A method for detecting staphylococcus aureus in milk based on crisper-cas14a

Abstract

The application discloses a method for detecting Staphylococcus aureus based on CRISPR-Cas14a, introduces trans cleavage activity of CRISPR / Cas14a into an electrochemical biosensor, combines signal amplification catalyzed by a Zr-based metal-organic framework@gold-platinum (PCN-222@AuPt) nanometer enzyme, and establishes an accurate and ultra-sensitive Staphylococcus aureus detection method. 1 Under the optimized conditions, the linear detection range of Staphylococcus aureus is 5*10 7 CFU / mL, and the detection limit is 10 CFU / mL. The application can provide a new electrochemical biosensor analysis method for Staphylococcus aureus detection, can provide a new idea for research on a food source pathogenic bacteria food field rapid detection method, and has certain reference value for practical sensor device development and research.

Description

Technical Field

[0001] This invention belongs to the field of food testing, specifically involving a method for detecting Staphylococcus aureus in milk based on CRISPR-Cas14a. Background Technology

[0002] To date, over 250 different foodborne illnesses have been identified, primarily caused by pathogenic microorganisms, including various foodborne bacteria, viruses, and parasites. Foodborne pathogens mainly infect humans through contaminated meat, dairy products, and raw fruits and vegetables. The main symptoms after infection include nausea, vomiting, abdominal pain, abdominal cramps, diarrhea, and fever. Severe cases can lead to uremia and even death. Therefore, the detection of foodborne pathogens is crucial for preventing their harmful effects.

[0003] Currently, the three most commonly used methods for detecting pathogenic bacteria are: traditional plate counting, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). Plate counting is the "gold standard" for detecting Staphylococcus aureus; however, this method typically takes 2-3 days or longer. ELISA can shorten the analysis time to 24 hours, but its sensitivity is insufficient to detect low levels of pathogens. Polymerase chain reaction (PCR) detects Staphylococcus aureus at the DNA level, offering high sensitivity and selectivity, but because it amplifies inactive or dead cells, it can lead to false positives.

[0004] Therefore, establishing a simple, rapid, efficient, and low-cost detection method for pathogenic bacteria (Staphylococcus aureus) has become an urgent need for rapid on-site food testing. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting Staphylococcus aureus in milk based on CRISPR-Cas14a.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for detecting Staphylococcus aureus in milk based on CRISPR-Cas14a, comprising,

[0009] Preparation of MIL-101(Cr) / PPy@Au nanocomposite suspension;

[0010] Preparation of PCN-222@AuPt nanozymes;

[0011] The glassy carbon electrode (GCE) surface was polished, and the MIL-101(Cr) / PPy@Au suspension was drop-coated onto the polished GCE surface to obtain MIL-101(Cr) / PPy@Au / GCE.

[0012] P-ssDNA was added to MIL-101(Cr) / PPy@Au / GCE and incubated to obtain the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor;

[0013] Mix cDNA and aptamer solutions, add hybridization buffer, incubate, anneal to room temperature, and incubate again to obtain aptamer / cDNA complex.

[0014] The test sample containing live Staphylococcus aureus was added to the aptamer / cDNA complex to form a mixture. The mixture was centrifuged, and the supernatant was added to the Cas14a / sgRNA complex. After incubation, the activated CRISPR / Cas14a system was obtained.

[0015] After the activated CRISPR / Cas14a system was added to the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor, the PCN-222@AuPt suspension was added to the biosensor surface. After incubation, the surface was rinsed with sterile, enzyme-free water, and the resulting electrode was immersed in a PBS solution containing 1.0 mM H2O2. The electrode was placed in a three-electrode system of an electrochemical workstation, and the electrochemical signal of the electrode was measured by the DPV method. By controlling the concentration of viable Staphylococcus aureus, the electrochemical signal response value of the electrode was measured using the above method, and a linear relationship between the current signal response value and the logarithm of the Staphylococcus aureus concentration was established.

[0016] After pretreatment of the milk to be tested, the electrochemical signal of the electrode is measured by the above treatment method. Substituting this signal into the linear relationship, the concentration of Staphylococcus aureus in the milk can be detected.

