Method for detecting staphylococcus aureus based on combination of screen-printed electrode and CRISPR / Cas13a

Abstract

The invention is applicable to the technical field of biological detection, and provides a method for detecting staphylococcus aureus based on combination of a screen-printed electrode and CRISPR / Cas13a, a probe containing a DNA / RNA hybrid chain is combined with a magnetic bead to form a simple composite structure, when the Cas13a recognizes 16sRNA of S. aureus, trans-cleavage activity can be immediately activated, the probe chain is rapidly cut off, the cut-off amount depends on the activated amount, and when the Cas13a recognizes 16sRNA of S. aureus, the Cas13a can be used for detecting staphylococcus aureus. And after transient magnetic separation, a result is observed on the screen-printed electrode. The method has ultrahigh specificity: the CRISPR / crRNA sequence ensures that staphylococcus aureus and other microorganisms can be accurately distinguished; the trans-cutting of Cas13a provides chemical amplification, and the magnetic separation purification greatly reduces background signals caused by non-specific adsorption, so that the signal-to-noise ratio and the sensitivity are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (SAA) is a common Gram-positive coccus, widely found in the natural environment and on the nasal cavity and skin surface of approximately 30% of healthy individuals. Normally, it is a harmless commensal organism, but certain strains can produce potent toxins and invasive enzymes, transforming into dangerous pathogens that can cause a range of diseases, from minor skin infections (such as boils and carbuncles) to life-threatening bacteremia, pneumonia, endocarditis, and toxic shock syndrome. Its pathogenicity primarily stems from various virulence factors, such as hemolysins, leukocidins, enterotoxins (causing food poisoning), and surface adhesion proteins. Furthermore, the main challenge in the clinical treatment of this bacterium lies in its strong drug resistance, especially the emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA). Routine detection of Staphylococcus aureus typically begins with traditional culture methods, which are the reliable gold standard: after culturing samples on blood agar plates, suspicious colonies with typical golden-yellow pigment and hemolytic zones are observed, followed by confirmation by Gram staining microscopy (observing grape-like arrangements of Gram-positive cocci) and the crucial plasma coagulase test. To meet the need for rapid screening, immunological methods such as colloidal gold test strips or latex agglutination tests can detect specific antigens within minutes. Laboratories widely employ more efficient and precise techniques, such as automated biochemical identification systems and real-time fluorescent PCR (which can simultaneously detect drug resistance genes). However, these methods often suffer from problems such as analytical complexity, high cost, and low throughput.

[0003] Clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have opened up new avenues for molecular diagnostics. CRISPR / Cas13, after arbitrary cleavage of non-target nucleic acids (a phenomenon known as trans-nucleic acid activity), has shown great potential as a biosensor. The CRISPR / Cas12a system, with its inherent high resolution, simplicity, and self-signal amplification capabilities, offers new opportunities for Staphylococcus aureus detection methods. In the field of bacterial detection, the CRISPR / Cas13a biosensor represents a new generation of molecular diagnostic paradigm that combines ultra-high sensitivity with precise specificity. Its core design aims to convert trace amounts of bacterial-specific nucleic acid signals into strong and readable physical signals. The entire detection process begins with the precise selection and acquisition of target bacterial nucleic acids: Unlike viruses, bacteria have more complex cellular structures, so it is usually necessary to first lyse the bacteria through chemical or mechanical means to release their total RNA; the detection targets are usually selected from highly conserved and copy-rich ribosomal RNAs (such as 16S rRNA), or mRNA transcripts of key virulence factor genes and drug resistance genes (such as the mecA gene of Staphylococcus aureus and the rpoB gene of Mycobacterium tuberculosis); in order to improve detection sensitivity and eliminate the need for large instruments, the target RNA in the sample is often pre-amplified by isothermal amplification techniques (such as RPA or LAMP) to generate sufficient RNA sequences that can be recognized by the Cas13a system. However, while the pre-amplification step brings high sensitivity, it also increases the complexity of operation, the risk of contamination, the possibility of misinterpretation of results, and raises the cost and equipment threshold. Therefore, a cutting-edge research direction in the current CRISPR detection field is to develop "amplification-free" direct detection strategies, aiming to overcome the above-mentioned shortcomings and achieve faster, more robust, and more convenient on-site molecular diagnostics.

