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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI 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 current monitoring and control of drinking water and food. The developed portable biosensor provides matching reagents

Method used

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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
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of the bioluminescence rapid detection E.coliO157:H7 kit

[0029] Bioluminescence rapid detection E.coliO157:H7 kit includes 5 bottles of immunomagnetic beads coated with antibodies, 100 μl in each bottle; standard ATP solution (1×10 -7 mol / l) 1 vial, 100 μl each; 1 vial of microbial lysate, 10 ml; 5 vials of detection buffer, 20 ml each; 5 vials of bioluminescence reagent, 1 ml each. Wherein, ① 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, magnetically separate and wash twice, then resuspend in 250μl 2-(N-morpholino) Add 500 μg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 750 μg N-hydroxysuccinimide (NHS) to ethanesulfonic acid (MES), 37°C Activate the carboxyl groups on the surface of the magnetic beads for 15 minutes, then wash twice with MES, add 50ug of antibody after resuspensi...

Embodiment 2

[0035] Example 2: Detection of Escherichia coli O157:H7 in food using bioluminescent method rapid detection of special pathogenic bacteria kit

[0036] Adopt the detection kit in embodiment 1 to be used for the detection of embodiment 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, carry out equal dilution to the ATP standard, and the concentration range is 10 -12 -10 -7 mol / l. A bioluminescent agent is added for detection, and the relationship between the concentration of the ATP standard substance and its luminescence value is obtained.

[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 sufficient binding.

[0041] d) adding a small amount of microbial lysate, and reacting at room temperature for 1 min.

[0...

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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.

Description

technical field [0001] The invention relates to a detection method and a detection kit for bioluminescent detection of special pathogenic bacteria. More precisely, the present invention uses a bioluminescence method to quickly detect the total amount of microorganisms or the number of specific microorganisms and the detection kit, which belong to microorganisms detection field. Background technique [0002] Rapid detection and identification of pathogenic bacteria is an important issue in today's society, whether it is in clinical diagnosis, foodborne pathogen monitoring, or even biological weapon prevention. There are many kinds of foodborne pathogenic microorganisms, and the lack of sensitive, convenient and specific rapid detection technology is one of the main reasons why food safety and people's health cannot be effectively guaranteed. Due to the lack of powerful monitoring technology, according to the WHO report, the reported incidence of foodborne diseases in countri...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569
Inventor 葛玉卿金庆辉周洪波赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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