[0017] As a preferred embodiment of the method described in this invention, the preparation of the MIL-101(Cr) / PPy@Au nanocomposite suspension includes,

[0018] Mix 5.0 g of MIL-101(Cr), 0.85 g of PVP and 40 mL of ethanol with 210 mL of double-distilled water, sonicate at room temperature for 10 min, then add 0.3 mL of pyrrole monomer and stir at room temperature for 20 min.

[0019] Then, 3.29 g of (NH4)2S2O8 was added and the reaction was continued for 4 h to obtain a mixed solution;

[0020] Add 50 mL of AuNPs to the above mixture, stir at 25°C for 10–12 h, centrifuge, and wash to obtain the MIL-101(Cr) / PPy@Au nanocomposite material.

[0021] As a preferred embodiment of the method described in this invention, the preparation of PCN-222@AuPt nanozyme includes,

[0022] Disperse 200 mg of ZrOCl2·8H2O and 50 mg of TCPP in a mixture of 50 mL of DMF and 4 mL of acetic acid.

[0023] The resulting mixture was stored at 120°C for 48 h, then washed three times with DMF, double-distilled water and ethanol, and vacuum dried for 12 h to obtain purple powder PCN-222.

[0024] 20 mg of PCN-222 was ultrasonically dispersed in 20 mL of double-distilled water, and 20 mL of 0.05 mol / L NaBH4 solution was added. The mixture was stirred at room temperature for 20 min to obtain the stirred mixture.

[0025] Add 10 mL of H2PtCl6·6H2O solution and 10 mL of HAuCl4·3H2O solution dropwise to the stirred mixture, stir at room temperature for 20-30 min, add 4 mL of NaBH4 solution, and stir at room temperature for 30 min.

[0026] The resulting mixture was centrifuged, washed, and dried to obtain PCN-222@AuPt nanozyme.

[0027] In a preferred embodiment of the method described in this invention, the MIL-101(Cr) / PPy@Au suspension is drop-coated onto the polished GCE surface, wherein the concentration of the MIL-101(Cr) / PPy@Au suspension is 1.0 mg / mL and the addition amount is 5–15 μL.

[0028] In a preferred embodiment of the method described in this invention, the addition of P-ssDNA to MIL-101(Cr) / PPy@Au / GCE is wherein the concentration of P-ssDNA is 10 μM and the amount of P-ssDNA modification is 15 μL.

[0029] In a preferred embodiment of the method described in this invention, the Cas14a / sgRNA complex has a concentration ratio of Cas14a to sgRNA of 1:1.5.

[0030] In a preferred embodiment of the method described in this invention, the activated CRISPR / Cas14a system is added to the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor for reaction, wherein the reaction temperature is 37°C and the reaction time is 45 min.

[0031] In a preferred embodiment of the method described in this invention, the electrode is immersed in a PBS solution, wherein the PBS concentration is 0.1M, contains 1.0mM H2O2, and has a pH of 7.2; and a PCN-222@AuPt suspension is added to the biosensor surface, wherein the concentration of the PCN-222@AuPt suspension is 1.0mg / mL.

[0032] In a preferred embodiment of the method described in this invention, the electrochemical signal of the electrode is measured using the DPV method, wherein the DPV measurement parameters are:

[0033] The test solution was a 0.1M PBS solution with pH 7.2 containing 1.0 mM H2O2;

[0034] The measured potential is -0.2 to 0.6V, the pulse amplitude is 0.05V, the pulse width is 0.05s, and the pulse period is 0.5s.

[0035] In a preferred embodiment of the method described in this invention, the milk to be tested includes cow's milk and goat's milk.

[0036] Beneficial effects of this invention:

[0037] (1) This invention designs an aptamer-based Cas14a electrochemical biosensor for the detection of Staphylococcus aureus. This biosensor utilizes a MIL-101(Cr) / PPy@Au composite material and a PCN-222@AuPt nanozyme to amplify the signal. The MIL-101(Cr) / PPy@Au composite material promotes accelerated electron transfer and provides abundant binding sites for single-stranded DNA. The high affinity between the aptamer and the target, along with the cDNA-specific triggering of Cas14a trans-cleavage activity, gives this electrochemical biosensor high accuracy and sensitivity. Furthermore, the PCN-222@AuPt nanozyme successfully catalyzes the reduction of H2O2, generating a strong electrochemical signal. This electrochemical biosensor has a wide detection range for Staphylococcus aureus (5×10⁻⁶). 1 ~5×10 7It has a low detection limit (10 CFU / mL) and good selectivity, repeatability and stability.

