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Colloidal gold test strip for detecting staphylococcus aureus and preparation method

A technology of colloidal gold test paper and staphylococcus, which is applied in the field of medical and sanitary products, can solve the problems of low sensitivity, long time, easy to be polluted, etc., and achieve the effect of high sensitivity detection and high detection accuracy

Pending Publication Date: 2020-04-28
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional biological culture method takes a long time, PCR requires a clean environment, is easily contaminated, and is not suitable for on-site rapid detection, ELISA requires multiple steps, and the sensitivity is not high, so it is not suitable for on-site rapid screening

Method used

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  • Colloidal gold test strip for detecting staphylococcus aureus and preparation method
  • Colloidal gold test strip for detecting staphylococcus aureus and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the preparation of streptavidinized colloidal gold

[0025] (1) Preparation of colloidal gold (AuNP) solution: 50mL of 0.01% HAuCl 4 Add the solution into a round-bottomed flask, and stir until it boils while heating; then, add 5mL of 1% sodium citrate to the above solution under stirring, and continue to boil for 10 minutes after the solution turns wine red. After stopping heating, continue to Stir for 15 minutes to obtain a colloidal gold (AuNP) solution, which is stored at 4°C in the dark.

[0026] (2) Preparation of biotinylated colloidal gold: add 0.1mol / L K to 1mL of colloidal gold solution 2 CO 3 (5 μL) and mix well, add 8 μg streptavidin (SA) after 5 min, shake at room temperature for 30 min. Add 10% BSA (100μL) to block for 30min, centrifuge at 4°C (10×10 3 rpm, 10min), to obtain a colloidal gold-streptavidin (AuNP-SA) probe. Discard the supernatant, and resuspend the AuNP-SA probe with 100 μL boric acid buffer (0.2 mol / L, pH9.0).

Embodiment 2

[0027] Embodiment 2, the preparation of a kind of colloidal gold test strip that detects Staphylococcus aureus

[0028] Such as figure 1 As shown, a colloidal gold test strip for detecting Staphylococcus aureus includes a PVC base plate 1, a nitrocellulose membrane 2, a binding pad 3, an absorbent paper 4, and a sample pad 5;

[0029] The nitrocellulose membrane 2 is bonded to the middle part of the PVC bottom plate 1; the bonding pad 3 and the absorbent paper 4 are respectively pasted on the two ends of the nitrocellulose membrane 2, and the sample pad 5 is pasted on the On the binding pad 3; the streptavidinized colloidal gold prepared in Example 1 is immobilized on the binding pad 3.

[0030] The nitrocellulose membrane 2 is provided with a control line 6 and a test line 7 sequentially from the end close to the absorbent paper 4, and the test line 7 is coated with a Staphylococcus aureus monoclonal antibody. The control line 6 is sprayed with single-stranded DNA complemen...

Embodiment 3

[0034] Embodiment 3, a kind of sensitivity detection of the colloidal gold test paper strip that detects Staphylococcus aureus

[0035] (1) Culture and count of Staphylococcus aureus

[0036] Staphylococcus aureus was enriched with L-B medium for 24 hours at 37°C. Then serial dilutions were performed with PBS buffer. The number of bacteria was obtained by plate counting method: 100 microliters of dispersions with different dilution gradients were evenly coated on crystal violet neutral red bile salt agar (VRBA) plates, and after culturing at 37°C for 48 hours, count the total number of colonies grown To determine the number of bacteria at the specified dilution, expressed in CFU / mL.

[0037] (2) Binding of biotinylated aptamer to Staphylococcus aureus

[0038] Mix 100 μL of biotinylated Staphylococcus aureus aptamer (Biotinylated aptamer) and 1000 μL at concentrations of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 , 10 1 Mix well with 1 CFU / mL Staphylococcus aureus sam...

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Abstract

The invention discloses a colloidal gold test strip for detecting staphylococcus aureus. A bottom plate is included; a nitrocellulose membrane, a conjugate pad, absorbent paper and a sample pad are fixed on the bottom plate; and streptavidin colloidal gold is immobilized on the conjugate pad, a test line and a control line are sequentially arranged on the nitrocellulose membrane from front to back, a staphylococcus aureus monoclonal antibody is immobilized on the test line, and single-stranded DNA complementary with a staphylococcus aureus aptamer is immobilized on the control line. The colloidal gold test strip can realize instant detection of staphylococcus aureus, detection time is shortened to 5-10 minutes, and large instruments and equipment and complex experimental steps are not needed. Staphylococcus aureus can be qualitatively recognized by comparing color changes of the colloidal gold test strip of a system before and after reaction, and quantitative detection can also be performed by reading data of a colloidal gold card reader.

Description

technical field [0001] The invention relates to the technical field of medical and sanitary products, in particular to a colloidal gold test strip for detecting Staphylococcus aureus and a method for preparing the test strip. Background technique [0002] According to the report of the World Health Organization, 420,000 people die from food-borne diseases every year in the world, and the vast majority of food-borne diseases are caused by food-borne pathogens. Staphylococcus aureus is the most common food-borne pathogen. It has significant growth ability and the ability to produce staphylococcal enterotoxins under different conditions in food, leading to staphylococcal food poisoning. Food is easily contaminated during processing, and milk is a good substrate for the growth of Staphylococcus aureus, so dairy products are a common source of poisoning. The development of effective methods to detect Staphylococcus aureus is of great significance for the prevention of foodborne ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558G01N33/52
CPCG01N33/577G01N33/56938G01N33/558G01N33/52
Inventor 曾令文陆琼
Owner FOSHAN UNIVERSITY
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