[0004] Electrochemical screen-printed electrode sensors, combining the high sensitivity of electrochemical analysis with the scalability and miniaturization advantages of screen printing technology, have become a core component for building point-of-care testing platforms. They are particularly suitable for integration with biometric components such as CRISPR, and their core advantages are reflected in four aspects: First, they employ mature thick-film printing technology, enabling mass production of disposable electrodes with uniform performance and low cost, significantly reducing the cost per test and avoiding cross-contamination. Second, the electrode miniaturizes and integrates the working electrode, reference electrode, and counter electrode onto a single card-type chip, requiring a very small sample volume (typically 20-50 microliters), and the overall device is easily portable, laying the hardware foundation for rapid on-site detection. Third, the surface of its carbon-based electrode materials is easily modified with diverse chemical or biological methods (such as depositing nanomaterials, immobilizing enzymes, or antibodies), allowing for flexible customization of highly selective sensing interfaces for different targets. Finally, when used in conjunction with a portable electrochemical workstation, this sensor can complete detection within minutes, is easy to operate, and requires no large instruments or specialized laboratory environment. These advantages have been fully demonstrated in recent cutting-edge research. For example, in the field of environmental and agricultural monitoring, researchers have used electrodeposited platinum particles to modify screen-printed electrodes and utilized the specific electrocatalytic effect of platinum on ammonium ions to achieve rapid on-site analysis of ammonium nitrogen in soil within 5 minutes, providing a new method for precision agriculture. In food safety monitoring, based on screen-printed carbon nanotube electrodes, acetylcholinesterase was immobilized through layer-by-layer assembly technology, successfully constructing a highly sensitive biosensor for detecting organophosphorus pesticides, with a detection limit of 10 ng / mL for chlorpyrifos. In addition, by modifying the working electrode surface with bismuth or mercury films, screen-printed electrodes can simultaneously and with high sensitivity detect trace heavy metals lead and cadmium in drinking water, with detection limits as low as µg / L or even ng / L, fully meeting the standards of the World Health Organization. These examples collectively demonstrate the enormous potential of electrochemical screen-printed electrode sensors in achieving rapid, low-cost, portable, and high-performance detection, and are key carriers for promoting the transition of various biosensors, including CRISPR diagnostic technology, from the laboratory to practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a, aiming to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a includes the following steps: Step 1: Prepare crRNA; Step 2: Preparation of magnetic bead incubation buffer I and preparation of probe complex: 2.1 Weigh Tris powder, EDTA powder and NaCl powder, add ultrapure water and stir magnetically until completely dissolved; then add Tween-20, shake thoroughly to mix, and slowly adjust the pH of the solution to 7.5 with HCl; filter and bring the filtered solution to a final volume. 2.2 Take 10 mg / mL streptavidin-modified magnetic beads, wash several times with magnetic bead incubation buffer I, and magnetically separate them after each wash, discarding the supernatant to remove impurities; add 1 mM PBS buffer to the washed magnetic beads, resuspend them, add 5 μM biotin-labeled probe solution, and mix thoroughly; incubate at 37℃ for 1 h to form a magnetic bead-probe complex by utilizing the specific binding of biotin to streptavidin SA; after incubation, wash several times with buffer I, magnetically separate them, and set aside for later use; Step 3: Strain culture and preparation; Step 4: 16S rRNA extraction; Step 5: Differential pulse voltammetry (DPV) detection: 5.1 Construct an activation reaction system comprising: 1× reaction buffer, 1 μM Cas13a protein, 1 μM crRNA and 1 μM target, with enzyme-free water added to make up the volume; pre-incubate at 37℃ for 30 min to activate the trans-cleavage activity of Cas13a; then add the prepared magnetic bead-probe complex to the system and continue incubation at 37℃ for 30 min to allow the activated Cas13a to cleave the probe on the surface of the magnetic beads; 5.2 After the reaction, the reaction system was washed several times with magnetic bead incubation buffer I, and the free probe and impurities were removed by magnetic separation. The magnetic bead complex was resuspended in 0.1 mM PBS buffer and transferred to the three-electrode system of the electrochemical workstation. The working electrode was a glassy carbon electrode, the reference electrode was a saturated calomel electrode, and the counter electrode was a platinum wire electrode. The DPV detection parameters were set as follows: potential range -0.2~0.2 V, pulse amplitude 50 mV, and pulse width 50 ms. The voltammetric curve was recorded, and the target concentration was quantitatively analyzed based on the peak current change.

