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Staphylococcus aureus detection kit and manufacturing method thereof

A technology for detecting kits and staphylococci, applied in the field of kits, can solve the problems of high cost, low sensitivity, complicated instruments and equipment, etc.

Pending Publication Date: 2020-06-23
赵薇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection methods of Staphylococcus aureus in my country are mainly traditional microbial culture and biochemical identification. These methods are cumbersome and time-consuming, and it is difficult to meet the requirements of rapid detection.
[0003] In recent years, in order to speed up detection and improve the accuracy of detection results, researchers have developed many alternative methods, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), enzyme-linked immunosorbent assay (ELISA), etc., but these methods have the disadvantages of high cost, complex equipment or low sensitivity, which limits their wide application and is not conducive to the on-site detection of samples.

Method used

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  • Staphylococcus aureus detection kit and manufacturing method thereof
  • Staphylococcus aureus detection kit and manufacturing method thereof
  • Staphylococcus aureus detection kit and manufacturing method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0031] The present invention also provides a preparation method of a staphylococcus aureus detection kit, comprising:

[0032] Step 1: Preparation of magnetic beads coated with gold nanoparticles

[0033] FeCl 3 ·6H 2 O and FeCl 2 4H 2 O dissolved in deionized water, the FeCl 3 ·6H 2 O and FeCl 2 4H 2 The molar concentration ratio of O is preferably 2:1, stir vigorously, add NaOH drop by drop, the concentration of NaOH is preferably 1M, stir well, and make the pH of the solution 12; use a permanent magnet to separate the black solid matter in the mixture, and clean it. Described cleaning is to preferably wash 3-5 times with deionized water, rinse 2 times with deionized water after perchloric acid rinse 1 time;

[0034] Place the solid product in an environment of 37°C, the standing time is preferably 2 hours, and then rinse and dry, preferably use deionized water to thoroughly rinse the black product 3 times during the washing, and the drying time is preferably 60-65 ...

Embodiment 1

[0050] Step 1: Preparation of immunomagnetic bead probes coated with gold nanoparticles

[0051] FeCl with a molar concentration ratio of 2:1 3 ·6H 2 O and FeCl 2 4H 2 Dissolve O in deionized water and stir vigorously; add NaOH with a concentration of 1M dropwise and stir well to make the pH of the solution 12; use a permanent magnet to separate the black solid matter in the mixture, wash it with deionized water three times, and rinse with perchloric acid Rinse once with deionized water twice; place the solid product at 37°C for 2 hours; rinse the black product thoroughly with deionized water three times and dry it in a vacuum oven at 60°C overnight.

[0052] Weigh 20 mg of the dried black solid product and dissolve it in 5 mL of deionized water, ultrasonically until uniformly dispersed; add 5 mL of LEDTA-NaOH solution and 14 mL of CTAB solution, and mix evenly; add 3 mL of chloroauric acid solution with a final concentration of 1 mM and a final concentration of 0.1M 0.6mL...

Embodiment 2

[0062] Example 2 Detection of Staphylococcus aureus based on magnetic separation and nano-gold etching technology

[0063] The detection flow chart is as figure 1 As shown: Take 100 μL of the test solution, 50 μg of the immunomagnetic bead probe, 175 μL of the gold nanoparticle probe and 100 μL of PBS and react at room temperature for 30 minutes; magnetic separation, absorb 100 μL of the supernatant liquid and add the etching solution, and react for 30 minutes; add TMB 50 μL, 2 μL hydrogen peroxide, react for 5 minutes; add stop solution and react for 5 minutes, and measure the absorption spectrum with a UV spectrophotometer.

[0064] Experimental results show that the detection method of the present invention is stable, the minimum detection concentration is as low as 10cfu / mL, the detection time is short, and the detection can be completed in 70 minutes, which meets the requirements of rapid detection. Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes, E...

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Abstract

The invention provides a staphylococcus aureus detection kit and a manufacturing method thereof, and belongs to the field of kits. The kit is coated with an immunomagnetic bead probe of gold nanoparticles, a gold nanoparticle probe, an etching solution and a standard bacteria solution. The immunomagnetic bead probe coated with the gold nanoparticles is prepared by the following steps: preparing magnetic beads coated with the gold nanoparticles; and coupling the magnetic beads with staphylococcus aureus specific chicken egg yolk antibody IgY. The gold nanoparticle probe is obtained by firstly synthesizing gold nanoparticles and coupling the gold nanoparticles with a staphylococcus aureus specific aptamer. The kit manufactured in the invention has advantages of strong specificity, high sensitivity and short detection time during staphylococcus aureus detection, a minimum detection concentration is as low as 10cfu / mL, and the kit can be used for detecting the staphylococcus aureus in food.

Description

technical field [0001] The invention belongs to the field of kits, and specifically provides a staphylococcus aureus detection kit and a preparation method thereof. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, Staphylococcus aureus) is an important food-borne pathogen, which can contaminate food through various channels and cause poisoning and other food-borne diseases. The main acute symptoms are nausea and vomiting , can lead to death in severe cases. Therefore, it is of great significance to develop a simple, sensitive and specific detection method for S. aureus. At present, the detection methods of Staphylococcus aureus in my country are mainly traditional microbial culture and biochemical identification. These methods are cumbersome and time-consuming, and it is difficult to meet the requirements of rapid detection. [0003] In recent years, in order to speed up detection and improve the accuracy of detection results, researchers have de...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/569G01N33/543
CPCG01N33/587G01N33/56938G01N33/54326G01N2469/10Y02A50/30
Inventor 赵薇赵超王娟李可维
Owner 赵薇
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