[0038] (2) The biosensor of the present invention successfully distinguishes between live and dead bacteria without the need for nucleic acid extraction. This strategy will provide a new detection platform for a variety of foodborne pathogens by targeting and introducing specific aptamer sequences. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0040] Figure 1 This is a schematic diagram illustrating the principle of the electrochemical biosensor for detecting Staphylococcus aureus in this invention.

[0041] Figure 2 In this embodiment of the invention, 5 mM [Fe(CN)6] containing 0.1 M KCl was used. 3- / 4- The CV curves and EIS spectra in the figure are shown, where a and a' represent naked GCE, b and b' represent MIL-101(Cr) / PPy@Au / GCE, and c and c' represent P-ssDNA / MIL-101(Cr) / PPy@Au / GCE.

[0042] d, d' is PCN-222@AuPt / P-ssDNA / MIL-101(Cr) / PPy@Au / GCE, e, e' is Cas14a-sgRNA / PCN-222@AuPt / P-ssDNA / MIL-101(Cr) / PPy@Au / GCE.

[0043] Figure 3 The figure shows the results of the optimization of experimental conditions in the embodiments of the present invention. In the figure, A is the amount of MIL-101(Cr) / PPy@Au composite material, B is the amount of P-ssDNA, C is the concentration ratio of CRISPR / Cas14a to sgRNA, and CRISPR / Cas14a / sgRNA reaction time.

[0044] Figure 4 This invention presents the DPV signal graphs of the electrochemical biosensor for different concentrations of Staphylococcus aureus and the linear relationship between the DPV signal and the logarithm of the Staphylococcus aureus concentration in this embodiment of the invention; wherein, A represents the DPV signal of the electrochemical biosensor for different concentrations of Staphylococcus aureus (0, 5, 5 × 10⁻⁶). 1 5×10 25×10 3 5×10 4 5×10 5 5×10 6 5×10 7 and 5×10 8 B represents the DPV signal (CFU / mL) and the linear relationship between the DPV signal and the logarithm of the Staphylococcus aureus concentration.

[0045] Figure 5 The electrochemical signals of the electrochemical biosensor constructed in the embodiments of the present invention for different targets are shown.

[0046] Figure 6 This is a graph showing the repeatability (A) and stability (B) evaluation of the electrochemical biosensor in this embodiment of the invention. Detailed Implementation

[0047] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0048] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0049] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0050] Experimental materials for this invention:

[0051] Table 1 lists the sequences of all oligonucleotides synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

[0052] Table 1

[0053]

[0054] Chloroauric acid (HAuCl4·3H2O), chloroplatinic acid hexahydrate (H2PtCl6·6H2O), hydrochloric acid (HCl), ascorbic acid (AA), N,N-dimethylformamide (DMF), polyethyleneimine (PPy, Mw = 70,000), zirconium dioxide chloride octahydrate (ZrOCl2·8H2O), and tetrakis(4-carboxyphenyl)porphyrin (TCPP) were purchased from Ala Reagent Co., Ltd. (Shanghai, China).

[0055] RNase inhibitors were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cas14a protein was purchased from Megabio Biotechnology Co., Ltd. (Guangzhou, China).

[0056] Culture of bacteria in this invention:

[0057] Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, and Bacillus subtilis strains stored at -80℃ were cultured in LB medium at 37℃ for 12 h.

[0058] Bacterial cells were then collected by centrifugation at 10,000 rpm for 5 min and washed three times with sterile physiological saline. The OD600 value of the bacterial suspension was determined by UV-Vis spectroscopy. The bacterial count was estimated using the conventional plate count method.

