CRISPR/Cas12a-RCA electrochemical sensor detection system and application thereof

An electrochemical and sensor technology, applied in the field of rapid detection of food-borne pathogens, can solve the problems of unsuitable rapid detection of food-borne pathogens and complicated detection process, and achieve shortened detection time, high detection sensitivity, and increased sensitivity Effect

Pending Publication Date: 2021-11-23
GUANGDONG OCEAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods need to extract and amplify the nucleic acid of the pathogen, generate a specific DNA sequence to activate CRISPR / Cas12a, and digest the specific nucleic acid to generate a detection signal. The detection process is relatively complicated and is not suitable for food-borne pathogens. It is of great significance to establish a sensitive and simple rapid detection method for Escherichia coli O157:H7

Method used

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  • CRISPR/Cas12a-RCA electrochemical sensor detection system and application thereof
  • CRISPR/Cas12a-RCA electrochemical sensor detection system and application thereof
  • CRISPR/Cas12a-RCA electrochemical sensor detection system and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 Preparation of Reagents Required for Electrochemical Sensing and Detection and Its Feasibility Analysis

[0054] The inventive principle of the present invention is as figure 1 As shown, it uses the RCA reaction to form a functional RCA product under the action of the 5'-phosphorylated linear padlock probe and the ligation probe, which is rich in a large number of repeat units that can specifically bind to Escherichia coli O157:H7 The aptamer sequence and the target ssDNA sequence that are specifically complementary to crRNA to initiate the endonuclease activity of the CRISPR / Cas12a-RCA enzyme cutting system.

[0055] After treating the Au electrode of the electrochemical biosensor, the MB-DNA hairpin probe rich in T sequence and modified with the electrochemical signaling molecule methylene blue (MB) was immobilized on the Au electrode through the Au-S bond, and The active site was blocked with MCH (6-mercapto-1-hexanol) to reduce non-specific adsorption in ...

Embodiment 2

[0092] Example 2 Optimization of Electrochemical Sensing Detection Conditions

[0093]During the research process of the present invention, it was found that the incubation time of antibody magnetic beads and the target-Escherichia coli O157:H7 and the RCA product to form a sandwich structure and the incubation time of CRISPR / Cas12a enzyme cleavage have a great influence on the detection of CRISPR / Cas12a-RCA electrochemical biosensor Effects will matter. Due to the high concentration of antibody magnetic beads and RCA product, non-specific coupling phenomenon will occur, which will cause some false positives for negative results. The amount of RCA product and the subsequent CRISPR / Cas12a enzyme digestion system will also be affected. Therefore, after determining the feasibility of the method, it is necessary to find the optimal detection amount of antibody magnetic beads and RCA products, and finally optimize the concentration of important Cas12a and crRNA in the CRISPR / Cas12a...

Embodiment 3

[0107] Example 3 Electrochemical Sensing Detection Sensitivity and Specificity Analysis

[0108] Under the optimal test condition optimized, the present invention utilizes DPV response to analyze the sensitivity of electrochemical sensor, Escherichia coli O157:H7 is diluted into 10,10 according to 10 times gradients 2 、10 3 、10 4 、10 5 、10 6 、10 7 CFU mL -1 . The result is as Figure 7 as shown, Figure 7 A shows the corresponding relationship between the DPV signal and the logarithm of the concentration of E. coli O157:H7. It can be seen from the figure that the oxidation peak current decreases with the increase of the concentration of the target bacteria. Figure 7 The results of B show that the ΔI% of the DPV response is related to the target bacteria concentration (logarithmic scale) between 10 and 10 7 CFU mL -1 There is a good linear relationship in the range (R 2 =0.9942), the corresponding regression equation is Y=10.71X+18.90, wherein Y and X represent the ...

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Abstract

The invention discloses a CRISPR/Cas12a-RCA electrochemical sensor detection system and application thereof. According to the present invention, the CRISPR/Cas12a-RCA electrochemical sensor detection system can be used for the detection of Escherichia coli O157: H7, has characteristics of simple operation and low cost, can accurately distinguish Escherichia coli O157: H7 from different bacteria, and has strong detection specificity; the detection concentration range is also wide, a good linear relation is achieved within the range of 10-107 CFU.mL <-1 >, the detection sensitivity is high, the lowest detection limit is 10 CFU.mL <-1 >, and the detection result is accurate and reliable. The detection method is not only suitable for detecting escherichia coli O157: H7 in food, but also has huge application potential in detection of other pathogenic bacteria or clinical diagnosis.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of food-borne pathogenic bacteria. More specifically, it relates to a CRISPR / Cas12a-RCA electrochemical sensor detection system and its application. Background technique [0002] Escherichia coli O157:H7 (E.coli O157:H7) is a food-borne pathogen that can cause intestinal diseases and even death in humans. It has attracted widespread attention because of its low infection dose and strong pathogenicity. Currently, the main methods for detecting E. coli O157:H7 include bacterial isolation, immunological detection and molecular biology detection. Biosensor detection methods in molecular biology methods have the characteristics of rapidity, sensitivity, simple operation, and low requirements for operators. [0003] CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats Related System) is an adaptive immune system found in bacteria and archaea. Biotechnology for gene editing. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/30G01N27/327G01N27/48
CPCG01N27/301G01N27/3275G01N27/48
Inventor 陈志宝马丽葛叶姚秋成陈进军徐春厚
Owner GUANGDONG OCEAN UNIVERSITY
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