Rapid pathogenic microorganism detection method

A technology of pathogenic microorganisms and detection methods, which is applied in the field of unlabeled SERS detection of pathogenic microorganisms, can solve problems such as complex processes, and achieve the effects of simple operation, excellent enrichment performance, and avoiding the decline in recognition ability

Pending Publication Date: 2020-12-22
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former requires the modification of additional signaling molecules, and usually requires the use of immunological means to detect microorganisms, and the whole process is relatively complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Taking Staphylococcus aureus as an example, a construction process for rapid detection of Staphylococcus aureus is provided.

[0026] 1. Preparation of magnetic bead-aptamer-Staphylococcus aureus@Ag composite structure

[0027] (1) Denaturation of aptamers:

[0028] The selected Staphylococcus aureus aptamer sequence is: 5'-Biotin-(CH 2 ) 6 - TCCCT ACGGC GCTAACCTCC CAACC GCTCC ACCCT GCCTC CGCCT CGCCA CCGTG CTACA AC-3'.

[0029] The above-mentioned aptamers were dispersed in PB solution (0.01M, pH7.4), then heated at 95°C for 2min, allowed to cool naturally, and finally stored in a refrigerator at -20°C.

[0030] (2) Synthesis of magnetic bead-aptamer-Staphylococcus aureus composite structure

[0031] 1mL Staphylococcus aureus (10 7 CFU / mL, prepared by PB (0.01M, pH7.4), add 500nM aptamer solution, mix well, and incubate at 4°C for 20min; then, add 10μL streptavidin functionalized magnetic beads (30mg / mL , particle size about 50nm), and incubated with shaking at ro...

Embodiment 2

[0038] Testing of Staphylococcus aureus mock samples

[0039] First, the room temperature milk (3000rpm, 5min) purchased from the supermarket was centrifuged to remove impurities. Then it was diluted 10 times with PB solution for treatment. Add 1,000 and 10,000 CFU / mL Staphylococcus aureus standard solutions therein, mix well, and perform the Staphylococcus aureus detection shown in Example 1. By measuring it at 735cm -1 According to the working curve obtained in Example 1, it is calculated that the concentration of Staphylococcus aureus in the simulated sample is 968.2 and 10121 CFU / mL, and then the recoveries of the two samples are respectively 96.82 % and 101.21%. Therefore, it can be verified that the method has good feasibility and accuracy in the detection of actual samples.

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PUM

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Abstract

The invention discloses a rapid pathogenic microorganism detection method. The method comprises the following steps: untwisting an aptamer into a single chain, preparing a magnetic bead-aptamer-targetmicroorganism, preparing a magnetic bead-aptamer target-microorganism-coated Ag, drawing a standard working curve, and detecting a to-be-detected sample. A rapid and efficient magnetic capture and separation technology is combined with an Ag nanoparticle in-situ enhanced pathogenic microorganism intrinsic Raman signal synthesized by aptamer mediation, and rapid detection of pathogenic microorganisms is realized by virtue of a portable Raman spectrometer. The construction process and the detection process of the sensor do not need large instruments, the operation is simple, the whole process can be completed within 1 hour, the sensitivity is high, and the selectivity is good.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a label-free SERS detection method for pathogenic microorganisms based on magnetic capture separation technology, aptamer-mediated-microorganism surface in-situ synthesis of Ag nanoparticles. Background technique [0002] Pathogenic microorganisms are the primary cause of foodborne diseases, which not only seriously endanger people's lives and health, but also cause large economic losses. Therefore, in order to reduce or control the occurrence of foodborne diseases, reduce or avoid their serious impact and harm, it is very necessary to detect the content of pathogenic microorganisms in food or agricultural products before they enter the market. [0003] Common pathogenic microorganisms in food and agricultural products mainly include Staphylococcus aureus, Salmonella, Escherichia coli, Listeria monocytogenes, etc. The common detection methods mainly include plate ...

Claims

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Application Information

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IPC IPC(8): G01N21/65G01N1/28
CPCG01N21/658G01N1/30
Inventor 王利华白向茹吴文辉邵志勇李婷婷郑思洁战艺芳董军陈洁王佳慧
Owner WUHAN ACADEMY OF AGRI SCI
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