[0059] The electrochemical performance measurements in these embodiments of the invention were performed using a CHI600E electrochemical workstation (Shanghai, Chenhua). The CHI600E electrochemical workstation comprises a traditional three-electrode system: a glassy carbon electrode (GCE) as the working electrode, Ag / AgCl as the reference electrode, a platinum wire electrode as the auxiliary electrode, and 5 mM [Fe(CN)6] as the electrode. 3- / 4- The solution acts as an electrolyte.

[0060] Example 1

[0061] (1) Synthesis of MIL-101(Cr) / PPy@Au composite nanomaterials:

[0062] MIL-101(Cr) was synthesized using a simple solvothermal method, the specific process of which is as follows:

[0063] First, 2.4 g of Cr(NO3)3·9H2O, 996 mg of terephthalic acid (TPA), and 1 mL of hydrofluoric acid (HF) were uniformly dispersed in 30 mL of ultrapure water. Second, the resulting light green solution was transferred to a high-pressure reactor and stored at 220 °C for 12 hours. Then, after cooling to room temperature, the suspension was centrifuged, and the green precipitate was washed several times with DMF and ethanol solutions. Finally, the obtained MIL-101(Cr) was vacuum dried for further experiments.

[0064] The AuNPs solution was obtained according to the classic Turkevich method, the specific process of which is as follows: 100 mL of 0.01% chloroauric acid solution was added to a beaker, heated to boiling, and then 1.5 mL of 1% trisodium citrate solution was quickly added. After boiling for 15 minutes, the solution was cooled to room temperature to obtain the AuNPs solution.

[0065] Detailed synthesis steps of MIL-101(Cr) / PPy@Au nanocomposite:

[0066] First, mix 5.0 g of MIL-101(Cr), 0.85 g of PVP and 40 mL of ethanol with 210 mL of double-distilled water; after sonicating at room temperature for 10 min, add 0.3 mL of pyrrole monomer to the above suspension and stir at room temperature for 20 min.

[0067] Then, 3.29 g of (NH4)2S2O8 was slowly added and the reaction was continued for 4 hours;

[0068] Then, 50 mL of the previously prepared AuNPs solution was added to the above mixture, stirred at 25 °C for 12 h, centrifuged, and washed to obtain the MIL-101(Cr) / PPy@Au nanocomposite material.

[0069] (2) Synthesis of PCN-222@AuPt nanozyme:

[0070] PCN-222@AuPt nanozymes were prepared by in-situ reduction using PCN-222 as the carrier of Au@Pt. The specific steps are as follows:

[0071] Disperse 200 mg of ZrOCl2·8H2O and 50 mg of TCPP in a mixture of 50 mL of DMF and 4 mL of acetic acid.

[0072] The resulting mixture was stored at 120°C for 48 h, then washed three times with DMF, double-distilled water and ethanol, and vacuum dried for 12 h to obtain a purple powder (PCN-222).

[0073] 20 mg of PCN-222 was ultrasonically dispersed in 20 mL of double-distilled water, and 20 mL of NaBH4 solution (0.05 mol / L) was added. The mixture was stirred at room temperature for 20 min.

[0074] Add 10 mL of H2PtCl6·6H2O (1%) solution and 10 mL of HAuCl4·3H2O (1%) solution dropwise into the solution and stir at room temperature for 30 min.

[0075] Finally, add 4 mL of NaBH4 solution (0.05 mol / L) and stir at room temperature for 30 min.

[0076] The resulting mixture was centrifuged, washed, and dried to obtain PCN-222@AuPt nanozyme.

[0077] (3) Construction of electrochemical biosensors:

[0078] First, polish the 3mm diameter GCE surface with 0.05μm Al powder;

[0079] Then, 15 μL of MIL-101(Cr) / PPy@Au suspension (1.0 mg / mL) was drop-coated onto the polished GCE surface to prepare MIL-101(Cr) / PPy@Au / GCE;

[0080] Two hours later, 15 μL of P-ssDNA (10 μM) was added to MIL-101(Cr) / PPy@Au / GCE and incubated at 37°C for 2 hours.

[0081] The prepared P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor platform was stored at 4°C for subsequent CRISPR / Cas14a reactions.

