Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence

A bioluminescent, pathogenic bacteria technology, applied in the field of microbial detection, to achieve the effect of strong specificity, high sensitivity, and easy operation

Inactive Publication Date: 2011-09-14
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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AI-Extracted Technical Summary

Problems solved by technology

[0008] The present invention aims at the lack of rapid, sensitive and reliable detection of pathogenic bacteria in the ...
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Abstract

The invention relates to a method and a detection kit for detecting special pathogenic bacteria by a bioluminescence method combined with immunomagnetic bead identification. According to the method of the invention, with monoclonal antibodies or polyclonal antibodies on an immunomagnetic bead, bacteria in a bacterial suspension or a sample are captured by antigen-antibody reactions; adenosine triphosphate (ATP) in the bacteria is released by adding a lysate; finally detecting the ATP content by fluorescein (D-Luciferin)-luciferase so as to determine whether specific microorganism is present in the sample, and determining the amount of the contained specific microorganism by the determination of an ATP standard curve. The kit provided by the invention comprises an immunomagnetic bead coating the antibodies, a microbial lysate, luciferase and a protective agent, a detection buffer, etc. With the invention, detection time can be shortened greatly (within 2 hours); no bacterium increasing or filtering is necessary; the operation is simple; the sensitivity is high; the specificity is high; and the invention is applicable to rapid field detection of food and environment samples, and basic popularization and application.

Application Domain

Material analysis

Technology Topic

Polyclonal antibodiesAtp content +15

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  • Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence
  • Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence
  • Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence

Examples

  • Experimental program(2)

Example Embodiment

[0028] Example 1: Construction of a kit for rapid detection of E.coli O157:H7 by bioluminescence method
[0029] The kit for rapid detection of E.coli O157:H7 by bioluminescence method includes 5 bottles of antibody-coated immunomagnetic beads, 100μl each; standard ATP solution (1×10 -7 mol/l) 1 bottle, 100μl per bottle; 1 bottle of 10ml microbial lysis solution; 5 bottles of detection buffer, 20ml per bottle; 5 bottles of bioluminescence reagent, 1ml per bottle. Among them, the preparation of reagents includes:
[0030] a) Immunomagnetic beads: Take 500μg of carboxyl magnetic beads in a 1.5ml centrifuge tube, add 1ml of washing buffer, mix well and sonicate for 15min, after washing twice with magnetic separation, resuspend in 250μl 2-(N-morpholino) Add 500μg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 750μg of N-hydroxysuccinimide (NHS) to ethanesulfonic acid (MES), 37℃ Activate the carboxyl group on the surface of the magnetic beads for 15 minutes, then wash twice with MES, add 50ug of antibody after resuspension, react at room temperature for 2 hours, couple the antibody to the surface of the magnetic beads to obtain immunomagnetic beads. Wash the coupled magnetic beads twice with PBS, add 50ul of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), and store in a refrigerator at 4°C.
[0031] b) Standard ATP reagent: Dilute the ATP standard sample (1×10 -7 mol/l) to obtain ATP standards of different concentrations.
[0032] c) Microbial lysate: use trichloroacetic acid as the ATP extractant with a concentration of 0.5% to 2%, and store it at 4°C after autoclaving (121°C, 30min).
[0033] d) Detection buffer: Tris-Acetate is used as the buffer. Weigh a certain amount of Tris base to make the concentration after constant volume is 0.1M, and adjust the pH value before constant volume. Insert the pH meter into the Tris solution, slowly add the acetic acid solution until the pH value is 7.75, then make the volume constant, and store it at 4°C after autoclaving (121°C, 30min).
[0034] e) Bioluminescence reagent: Dissolve 5mg bovine serum albumin and 5mg trehalose in 0.1M Tris-HAc buffer (pH=7.6-7.8), then add 1mg luciferin, 0.5mg luciferase, light Shake gently (not shake) to dissolve. Place it at room temperature for 1 hour before use to restore the temperature to room temperature, and store in a refrigerator at -20°C in the dark when not in use.

Example Embodiment

[0035] Example 2: Detection of Escherichia coli O157:H7 in food using a kit for rapid detection of special pathogenic bacteria using bioluminescence method
[0036] Using the detection kit in Example 1 for the detection in Example 2
[0037] The schematic diagram of the detection process is as figure 1 Shown. Specific steps are as follows:
[0038] a) Make a standard ATP curve first, that is, dilute the ATP standard in equal proportions, and the concentration range is 10 -12 -10 -7 mol/l. Add bioluminescent agent for detection, get the relationship between the concentration of ATP standard and its luminescence value.
[0039] b) The sample is then pretreated to prepare a suitable sample solution.
[0040] c) Add the immunomagnetic beads to the sample solution, incubate together, and remove the supernatant after fully binding.
[0041] d) Add a small amount of microbial lysate and react at room temperature for 1 min.
[0042] e) Add 100ul of detection buffer and 50ul of bioluminescence reagent, and then immediately put it in a luminescence detector to measure the luminescence value.
[0043] f) Calculation of the ATP content of the corresponding pathogenic bacteria in the sample: According to the measured luminescence value and the standard curve obtained in a), the corresponding ATP concentration and the number of pathogenic bacteria in the sample are calculated.
[0044] The results show that the detection limit of this method can reach 10 2 CFU·ml -1.
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