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Method for rapidly detecting Escherichia coli

A rapid technology for Escherichia coli, which is applied in biochemical equipment and methods, measuring devices, and microbial determination/inspection. Accuracy, shorter incubation time, better linearity

Pending Publication Date: 2020-02-28
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention will solve the problem of using 366nm excitation and 450nm emission combination in the prior art. After the MUG background is excited by 366nm, it also has a certain fluorescence intensity at 450nm. The bottom signal is inseparable, which leads to the technical problem that the existing method cannot detect the samples with extremely low concentration of E. coli in water samples. A method for rapid detection of E. coli is provided.

Method used

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  • Method for rapidly detecting Escherichia coli
  • Method for rapidly detecting Escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0027] 1. Preparation of MUG medium

[0028] 1L culture medium, weigh the following reagents:

[0029] 4-methylumbelliferone-β-D-glucuronide (MUG) 75.0mg;

[0030] Tryptone 10.0g;

[0031] Ammonium sulfate [(NH 4 ) 2 SO 4 ]5.0g;

[0032] Manganese sulfate (MnSO 4 )0.5 mg;

[0033] Zinc sulfate (ZnSO 4 )0.5 mg;

[0034] Magnesium Sulfate (MgSO 4 ) 100.0mg;

[0035] Sodium chloride (NaCl) 10.0g;

[0036] Calcium Chloride (CaCl 2 )50.0mg;

[0037] Sodium sulfite (Na 2 SO 3 ) 40.0 mg;

[0038] K H 2 PO 4 0.9g;

[0039] Na 2 HPO 4 6.2g;

[0040] Then take 5mL into a 10mL centrifuge tube, the number of centrifuge tubes is determined according to the experimental needs, and mark the centrifuge tubes with a marker pen.

[0041] 2. Inoculate E.coli ATCC 25922 in LB medium by aseptic operation, and cultivate to the stationary phase, which takes about 12 hours at 37°C and 220 rpm. The cultured E.coli was cleaned by centrifugation for 3 times with aseptic operati...

Embodiment 2

[0049] 1. Preparation of MUG medium

[0050] 1L culture medium, weigh the following reagents:

[0051] 4-methylumbelliferone-β-D-glucuronide (MUG) 50.0mg;

[0052] Tryptone 10.0g;

[0053] Sodium chloride (NaCl) 5.0g;

[0054] Then take 5mL into a 10mL centrifuge tube, the number of centrifuge tubes is determined according to the experimental needs, and mark the centrifuge tubes with a marker pen.

[0055] Steps 2-4 are the same as Steps 2-4 of Example 1.

[0056] 5. Incubate the culture in step 4 at 37°C for 18 hours, and then use H 2 SO 4 Adjust pH 6 and measure fluorescence intensity. Excite at 320nm, and read the emission peaks at 380nm and 450nm, respectively.

[0057] 6. According to the Escherichia coli concentration calculated in step 4, and the ratio of the emission peak peak value of the culture medium in step 4 measured in step 5 at 380nm and 450nm, draw a standard curve, see figure 2 .

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Abstract

The invention relates to a method for rapidly detecting Escherichia coli, belongs to the technical field of Escherichia coli detection, and solves the technical problems that 366nm excitation and 450nm emission are combined, a background MUG has a certain fluorescence intensity at 450nm after 366nm excitation, a product signal cannot be separated from a background signal when the concentration ofthe Escherichia coli is low and 4-MU yield is less, and a sample with very low concentration of the Escherichia coli in water samples cannot be detected by an existing method in the prior art. According to the method for rapidly detecting the Escherichia coli, by a ratio fluorescence method, reduction of an absorption peak of the background MUG and increase of an absorption peak of a product 4-MUcan be simultaneously utilized, and the ratio of absorption peak values of the background MUG and the product 4-MU and the concentration of the Escherichia coli are linearly fitted. Compared with a single method of 366nm excitation and 450nm emission, fitting linearity is better, detection accuracy is improved, incubation time of Escherichia coli detection can be shortened, and distinction can beachieved by the aid of ratio fluorescence when 4-MU yield cannot reach distinction degree.

Description

technical field [0001] The invention relates to the technical field of Escherichia coli detection, in particular to a method for rapidly detecting Escherichia coli. Background technique [0002] Coliform is an internationally recognized indicator bacterium for testing the epidemiological safety of various foods, medicines, and environmental water quality, and it is the best indicator bacterium for fecal contamination. How to detect Escherichia coli, especially how to detect low concentration Escherichia coli is of great significance. [0003] Traditional E.coli detection methods mainly include multi-tube fermentation, membrane filtration method and plate counting method (GB4789.3-2016 replaces GB / T4789.32-2002), these methods have the disadvantages of cumbersome operation, long time-consuming and low sensitivity . In recent years, scientists have developed many rapid detection methods, which are mainly divided into three categories: molecular biology methods, immunoassay t...

Claims

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Application Information

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IPC IPC(8): C12Q1/10C12Q1/06G01N21/64
CPCC12Q1/06C12Q1/10G01N21/6428
Inventor 刘玲吴娓娓董绍俊
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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