[0007] The embodiments of this invention have extremely high specificity, guaranteed by the CRISPR / crRNA sequence, and can accurately distinguish Staphylococcus aureus from other microorganisms; it can achieve dual signal amplification and noise reduction, with the trans-cleavage of Cas13a providing chemical amplification and magnetic separation purification greatly reducing the background signal caused by non-specific adsorption, thereby significantly improving the signal-to-noise ratio and sensitivity. Attached Figure Description

[0008] Figure 1 A flowchart illustrating the detection method for Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a provided in this embodiment of the invention; Figure 2 Feasibility analysis diagram provided for embodiments of the present invention; Figure 3 The parameter optimization results provided for the embodiments of the present invention are as follows: A is the concentration of Cas13a; B is the concentration of crRNA; C is the particle size of magnetic beads; D is the volume of magnetic beads; E is the incubation time between magnetic beads and probe; and F is the concentration of probe. Figure 4 The specificity analysis results provided in the embodiments of the present invention; Figure 5 The linear relationship between the logarithm of Staphylococcus aureus concentration and the current signal is provided in the embodiments of the present invention. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] This invention provides a special DNA / RNA hybridization probe, whose DNA portion is pre-fixed on magnetic beads and labeled with an electroactive molecule (methylene blue); its RNA portion acts as a "sacrificial substrate" for Cas13a trans-cleavage. This composite structure is the basis for all subsequent signal transduction: when the target substance—Staphylococcus aureus 16S rRNA—is present in the sample, a series of cascade reactions occur: 1. Specific recognition and activation: The designed crRNA guides the Cas13a protein to accurately recognize and bind to the target 16S rRNA. 1. Once rRNA binds successfully, Cas13a undergoes a conformational change, activating its powerful, non-specific "trans-cleavage" activity; 2. Proximity cleavage and signal transduction: Activated Cas13a protein indiscriminately cleaves all surrounding single-stranded RNA. At this point, the RNA strand of the hybridization probe on the magnetic bead is rapidly cleaved. The key point is that one activated Cas13a can cleave hundreds or thousands of probe RNA strands. This is the first stage of signal amplification, and the number of cleavages is directly positively correlated with the concentration of target RNA in the sample (i.e., the number of activated Cas13a); 3. Magnetic separation purification and signal differentiation: A brief magnetic separation is performed after the reaction to reduce background noise. The core step of the process involves the removal of cleaved probe fragments (RNA strand breaks, DNA strands with detached electrochemical tags) from the supernatant. Uncleaved probes, however, remain firmly bound to the magnetic beads and are adsorbed by the magnet. This ensures that only the background signal from "unreacted" probes is detected, while the signal from "reacted" probes is effectively removed. 4. Electrochemical Quantitative Detection: Magnetic beads with remaining intact probes are dropped onto the screen-printed electrode surface for detection. The electroactive molecules labeled on the intact probes generate an electrochemical signal (e.g., the current peak measured by square wave voltammetry). The logic is: more initial target RNA → more Cas13a activation → more probe cleavage → fewer intact probes remaining on the magnetic beads after magnetic separation → lower electrochemical signal detected on the electrode. Therefore, by measuring the degree of signal attenuation, the content of Staphylococcus aureus in the original sample can be accurately calculated.

