Staphylococcus aureus rapid detection method based on enzyme digestion protection aptamer sensor

An aptamer sensor, staphylococcus technology, applied in biochemical equipment and methods, microbial determination/inspection, instruments, etc., can solve the problems of affecting the sensing accuracy and complicating the detection process, to simplify the operation steps, shorten the Simple effect of detection time and sample preparation

Active Publication Date: 2020-08-07
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most pathogenic aptamer sensors output recognition signals through magnetic force separation, which will complicate the detection process and seriously affect the sensing accuracy.

Method used

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  • Staphylococcus aureus rapid detection method based on enzyme digestion protection aptamer sensor
  • Staphylococcus aureus rapid detection method based on enzyme digestion protection aptamer sensor
  • Staphylococcus aureus rapid detection method based on enzyme digestion protection aptamer sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Using Gel Electrophoresis Analysis to Verify the Working Principle of Enzyme Cut Protection Aptamer Sensor

[0025] The results of gel electrophoresis analysis were as figure 2 shown. It can be seen from the figure that the molecular beacon MB can be self-quenched when there is no aptamer SA43 (line 2, the fluorescence is dim); when MB is combined with SA43, the product band lags behind and the signal is significantly enhanced (line 3), indicating that SA43 Hybridized successfully with MB. When exonuclease I and exonuclease III were added, the band of SA43 disappeared (line4,6), indicating that the aptamer would be efficiently digested by exonuclease. And because the exonuclease has been inactivated before adding MB, Molecular Beacon MB will not be cleaved. When S. aureus is present, the strain can protect SA43 from complete exonuclease cleavage (line 5,7), but SA43 will be truncated because the band moves faster compared to the original aptamer (line 7)...

Embodiment 2

[0026] Embodiment 2: Optimization of reaction conditions

[0027] Take a certain amount of washed and resuspended Staphylococcus aureus bacteria liquid, mix with a certain volume and concentration of aptamer SA43, and react for 30 minutes, then add a certain concentration of exonuclease I and III to digest for 30 minutes, and then place it at 90°C After inactivating the enzyme activity for 5 minutes, add a certain amount of molecular beacon MB to the above mixture, mix well, place it at 90°C for 5 minutes, and measure the fluorescence signal after standing at room temperature for 10 minutes. The present invention optimizes the concentration of exonuclease I and exonuclease III, the concentration of molecular beacon MB, and the addition ratio of aptamer SA43 and MB. The optimization results were as follows: the amount of exonuclease I added was 0.16U / μL, the amount of exonuclease III added was 1.6U / μL, the ratio of SA43 to MB was 2:1, and the concentration of MB was 1 μM.

Embodiment 3

[0028] Embodiment 3: the drafting of Staphylococcus aureus standard curve

[0029] Preparation of Staphylococcus aureus suspensions with different concentration gradients: 0, 10, 10 2 ,10 3 ,10 4 ,10 5 ,10 6 ,10 7 ,10 8 ,10 9 ,10 10 ,10 11 CFU / mL, take 8 μL of the above bacterial suspension, mix with 8 μL of 20 μM aptamer SA43 solution, 8 μL of exonuclease I buffer (10x) and 46 μL of water, combine at 37°C for 30 min, then add 2 μL of exonuclease I Enzyme I (6.4U / μL) and exonuclease III (64U / μL) solution, mix well, place at 37°C for enzyme digestion for 30min, after digestion, place the mixture at 90°C for 5min to inactivate the enzyme, then Take 8 μL of 10 μM MB solution and add it to the above mixture, mix well, place it at 90°C for 5 minutes and let it stand at room temperature for 10 minutes, then measure the fluorescence signal. The experimental results are in 10 2 to 10 7 In the bacterial solution concentration in the interval, the fluorescence intensity and ...

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Abstract

The invention discloses a staphylococcus aureus rapid detection method based on an enzyme digestion protection aptamer sensor. The aptamer sensor comprises two DNA sequences: a nucleic acid aptamer SA43 and a molecular beacon MB. The action mechanism of the method is the enzyme digestion protection effect of the aptamer-staphylococcus aureus conjugate (figure 1): the aptamer SA43 can quickly recognize and be specifically bonded to staphylococcus aureus so as to prevent enzyme digestion by an excision enzyme. Furthermore, the aptamer can be stably hybridized with a molecular beacon MB, a hairpin structure of the MB is opened, and a fluorescence signal is remarkably improved. On the contrary, when the sample does not contain staphylococcus aureus, SA43 is easily cut off by the excision enzyme, so that SA43 cannot be hybridized with MB. At the moment, MB is of a head-tail self-hybridization structure, and fluorescence quenching is achieved. Staphylococcus aureus can be quantitatively detected through a linear curve of fluorescence intensity and bacterium concentration. The method has the advantages that the aptamer is easy to synthesize, culture-free, separation-free, high in sensitivity and the like.

Description

technical field [0001] The invention belongs to the field of food safety, and in particular relates to a rapid detection method for Staphylococcus aureus based on an enzyme-cut protection aptamer sensor. Background technique [0002] Staphylococcus aureus is easy to cause suppurative infection in humans. It is a major food-borne Gram-positive pathogenic bacteria. It is not strict to the growth environment. Staphylococcus aureus contamination can cause food poisoning. Staphylococcus foodborne disease is one of the most common foodborne diseases. According to statistics, global food poisoning caused by Staphylococcus aureus accounts for about 25% of bacterial foodborne diseases, seriously affecting human food safety and economic development. Therefore, it is particularly important to establish a rapid analytical method for Staphylococcus aureus, which will help to effectively control foodborne pathogens, thereby promoting food safety and public health. [0003] At present, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/14C12Q1/06G01N21/64
CPCC12Q1/6825G01N21/6428C12Q2521/319C12Q2525/205C12Q2563/107C12Q2565/607Y02A50/30
Inventor 邓锐杰卢云浩杨林子
Owner SICHUAN UNIV
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