escherichia coli O157:H7 (British Type Culture Collection) in 5 mL of LB medium, and culture overnight at 37°C. Take 1 mL of bacterial liquid in a 1.5 mL centrifuge tube, serially dilute ten times with PBS, and count the bacterial liquid concentration as 4.78×10 8 , 4.78×10 7 , 4.78×10 6 , 4.78×10 5 , 4.78×10 4 , 4.78×10 3 and 4.78×10 2CFU / mL. Take 500 μL of different concentrations of bacterial liquid and use the boiling water bath method to extract genomic DNA. The logarithm of the number of live bacteria obtained by plate counting is the abscissa, and the Ct value of the fluorescent quantitative PCR is used as the ordinate to draw the E. coli O157:H7 standard curve: y=-3.27x+41.803, see image 3 .

Embodiment 3

[0031] Example 3. Quantitative detection of live bacteria of Escherichia coli O157:H7 in food

[0032] 1) Food sample pretreatment

[0033] Weigh 1g or 1mL of food samples without objective bacteria, add 9mL of PBS phosphate buffer solution, grind to make a homogenate, add different concentrations of Escherichia coli O157:H7 to ensure a concentration of 10 7 -10 2 CFU / mL, centrifuge at 800rpm for 5 minutes, remove large food residues, and take the supernatant (this process must be performed aseptically)

[0034] 2) Magnetic bead enrichment

[0035] Take 980 μL of the bacterial suspension in step 1) and add it to a sterilized centrifuge tube, add 0.05 mg of immunomagnetic beads coupled with E.coli O157:H7 antibody, the particle size of the magnetic beads is 180 nm, and place it on a rotary mixer at room temperature at 10 rpm After rotating for 45 minutes, insert the centrifuge tube into a magnetic stand for separation for 3 minutes, suck out the supernatant with a pipette, a...

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Abstract

The invention discloses a fluorescence quantitative PCR detection method for escherichia coli O157:H7. aA pair of specific primers amplified with a 20 bp section is designed aiming at the flic of escherichia coli O157:H7. The detection method comprises the following steps: firstly, separating escherichia coli O157:H7 from an actual sample by adopting a magnetic bead enriching method, then eliminating the disturbance brought by dead bacteria in the food sample by the treatment of sodium deoxycholate (SD) and propidium monoazide (PMA), and finally amplifying the live bacteria through a PCR amplification method. The detection method has the advantages of high sensitivity, accurate quantitation, strong specificity, short detection time, simple operation process, and no disturbance caused by the residual DNA or dead bacteria in the food sample.

Description

technical field [0001] The invention belongs to the field of microorganism detection, in particular to a method for rapidly detecting Escherichia coli O157:H7 in food. technical background [0002] Escherichia coli O157:H7 is a food-borne pathogen that was discovered and recognized in the past 30 years. According to the serotype classification, it belongs to Entero Hemorrhagic Escherichia coli (EHEC). It is mainly transmitted by food, and the main hosts are livestock and poultry such as cattle and chickens. Humans may cause hemorrhagic colitis (Hemorrhagic colitis HC) and hemolytic uremie syndrome (Hemolytic uremie syndrome HUS) if they ingest less than 10 live bacteria. threaten. Pasteurization and refrigerated storage are two common methods to reduce the contamination of food by E. coli O157:H7. However, due to the food matrix and E. coli O157: H7 autoimmune mechanisms often have a protective function against bacteria, so E. coli O157: H7 often contaminates food. Since...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06G01N21/64
CPCC12Q1/04C12Q1/6848C12Q1/6851C12Q1/686C12Q2545/114C12Q2549/125C12Q2531/113
Inventor 李林杨晓慧许恒毅张勇攀
Owner WUXI ZODOLABS BIOTECH
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