[0011] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0012] Example 1, such as Figure 1 The flowchart shown is a method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a according to an embodiment of the present invention, including the following steps: Step 1: Preparation of Cas13a-crRNA: 1.1 Mix 2 μL of Cas13a-crRNA template (its sequence is shown in SEQ ID NO.2) and 2 μL of 100 μM T7 promoter in 14 μL of enzyme-free water. Incubate the mixture at 95 °C for 5 min, then gradually cool it to 25 °C and let it stand for 2 h. 1.2. Following the instructions of the HiScribe™ T7 RNA Synthesis Kit, 8 μL of 25 mM rNTP mixture and 2 μL of T7 RNA polymerase were mixed and transcribed at 37 °C for 16 h to transcribe a large amount of target crRNA. 1.3. The crRNA template was degraded with DNase I at 37 °C for 15 min. The obtained crRNA was then purified using the Monarch® RNA Cleanup Kit and redissolved in DNase-free / RNase-free water to obtain Cas13a-crRNA (the sequence of which is shown in SEQ ID NO.3). 1.4. Measure the concentration of Cas13a-crRNA using a NanoDrop 1000 (Waltham, MA, USA) and store at -80°C before use; Step 2: Preparation of magnetic bead incubation buffer I and preparation of probe complex: 2.1 Accurately weigh 0.06057g Tris powder, 0.014612g EDTA powder and 2.922g NaCl powder, place them in a 100mL beaker, add 40mL ultrapure water, stir magnetically until completely dissolved, then add 20μL Tween-20, shake thoroughly to mix, slowly adjust the pH of the solution to 7.5 with HCl, filter the solution using a 0.22 μm filter membrane to remove impurities, transfer the filtered solution to a 50mL volumetric flask, dilute to the mark with ultrapure water, and store sealed at 4℃ for later use; 2.2 Take 10 mg / mL streptavidin-modified magnetic beads and wash them three times with magnetic bead incubation buffer I (discard the supernatant after each wash) to remove impurities; add 10 μL of 1 mM PBS buffer to the washed magnetic beads, resuspend them, and then add 10 μL of 5 μM biotin-labeled probe (DHX-Rorpter)-MB (its sequence is shown in SEQ ID NO.4) solution and mix thoroughly; incubate at 37℃ for 1 h to form a magnetic bead-probe complex by utilizing the specific binding of biotin to streptavidin (SA); after incubation, wash three times with buffer I, and then dissolve them magnetically for later use; Step 3, Strain Cultivation and Preparation: 3.1 Target bacterial strains (Staphylococcus aureus and Salmonella typhimurium) and non-target pathogens were selected and inoculated into LB medium. The cultures were incubated at 37℃ and 180 rpm for 12 h in a shaker to induce the logarithmic growth phase. The absorbance (OD) of the bacterial culture at 600 nm was measured using a UV-Vis spectrophotometer. 600 ), and convert the bacterial concentration (OD) according to the standard curve. 600 When ≈1, the bacterial concentration is approximately 10. 9 (CFU / mL) 3.2. Take an appropriate amount of bacterial culture and place it in a centrifuge tube. Centrifuge at 6000 r / min for 10 min. Discard the supernatant. Wash the precipitate once with 1 mM PBS buffer (pH 7.4). Then dilute it with the same PBS buffer to the required concentration. Store at 4℃ for later use. All pathogen preparation operations are performed in a biosafety cabinet. Experimental waste is sterilized by autoclaving at 121℃ for 20 min before disposal to ensure biosafety. Step 4, 16S rRNA extraction: 4.1 Take the prepared bacterial culture, centrifuge at 6000 r / min for 5 min, and discard the supernatant; add 500 μL of Trizol reagent to the precipitate, and let it stand at room temperature for 5-10 min to completely separate the nucleoprotein from the nucleic acid; if the sample contains abundant protein, polysaccharide or fat, it needs to be centrifuged at 12000 r / min for 10 min to remove impurities. 4.2. Following the instructions of the bacterial RNA extraction reagent kit, add 0.2 mL of chloroform to the lysis buffer, vortex thoroughly for 30 s, let stand at room temperature for 3 min, and then centrifuge at 4℃ and 12000 r / min for 10 min. After the sample separates into layers, carefully aspirate the upper aqueous phase (containing RNA) into a new sterile centrifuge tube, avoiding aspiration of the middle layer and the lower organic phase. 4.3 Add 1 / 2 volume of anhydrous ethanol to the upper aqueous phase, invert and mix well to promote RNA precipitation; 4.4 Place the adsorption column into the collection tube, transfer the above mixture and the resulting translucent fibrous suspension into the adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 12000 r / min for 3 minutes, and discard the waste liquid in the collection tube.

