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

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Problems solved by technology

Although immunoassay technology has been greatly improved in the above aspects, the preparation

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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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Example Embodiment

[0037] Example 1: Establishment of a colorimetric sensor detection method for Staphylococcus aureus based on aptamer recognition-HCR reaction, including the following specific steps:

[0038] (1) Preparation of DNA-modified gold nanoprobes: use 1 mL of synthetic 20 nm gold nanoparticles with K 2 CO 3 The pH of the solution was adjusted to 8.0, and it was concentrated by centrifugation at 10000 r / min by 10 times; 10 μL of sulfhydryl modified aptamer 2 (SH-apt2, 1 μM) and priming chain (TP, 5 μM) were taken respectively Add to 4 μL of tris-(2-formylethyl)phosphine hydrochloride (TCEP, 1 mM) solution, react for 1 h in the dark, then add to the colloidal gold solution, couple reaction for 2 h, then add 10 uL dATP (100 μM) solution, react at room temperature for 1 h, add NaCl solution to make the final concentration 80 mM, aging at 4°C overnight; centrifuge at 1000 rpm / min at 4°C for 10 min, discard the supernatant, and obtain Apt 1-AuNPs- TP probe, suspended in 100 μL PBS for reconst...

Example Embodiment

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

[0045] (1) Preparation of the sample to be tested: Aseptically take 25 mL of the milk sample and add it to the mass bag, then add 25 mL of sterile saline, homogenize at 9000 r / min for 2 minutes to obtain the sample solution to be tested.

[0046] (2) Sample detection: Take 50μL of Apt1-MB (10 mg / mL), add it to the wells of the microtiter plate, add 50μL of the test solution, incubate at 37 ℃ for 30 min, magnetically separate, wash with PBS, and resuspend in 100 μL Add 50 μL of 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 minutes, magnetically separate, wash with PBS, add to each well 100 μL TMB was added for 10 min in the dark for color development, and finally 100 μL stop solution (2.0 mol / L H 2 SO 4 ), the absorbance v...

Example Embodiment

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

[0048] Take the PBS buffer containing Apt1-MB (10 mg / mL) and add it to the wells of the microtiter plate, 50μL per well, and add 50μL to the concentration of 10 4 Cfu / mL of Salmonella Typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, Shigella dysentery and other standard bacterial suspensions were incubated at 37 ℃ for 1 h. Magnetic separation, wash with PBS, resuspend in 100 uL PBS solution, add 50uL HCR reaction product, incubate at 37℃ for 30 min, magnetic separation, wash with PBS. 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, add it for 10 min in the dark and color, and finally add 100 μL stop solution (2.0 mol / L H 2 SO 4 ), the absorbance at 450nm measured by the microplate reader. by Figure 5 It can be seen that ...

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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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