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Staphylococcus aureus colorimetric sensation detection method based on aptamer recognization-HCR (hybridization chain reaction) and application of method

A Staphylococcus, colorimetric sensing technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, color/spectral property measurement, etc., can solve the problems of high cost, easy inactivation of antibodies, etc. Reduce matrix interference and overcome the effect of long preparation cycle

Pending Publication Date: 2020-06-05
YANGTZE NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although immunoassay technology has been greatly improved in the above aspects, the preparation of antibodies requires immunization of animals, high cost, and easy inactivation of antibodies

Method used

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  • Staphylococcus aureus colorimetric sensation detection method based on aptamer recognization-HCR (hybridization chain reaction) and application of method
  • Staphylococcus aureus colorimetric sensation detection method based on aptamer recognization-HCR (hybridization chain reaction) and application of method
  • Staphylococcus aureus colorimetric sensation detection method based on aptamer recognization-HCR (hybridization chain reaction) and application of method

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: The establishment of the Staphylococcus aureus colorimetric sensing detection method based on the aptamer recognition-HCR reaction, including the following specific steps:

[0038] (1) Preparation of DNA-modified gold nanoprobes: 1 mL of synthesized 20 nm gold nanoparticles was washed with K 2 CO 3 The pH of the solution was adjusted to 8.0, and it was concentrated 10 times by centrifugation at 10,000 r / min; 10 μL of sulfhydryl-modified aptamer 2 (SH-apt2, 1 μM) and priming strand (TP, 5 μM) were taken respectively Added to 4 μL tris-(2-formylethyl)phosphine hydrochloride (TCEP, 1 mM) solution, reacted in the dark for 1 h, added to the colloidal gold solution, coupled for 2 h, then added 10 uL dATP (100 μM) solution, reacted at room temperature for 1 h, added NaCl solution to make the final concentration 80 mM, aged at 4 °C overnight; centrifuged at 1000 rpm / min, 4 °C for 10 min, and discarded the supernatant to obtain Apt 1-AuNPs- TP probe, suspended in...

Embodiment 2

[0044] Example 2: Detection of milk samples by colorimetric sensing of Staphylococcus aureus based on aptamer recognition-HCR reaction

[0045] (1) Preparation of samples to be tested: 25 mL of milk samples were taken aseptically and added to the quality bag, then 25 mL of sterile saline was added, and homogenized at 9000 r / min for 2 min to obtain the sample solution to be tested.

[0046] (2) Sample detection: Take 50 μL of Apt1-MB (10 mg / mL), add it to the microplate well, add 50 μL of the solution to be tested, incubate at 37 °C for 30 min, magnetically separate, wash with PBS, and resuspend in 100 μL Add 50 μL HCR reaction product to PBS solution, incubate at 37°C for 30 min, magnetically separate, wash with PBS, add 100 μL SA-HRP (0.5 μg / mL) solution, react at room temperature for 30 min, magnetically separate, wash with PBS, add to each well 100 μL TMB was added for 10 min to develop color in the dark, and finally 100 μL stop solution (2.0 mol / L H 2 SO 4 ), measured th...

Embodiment 3

[0047] Embodiment 3: Specific verification of Staphylococcus aureus colorimetric sensing detection method based on aptamer recognition-HCR reaction

[0048] Add the PBS buffer solution containing Apt1-MB (10 mg / mL) to the wells of the microtiter plate, 50 μL per well, and add 50 μL at a concentration of 10 4 Cfu / mL suspensions of standard bacterial strains such as Salmonella typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, Shigella dysenteriae, etc., incubated at 37 °C for 1 h. Magnetic separation, PBS washing, resuspended in 100 uL PBS solution, adding 50 uL HCR reaction product, incubate at 37°C for 30 min, magnetic separation, PBS washing. Add 100 uL SA-HRP (0.5ug / mL) solution, react at room temperature for 30 min, magnetically separate, and wash with PBS. Add 100 uL TMB to each well, develop color in the dark for 10 min, and finally add 100 μL stop solution (2.0 mol / L H 2 SO 4 ), and measure the absorbance at 450 nm with a microplate reader. Depend on ...

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Abstract

The invention provides a staphylococcus aureus colorimetric sensation detection method based on aptamer recognization-HCR (hybridization chain reaction). The detection method comprises the following steps: (1) preparing a DNA (deoxyribonucleic acid) functionalized nano golden probe; (2) preparing aptamer modified magnetic nanoparticles; (3) synthesizing a hairpin probe for a hybridization chain reaction system; (4) preparing an HCR product; and (5) detecting a target. The method has the advantages of constant temperature amplification, high sensitivity, low cost, and the like, and has good application prospects.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to an aptamer recognition-HCR reaction-based colorimetric sensing detection method for Staphylococcus aureus and an application thereof. Background technique [0002] With Staphylococcus aureus (S.au) ubiquitous in nature, it is one of the main pathogens causing foodborne diseases, often contaminating milk and dairy products, meat and meat products, poultry and egg products, salads, Pastry and other foods. Staphylococcus aureus can secrete enterotoxins (Staphylococcalenterotoxins, SEs) after breeding at 20-37°C for 4-8 hours, which is the main pathogenic factor causing Staphylococcus aureus food poisoning. According to statistics, food poisoning incidents caused by Staphylococcus aureus enterotoxin in the world account for about 25% to 45% of the total bacterial food poisoning incidents. [0003] At present, the identification and detection methods for foodborne pathogens are ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/14G01N21/78G01N21/31
CPCC12Q1/6844G01N21/78G01N21/31C12Q2531/119C12Q2525/205C12Q2525/301C12Q2563/137C12Q2563/143C12Q2563/155Y02A50/30
Inventor 卢春霞高晓旭
Owner YANGTZE NORMAL UNIVERSITY
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