[0013] 4.5 Add 500 μL of RPE Solution to the adsorption column, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 30 s, and discard the waste liquid; repeat this step once to enhance the washing effect.

[0014] 4.6 Add 30-50 μL of RNase-free ddH2O preheated to 65℃ to the center of the adsorption column, let stand at room temperature for 2 min, centrifuge and collect the eluent, which is the extracted 16S rRNA (as shown in SEQ ID NO.1 for partial specific sequence).

[0015] 4.7. Measure the absorbance of RNA at 260 nm and 280 nm using a nucleic acid protein detector, and calculate A. 260 / A 280 The ratio (within a acceptable range of 1.8 to 2.0) is used to verify RNA purity. The extracted RNA is aliquoted and frozen at -80°C to avoid repeated freeze-thaw cycles, or it can be used directly for subsequent reverse transcription experiments.

[0016] Step 5: Differential Pulse Voltammetry (DPV) Detection: 5.1 Construct a 38 μL activation reaction system containing 1× reaction buffer, 1 μM LwaCas13a protein, 1 μM Cas13a-crRNA and 1 μM 16S rRNA, and make up the volume with DNase / RNase-free water; pre-incubate at 37℃ for 30 min to activate the trans-cleavage activity of Cas13a, then add 2 μL of the prepared magnetic bead-probe complex to the system and continue incubation at 37℃ for 30 min to allow the activated Cas13a to cleave the probe on the surface of the magnetic beads; 5.2 After the reaction, the reaction system was washed three times with magnetic bead incubation buffer I to remove free probes and impurities by magnetic separation; the magnetic bead complex was resuspended in 50 μL of 0.1 mM PBS buffer and transferred to the three-electrode system of the electrochemical workstation (working electrode: glassy carbon electrode; reference electrode: saturated calomel electrode; counter electrode: platinum wire electrode); the DPV detection parameters were set as follows: potential range -0.2~0.2 V, pulse amplitude 50 mV, pulse width 50 ms; the voltammetric curve was recorded, and the target concentration was quantitatively analyzed based on the peak current change.

[0017] All oligonucleotides used in the embodiments of this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the oligonucleotide sequences are listed in Table 1. All primers were purified using high-performance liquid chromatography (HPLC). Table 1 Oligonucleotide Sequences

[0018] The reagents and instruments involved are shown in Tables 2 and 3: Table 2 Reagents

[0019] Table 3 Instruments and Equipment

[0020] according to Figure 1 As can be seen, the core principle of this invention is as follows: when the target Staphylococcus aureus RNA is absent in the reaction system, the Cas13a-crRNA complex is inactive, and the methylene blue-labeled single-stranded DNA probe immobilized on the working electrode surface of the screen-printed electrode remains intact, generating a strong methylene blue characteristic electrochemical redox peak; when the target RNA is present, the Cas13a-crRNA complex specifically binds to and activates the trans-cleavage activity of Cas13a. The activated Cas13a will indiscriminately cleave the single-stranded RNA or DNA probes adjacent to the electrode surface in the solution. The cleavage of the DNA probes on the electrode surface causes the methylene blue molecules to detach from the electrode surface or the electron transport pathway to be disrupted, thereby causing a significant decrease in the detected methylene blue electrochemical signal (peak current). The magnitude of the signal decrease is proportional to the concentration of the target RNA.

