Fluorescent signal enhancement method for detecting escherichia coli by enzyme substrate process

A technology of Escherichia coli and fluorescence signal, which is applied in the fields of biochemical equipment and methods, fluorescence/phosphorescence, and microbial determination/inspection, etc., can solve the problems of less 4-MU, inability to separate product signal and substrate signal, etc.

Inactive Publication Date: 2020-04-28
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can give the specific concentration value of Escherichia coli, but since the substrate MUG also has fluorescence intensity at the emission peak position of the product 4-MU, when the amount of Escherichia coli in the sample is very small, the amount of 4-MU produced is very small , the product signal cannot be separated from the substrate signal

Method used

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  • Fluorescent signal enhancement method for detecting escherichia coli by enzyme substrate process
  • Fluorescent signal enhancement method for detecting escherichia coli by enzyme substrate process
  • Fluorescent signal enhancement method for detecting escherichia coli by enzyme substrate process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Step 1. Purchase commercially available medium containing MUG enzyme substrate, 1L medium contains the following reagent components:

[0027] 4-Methylumbelliferone-β-D-glucuronide (MUG) 75.0mg

[0028] Tryptone 10.0g

[0029] Ammonium sulfate [(NH 4 ) 2 SO 4 ] 5.0g

[0030] Manganese sulfate (MnSO 4 ) 0.5mg

[0031] Zinc sulfate (ZnSO 4 ) 0.5mg

[0032] Magnesium Sulfate (MgSO 4 ) 100.0mg

[0033] Sodium chloride (NaCl) 10.0g

[0034] Calcium Chloride (CaCl 2 ) 50.0mg

[0035] Sodium sulfite (Na 2 SO 3 ) 40.0mg

[0036] K H 2 PO 4 0.9g

[0037] Na 2 HPO 4 6.2g

[0038] Step 2. Inoculate E.coli ATCC 25922 in LB medium by aseptic operation, cultivate to the stationary phase, and it takes about 12 hours at 37°C and 220 rpm. The cultivated E.coli was centrifuged and washed 3 times, and the OD was quantified 600 =0.2 is the bacterial solution A, and the bacterial solution A is diluted by 10 5 For bacterial solution B. Inoculate 50 μL of bacterial s...

Embodiment 2

[0044] Step 1. Purchase commercially available NA-MUG medium, and prepare the solution according to the instructions, 1L content:

[0045] Beef Extract 3.0g

[0046] Tryptone 5.0g

[0047] MUG 100mg

[0048] Step 2. is described with embodiment 1 step 2.

[0049] Step 3. Incubate the culture solution in step 2 at 44° C. for 12 hours.

[0050] Step 4. Divide each tube of the solution in step 3 into two parts, one of which is adjusted to pH=10 by adding NaOH (marked as pH10), and the other part is used as a control (marked as Normal) for normal fluorescence measurement.

[0051] Step 5. Measure the fluorescence intensity of the two solutions described in step 4. Excited at 366nm, read the emission peak at 450nm.

[0052] Step 6. Results see figure 2 : It can be seen from the fluorescence measurement results of the two solutions marked as Normal and pH10 that the value of the fluorescence intensity subtracted from the blank of the same concentration sample increases by nea...

Embodiment 3

[0054] Step 1. Purchase commercially available MMO-MUG medium, and prepare the solution according to the instructions, 1L content:

[0055] Ammonium sulfate 5.0g

[0056] Manganese sulfate 0.5mg

[0057] Zinc sulfate 0.5mg

[0058] Magnesium Sulfate 100mg

[0059] Sodium chloride 10.0g

[0060] Calcium chloride 50.0mg

[0061] Sodium sulfite 40.0mg

[0062] ONPG 500mg

[0063] MUG 75mg

[0064] HEPES 6.9g

[0065] HEPES sodium salt 5.3g

[0066] Amphotericin B 1.0mg

[0067] Solanium Extract 500mg

[0068] Step 2. is described with the step 2 of embodiment 1.

[0069] Step 3. Incubate the culture solution in step 2 at 35° C. for 26 hours.

[0070] Step 4. Divide each tube of the solution in step 3 into two parts, one of which is adjusted to pH=11 by adding KOH (marked as pH11), and the other part is used as a control (marked as Normal) for normal fluorescence measurement.

[0071] Step 5. Measure the fluorescence intensity of the two solutions described in step 4. ...

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Abstract

The invention relates to a fluorescent signal enhancement method for detecting escherichia coli by an enzyme substrate process, and belongs to the technical field of escherichia coli detection. An enzyme substrate targeted by the method provided by the invention is MUG, and the technical problems that a combination of 366 nm excitation and 450 nm emission is adopted in the prior art, after the MUGbackground is excited by 366 nm, the MUG background has certain fluorescence intensity at 450 nm, when the concentration of escherichia coli is very low, the yield of 4-MU is low, a product signal isinseparable from a background signal, so that water samples with an extremely low escherichia coli concentration cannot be detected by current methods are solved. The method for detecting the escherichia coli by the enzyme substrate process provided by the invention adopts the manner of adding one-step operation after the end of incubation and before fluorescence measurement; the operation is that the pH is adjusted to >=9 by using an alkaline reagent, the absorption peak of the product 4-MU can be obviously enhanced, the absorption peak of the substrate MUG changes little, so that the product signal can be separated from the background signal; and through the operation provided by the invention, the difference in fluorescence intensity between the product signal and the background signalis increased by five times, and the method is particularly suitable for detection of the escherichia coli with a very low concentration.

Description

technical field [0001] The invention relates to the technical field of Escherichia coli detection, in particular to a fluorescent signal enhancement method for detecting Escherichia coli with an enzyme substrate method. Background technique [0002] The detection of Escherichia coli is of great significance as an indicator microorganism of fecal pollution. The method of detecting E. coli based on metabolism is widely used because of its wide detection range and high accuracy. By selecting a suitable enzyme substrate, such as the use of E.coli can produce β-glucuronidase, decompose 4-methylumbelliferone-β-D-glucuronide (MUG) to generate fluorescent 4-methyl Umbelliferone (4-MU) was detected. Escherichia coli with this metabolic pathway can be detected. At present, there are two ways to realize the E. coli detection method based on the enzyme substrate method. One method is the multi-well plate method stipulated in the national standard law (HJ1001-2018). Under certain con...

Claims

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Application Information

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IPC IPC(8): C12Q1/10G01N21/64
CPCC12Q1/10G01N21/6486
Inventor 刘玲董绍俊
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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