Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence
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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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[0008] The present invention aims at the lack of rapid, sensitive and reliable detection of pathogenic bacteria in the
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[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 twic...
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[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 r...
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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...
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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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