[0021] Example 2: Verification of the feasibility of the detection method of this invention using differential pulse voltammetry: like Figure 2 As shown, a clear and strong methylene blue redox peak was observed in the blank control group without the addition of target Staphylococcus aureus RNA. Conversely, when the target RNA was added, the peak current in the DPV curve decreased significantly. This significant difference proves that the target RNA successfully activated Cas13a, thereby cleaving the DNA probe on the electrode surface, resulting in a weakening of the electrochemical signal, indicating that the method is feasible. In addition, no significant signal decrease was observed in the control groups lacking Cas13a protein or crRNA, respectively, confirming the indispensable role of Cas13a and crRNA in this sensor system.

[0022] Example 3: Optimization experiment of key reaction parameters: By adjusting the key reaction parameters and comparing the results, the following findings were obtained: like Figure 3 As shown in Figure A, when the concentration of Cas13a was adjusted within the gradient range of 0.125~1 μM, the current signal did not fluctuate significantly and remained at a similar level. This result indicates that the amount of Cas13a used within this concentration range can fully meet the basic requirements of the reaction, and its concentration change will not have a limiting effect on the signal output. like Figure 3 As shown in Figure B, the current signal remained stable when the crRNA concentration was increased from 0.125 μM to 1 μM; however, the signal dropped significantly when the concentration was increased to 2 μM. Based on the reaction principle, it is speculated that high concentrations of crRNA may consume the active components in the system through non-specific binding or interfere with the target binding process. Therefore, the appropriate amount of crRNA should be controlled in the low concentration range of 0.125~1 μM. like Figure 3 As shown in Figure C, the current signal corresponding to the 0.3μM particle size magnetic beads is significantly higher than that of the 1μM and 2.8μM groups, while the signal of the 2.8μM particle size group is significantly reduced. This difference is directly related to the specific surface area of ​​the magnetic beads: small particle size magnetic beads have a larger surface contact area, which can participate in the reaction-related binding process more efficiently, thereby enhancing the signal output efficiency. like Figure 3 As shown in Figure D, the influence of magnetic bead volume is presented: as the magnetic bead volume increases from 1 μL to 6 μL, the current signal shows a continuous downward trend. This is because excessive magnetic beads will cause a spatial crowding effect in the local reaction environment, reduce the effective collision probability of active components, and thus inhibit the occurrence of signal-related reactions. like Figure 3 As shown in Figure E, the signal gradually increases with time, reaching a peak at 80 min, and then slightly decreases at 100 min. This dynamic change reflects that the reaction has been fully carried out at 80 min, while an excessively long reaction time may lead to the degradation of active components or the accumulation of side reactions in the system. Therefore, 80 min is a better time to balance the sufficiency and stability of the reaction. like Figure 3 As shown in Figure F, the signal remained stable in the range of 5-9 μM and 11 μM, but the signal showed a significant decrease in the 10 μM concentration group. This specific fluctuation suggests that the probe may have unexpectedly interacted with other components in the system at the 10 μM concentration, interfering with the signal-related reaction process. Subsequent experiments should avoid this concentration.

