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A method for the determination of Staphylococcus aureus based on aptamer and strand displacement amplification reaction

A technology of strand displacement amplification and staphylococcus, which is applied in the fields of analytical chemistry, food safety and fermentation analysis, can solve the problems of low sensitivity, long detection time, complicated steps, etc., and achieves improved detection sensitivity, expanded detection range, and high sensitivity. Effect

Active Publication Date: 2021-11-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the traditional culture and biochemical detection methods take a long time to detect, and are easily affected by the colony shape and quantity of miscellaneous bacteria and the variety of Staphylococcus aureus, resulting in false negative results; the immunological method has a long test cycle and cumbersome steps, and various non-related antigens are easy to detect. Cause cross-reaction, enterotoxin protein agglutination produced during food heating will cause false positive or false negative test results; molecular biology methods are also easily affected by sample components and other factors
These commonly used methods for the detection of Staphylococcus aureus either require long-term processing, skilled staff, and expensive laboratory equipment, or have disadvantages such as instability, low sensitivity, and poor accuracy.

Method used

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  • A method for the determination of Staphylococcus aureus based on aptamer and strand displacement amplification reaction
  • A method for the determination of Staphylococcus aureus based on aptamer and strand displacement amplification reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 The drawing of Staphylococcus aureus concentration standard curve

[0039] Take 100 μL of streptavidin-modified magnetic beads and 100 μL of aptamer-cDNA complex in a shaker at 37°C for 220r·min -1 Incubate for 45min. Use PBS buffer to wash the magnetic beads. After magnetic separation, add ten-fold gradient dilution of Staphylococcus aureus bacteria solution to each EP tube, the concentration range is 7~7×10 7 CFU mL -1 . Shaking at 37°C 220r·min -1 Incubate for 45 min, wash the magnetic beads, and keep the supernatant after magnetic separation. Take 3 μL supernatant and add 0.0125 μmol L -1 Trigger primer, 0.1 U·μL -1 Bsm DNA polymerase, 0.25 U·μL -1 Nb.bpu10I nicking endonuclease, 250 μmol·L -1 Free deoxyribonucleoside triphosphate, 10 mmol L -1 Tris-HCl, 10 mmol L -1 MgCl 2 , 100 mmol·L -1 KCl, 0.1 mg·mL -1 BSA, mix well and incubate in a 37°C water bath for 100 min, and finally add 30 μL of SYBR Green I for staining for 40 min to measu...

Embodiment 2

[0040] Embodiment 2. Determination of Staphylococcus aureus content in the actual milk sample

[0041] In order to further verify the accuracy of this method in determining the content of Staphylococcus aureus in actual milk samples, a commercially available milk was selected, 12000 r min -1 Centrifuge for 15 min to precipitate protein, and filter the supernatant twice with a 0.22 μm aqueous filter membrane. Inject 80 μL of Staphylococcus aureus in a sterile environment for 10 2 to 10 6 CFU mL -1 Inoculate 20 μL of treated milk samples with serially diluted concentrations of . Take 100 μL of streptavidin-modified magnetic beads and 100 μL of aptamer-cDNA complex in a shaker at 37°C at 220 r min -1 Incubate for 45 min. Wash the magnetic beads, after magnetic separation, add the actual sample of milk after pretreatment, and shake at 37°C at 220 r min -1 Incubate for 45 min, wash the magnetic beads, and keep the supernatant after magnetic separation. Take 3 μL supernatant...

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Abstract

The invention relates to a determination method for Staphylococcus aureus based on an aptamer and a chain displacement amplification reaction, which belongs to the technical fields of analytical chemistry, food safety and fermentation analysis. In the present invention, a target recognition system is firstly constructed, through the high affinity between Staphylococcus aureus and the aptamer, cDNA can be competitively released from the double strand; Single-stranded DNA with a specific sequence; after adding a specific hairpin structure, the single-stranded DNA and the hairpin structure self-assemble in solution to form a specific hexagonal structure; finally add a fluorescent dye, the dye specifically binds to the DNA double strand, and releases fluorescent signal. The amount of Staphylococcus aureus in the sample was calculated using the linear relationship between the fluorescence signal and the number of Staphylococcus aureus. According to the principle of fluorescence resonance energy transfer, the invention correlates the fluorescence signal with the number of staphylococcus aureus, and realizes the direct determination of the content of staphylococcus aureus in food samples such as milk. The established detection method has the characteristics of short detection period, high sensitivity, low cost and strong specificity.

Description

technical field [0001] The invention relates to a method for determining Staphylococcus aureus based on aptamer and strand displacement amplification reaction, which can perform highly sensitive determination of the content of Staphylococcus aureus in food and fermentation samples, and belongs to analytical chemistry, food safety and fermentation Analyze technical fields. Background technique [0002] Staphylococcus aureus, a common human pathogen found in raw milk and raw meat, can produce a variety of endotoxins and cause various infections and foodborne diseases, such as sepsis, infective endocarditis (IE), and Osteoarthritis, skin and soft tissue, pleuropneumonia, etc., seriously threaten human health, and about 30% of the population are carriers of Staphylococcus aureus. At the same time, Staphylococcus aureus is also often a contaminating bacteria in the fermentation process. Therefore, the development of highly sensitive and accurate detection methods for Staphyloco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/14C12Q1/06
CPCC12Q1/6851C12Q1/689C12Q2521/301C12Q2525/205C12Q2525/301C12Q2563/107C12Q2537/1376
Inventor 周楠迪蔡蓉凤尹凡田亚平
Owner JIANGNAN UNIV