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Highly-sensitive and rapid method for detecting food-borne pathogenic bacteria based on signal cascade double-amplification system

A food-borne pathogenic bacteria, double-amplification technology, applied in measurement devices, instruments, material analysis by optical means, etc., can solve the problems of time-consuming, labor-intensive, false positives, unable to meet the purpose of immediate and sensitive detection, etc.

Inactive Publication Date: 2019-11-29
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although the traditional detection method is regarded as the gold standard, it is time-consuming and labor-intensive. The entire detection process takes about 4 to 7 days, which cannot meet the purpose of immediate and sensitive detection; detection methods based on immunology or molecular biology such as ELISA, PCR , LAMP and other methods have been greatly improved in sensitivity, but they are prone to false positives, so the specificity needs to be improved

Method used

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  • Highly-sensitive and rapid method for detecting food-borne pathogenic bacteria based on signal cascade double-amplification system
  • Highly-sensitive and rapid method for detecting food-borne pathogenic bacteria based on signal cascade double-amplification system
  • Highly-sensitive and rapid method for detecting food-borne pathogenic bacteria based on signal cascade double-amplification system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1——Salmonella typhimurium in the detection buffer system of immune HCR combined with SERS

[0053] 1 Preparation of immunomagnetic beads

[0054] 1.1 Take 200μg of carboxy magnetic beads in a 2mL centrifuge tube, remove the original preservation solution, add 200μL of MES buffer (pH6) to wash twice, and then activate with 200μL of freshly prepared EDC solution (10mg / mL) for 30 minutes;

[0055] 1.2 Wash once with PBST to avoid aggregation of magnetic beads, add 200 μL anti-Salmonella typhimurium rabbit polyclonal antibody solution (40 mg / mL) and incubate on a mixer for 3 hours, the magnetic beads without antibody are used as negative control;

[0056] 1.3 After washing once with PBST, store the immunomagnetic beads in 800 μL BSA blocking solution (mass fraction 1%, solvent 1×PBS buffer), and store them at 4°C for later use. For the characterization results, see figure 1 .

[0057] 2 Immunomagnetic beads capture Salmonella typhimurium

[0058] 2.1 Take 200 μL ...

Embodiment 2

[0069] Example 2—Immune HCR combined with SERS to detect Salmonella typhimurium in food system

[0070] 1 The preparation of immunomagnetic beads is the same as in Example 1

[0071] 2 Food sample pretreatment

[0072] 2.1 Take 10mL of commercially available ultra-high temperature sterilized milk, centrifuge at 8000rpm for 8 minutes, remove the upper layer of fat, and distribute 1.35mL in each tube into 2mL centrifuge tubes;

[0073] 2.2 Take 150 μL of a series of different concentrations of Salmonella typhimurium in a centrifuge tube containing 1.35 mL of sample, mix well, and obtain 1.5 mL of spiked sample;

[0074] 3 Spiked sample plate count

[0075] For the spiked sample with a large concentration of target bacteria, it was diluted with PBS buffer, and then 100 μL of 2 to 3 suitable continuous gradient samples were selected for each group, spread on LB solid medium, and incubated at 37°C for 24 hours. For counting, three groups of parallels were performed for each samp...

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Abstract

The invention discloses a highly-sensitive and rapid method for detecting food-borne pathogenic bacteria based on a signal cascade double-amplification system. The method mainly comprises the following steps: (1) coupling a polyclonal / monoclonal antibody of pathogenic bacteria to the surface of a magnetic bead to obtain an immunomagnetic bead; (2) mixing the immunomagnetic bead with a sample containing food-borne pathogenic bacteria, and specifically capturing the pathogenic bacteria through antigen-antibody reaction; (3) initiating a hybridization chain reaction by using the pathogenic bacteria aptamer modified by the initiation sequence and a DNA hairpin structure probe to obtain an HCR product (double-stranded DNA with a notch); and (4) adding Raman signal molecules and a Raman substrate into the HCR product, and carrying out corresponding SERS determination to realize cascade amplification and output of signals. The method has great significance in guaranteeing food safety and preventing food-borne diseases, and has great application potential for other biological analysis.

Description

technical field [0001] The invention relates to a method for highly sensitive and rapid detection of food-borne pathogens based on immune HCR combined with SERS, and belongs to the technical field of high-sensitivity and rapid detection of food-borne pathogens. Background technique [0002] In recent years, food safety problems caused by Salmonella are still serious. Because food contaminated by Salmonella does not show obvious sensory properties, it is easy to be accidentally eaten by humans and animals, resulting in foodborne diseases. Typically, intakes of more than 10 5 CFU of Salmonella can infect ordinary people, and for immunocompromised or sensitive people, 15-20 CFU can cause disease, so it is very important to develop a new detection method to achieve highly sensitive detection of Salmonella. [0003] Although the traditional detection method is regarded as the gold standard, it is time-consuming and labor-intensive. The entire detection process takes about 4 to ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N21/65
CPCG01N21/658G01N33/56916
Inventor 叶邦策左鹏李傲
Owner EAST CHINA UNIV OF SCI & TECH
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