[0023] Example 4: Specificity analysis of the reaction system: The selectivity of the system was verified by detecting the current signals corresponding to different targets: such as Figure 4 As shown, when the system is respectively with S. boydii, EHEC O157:H7, V. cholerae, V. alginolyticus, V. parahaemolyticus When co-incubated with non-target objects, the current signal remained at a high level (similar to the system's background signal); however, when reacting with the target object (positive group), the current signal showed a significant decrease. This result indicates that the system only produces a specific response to the target object, and there is no significant signal change for other closely related or unrelated objects, indicating that it has good specificity. This may be because the recognition elements in the system (such as crRNA) only specifically bind to the target sequence, thereby triggering a signal change, and there is no effective interaction with non-target sequences, thus effectively distinguishing the target object from other interfering objects. Figure 5 The linear relationship between the logarithm of Staphylococcus aureus concentration (lg CFU / mL) and the current signal was presented: the horizontal axis represents the logarithm of bacterial concentration (lg CFU / mL), and the vertical axis represents the current signal (A). The linear equation is Y = -1.083e-007*X +1.620e-006, and the correlation coefficient R0 is [value missing].2 = 0.97, indicating a good linear correlation. As can be seen from the data point distribution in the graph, its detection range covers the concentration range corresponding to lg(CFU / mL) = 1~7 (i.e., 10~10). 7 (CFU / mL); combining linear response and signal characteristics, the limit of detection (LOD) for Staphylococcus aureus using this method is 10 CFU / mL.

[0024] The detection method provided in this embodiment of the invention was compared with five different miRNA assay methods, and the results are shown in Table 4: Table 4 Comparison of different miRNA assay methods

[0025] As can be seen from the table, the detection method used in this invention has a low LOD of 10 CFU / mL, which is comparable to the sensitivity of electrochemical sensing and SERS methods, and far superior to fluorescence intensity and colorimetric methods. The detection time is only 1.0 h, which is significantly shorter than similar electrochemical sensing methods (2.5 h) and SERS methods (3.0 h), and only slightly longer than the fastest RAA-LFS method. It combines high sensitivity and high efficiency. This method provides a new technical approach for rapid detection of microorganisms. In scenarios such as food hygiene and clinical diagnosis, it can achieve rapid screening of pathogens and has important practical application value.

[0026] Table 5 shows the spiked recovery of Staphylococcus aureus in three actual food samples: orange juice, eggs, and milk. Figure 5 Actual recovery rate

[0027] Add 1×10 to each sample 5 1×10 6 1×10 7 After adjusting for the target bacteria concentration at CFU / mL, the recovery rates for orange juice samples were 95.71%–99.83%, for egg samples 92.12%–99.08%, and for milk samples 96.13%–100.57%. Overall, the recovery rates for different spiked concentrations in each sample were relatively high with minimal fluctuations, except for egg samples at 1×10⁻⁶. 7 The recovery rate of the CFU / mL spiked group was slightly lower (92.12%), which indicates that the detection method has good anti-interference ability in complex food matrices such as orange juice, eggs, and milk, and can stably achieve accurate detection of Staphylococcus aureus.

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a, characterized in that, Includes the following steps: Step 1: Prepare crRNA; Step 2: Preparation of magnetic bead incubation buffer I and preparation of probe complex: 2.1 Weigh Tris powder, EDTA powder and NaCl powder, add ultrapure water and stir magnetically until completely dissolved; then add Tween-20, shake thoroughly to mix, and slowly adjust the pH of the solution to 7.5 with HCl; filter and bring the filtered solution to a final volume. 2.2 Take 10 mg / mL streptavidin-modified magnetic beads, wash several times with magnetic bead incubation buffer I, and magnetically separate them after each wash, discarding the supernatant to remove impurities; add 1 mM PBS buffer to the washed magnetic beads, resuspend them, add 5 μM biotin-labeled probe solution, and mix thoroughly; incubate at 37℃ for 1 h to form a magnetic bead-probe complex by utilizing the specific binding of biotin to streptavidin SA; after incubation, wash several times with buffer I, magnetically separate them, and set aside for later use; Step 3: Strain culture and preparation; Step 4: 16S rRNA extraction; Step 5: Differential pulse voltammetry (DPV) detection: 5.1 Construct an activation reaction system comprising: 1× reaction buffer, 1 μM Cas13a protein, 1 μM crRNA and 1 μM target, with enzyme-free water added to make up the volume; pre-incubate at 37℃ for 30 min to activate the trans-cleavage activity of Cas13a; then add the prepared magnetic bead-probe complex to the system and continue incubation at 37℃ for 30 min to allow the activated Cas13a to cleave the probe on the surface of the magnetic beads; 5.2 After the reaction, the reaction system was washed several times with magnetic bead incubation buffer I, and the free probe and impurities were removed by magnetic separation. The magnetic bead complex was resuspended in 0.1 mM PBS buffer and transferred to the three-electrode system of the electrochemical workstation. The working electrode was a glassy carbon electrode, the reference electrode was a saturated calomel electrode, and the counter electrode was a platinum wire electrode. The DPV detection parameters were set as follows: potential range -0.2~0.2 V, pulse amplitude 50 mV, and pulse width 50 ms. The voltammetric curve was recorded, and the target concentration was quantitatively analyzed based on the peak current change.

2. The method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a according to claim 1, characterized in that, Step 1 specifically includes the following process: 1.1 Mix the crRNA template and 100 μM T7 promoter in enzyme-free water; incubate the mixture at 95 °C for 5 min, then gradually cool to 25 °C and let it stand for 2 h; 1.2 Mix 100mM dNTP buffer and T7 RNA polymerase; transcribe at 37 ℃ for 16 h to transcribe a large amount of target crRNA; 1.

3. Degrade the crRNA template with DNase I at 37 °C for 15 min; then purify the obtained crRNA using an RNA clearance kit and redissolve it in DNase / rNase-free water; 1.

4. Measure the concentration of crRNA using NanoDrop 1000 and store it before use.

3. The method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a according to claim 1, characterized in that, Step 3 is specifically... Includes the following processes: 3.1 Target strains of Staphylococcus aureus and Salmonella typhimurium, as well as non-target pathogens, were selected and inoculated into LB medium. The cultures were incubated at 37℃ with shaking at 180 rpm in a constant-temperature shaker to induce the logarithmic growth phase. The absorbance (OD) of the bacterial culture at 600 nm was measured using a UV-Vis spectrophotometer. 600 And calculate the bacterial concentration based on the standard curve; 3.

2. Take the bacterial culture and place it in a centrifuge tube. Centrifuge and discard the supernatant. Wash the precipitate with 1mM PBS buffer and then dilute it with PBS buffer to the required concentration.

4. The method for detecting Staphylococcus aureus based on screen-printed electrodes combined with CRISPR / Cas13a according to claim 1, characterized in that, Step 4 is specifically... Includes the following processes: 4.1 Take the prepared bacterial culture, centrifuge it, and discard the supernatant; add Trizol reagent to the precipitate, and let it stand at room temperature to completely separate the nucleoprotein and nucleic acid to obtain the lysis buffer; 4.2 Add chloroform to the lysis buffer, vortex thoroughly, let stand at room temperature, and then centrifuge; after separation, transfer the upper aqueous phase to a new sterile centrifuge tube. 4.3 Add 1 / 2 volume of anhydrous ethanol to the upper aqueous phase, invert and mix well to promote RNA precipitation; 4.4 Place the adsorption column into the collection tube, transfer the above mixture and the resulting translucent fibrous suspension into the adsorption column, let it stand at room temperature, centrifuge at 12000 r / min for 3 min, and discard the waste liquid in the collection tube. 4.5 Add RPE Solution to the adsorption column, let stand at room temperature, centrifuge at 10000 r / min for 30 s, discard the waste liquid, and repeat the operation. 4.6 Add RNase-free ddH2O preheated to 65℃ to the center of the adsorption column, let it stand at room temperature, and centrifuge to collect the eluent, which is the extracted 16S rRNA. 4.

7. Measure the absorbance of RNA at 260 nm and 280 nm using a nucleic acid protein detector, and calculate A. 260 / A280 The ratio is used to verify RNA purity.

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