[0082] (4) Detection of Staphylococcus aureus by electrochemical biosensors

[0083] Take 3 μL of cDNA (10 μmol) and 3 μL of aptamer solution (S. aureusaptamer aqueous solution, 10 μmol) into a 0.2 mL centrifuge tube, add 24 μL of hybridization buffer, mix well, and incubate at 95 °C for 5 min. Anneal at a rate of 2 °C / min until room temperature is reached. After incubation for 12 h, an aptamer / cDNA complex is formed.

[0084] Then, 30 μL of different concentrations (5 × 10⁻⁶) were added. 1 ~5×10 7 Live Staphylococcus aureus (CFU / mL) were added to the aptamer / cDNA complex and stored at 37°C for 2 h. The resulting mixture was centrifuged (12,000 rpm, 5 min), and 30 μL of the supernatant was added to a Cas14a / sgRNA complex containing 200 nM Cas14a and 300 nM sgRNA. The mixture was incubated at 37°C for 60 min to obtain the activated CRISPR / Cas14a system.

[0085] For the detection of Staphylococcus aureus, 20 μL of activated CRISPR / Cas14a system was added to the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor platform and reacted at 37℃ for 45 min. Then, 10 μL of PCN-222@AuPt suspension (1.0 mg / mL) was added to the electrode surface and incubated at 37℃ for 30 min. After gently rinsing with sterile enzyme-free water, the electrode was immersed in 0.1 M PBS (containing 1.0 mM H2O2, pH = 7.2), and the electrochemical signal of the electrode was measured using DPV technology.

[0086] The measurement parameters for DPV are as follows: the detection solution is 0.1M PBS (pH 7.2) containing 1.0mM H2O2; the measurement potential is -0.2 to 0.6V; the pulse amplitude is 0.05V; the pulse width is 0.05s; and the pulse period is 0.5s.

[0087] By controlling the concentration of live Staphylococcus aureus, the electrochemical signal response value of the electrode was measured using the above method, and a linear relationship between the current signal response value and the logarithm of the Staphylococcus aureus concentration was established.

[0088] After pretreatment of the milk to be tested, the electrochemical signal of the electrode is measured by the above treatment method. Substituting this signal into the linear relationship, the concentration of Staphylococcus aureus can be detected.

[0089] (5) Detection principle of electrochemical biosensors

[0090] Figure 1 This is a schematic diagram illustrating the principle of an electrochemical biosensor for detecting Staphylococcus aureus. The modification of the GCE surface with the MIL-101(Cr) / PPy@Au nanocomposite material not only increases the amount of oligonucleotides immobilized but also enhances the electron transfer capability of the electrode surface, thereby contributing to improved detection sensitivity and stability of the electrochemical biosensor.

[0091] Both ends are marked with -PO4 - The -SH ssDNA can be fixed to MIL-101(Cr) / PPy@Au / GCEP-ssDNA via Au-S bonds; complementary DNA (cDNA) is the activator that triggers the cleavage activity of Cas14a / sgRNA.

[0092] In the absence of Staphylococcus aureus, cDNA and aptamers can hybridize to form a double strand, the cleavage activity of Cas14a / sgRNA is not activated, and the P-ssDNA on the electrode surface remains intact.

[0093] PCN-222@AuPt nanozymes can be covalently immobilized on P-ssDNA / MIL-101(Cr) / PPy@Au / GCE via Zr-OP bonds. PCN-222@AuPt nanozymes generate strong DPV signals by catalyzing the decomposition of H2O2.

[0094] Conversely, in the presence of Staphylococcus aureus, the strong affinity between the aptamer and Staphylococcus aureus, such as spatial matching, electrostatic and hydrogen bonding effects, causes the cDNA to dissociate in solution. The freely distributed cDNA forms a Cas14a / sgRNA / cDNA ternary complex with Cas14a / sgRNA, the trans-cleavage activity of Cas14a is activated, and the P-ssDNA on the GCE surface is cleaved by Cas14a, which prevents the PCN-222@AuPt nanozyme from being attached to the electrode surface, thereby generating a weak electrochemical signal.

[0095] Therefore, the concentration of Staphylococcus aureus can be determined by monitoring the electrochemical signals on the electrode surface.

[0096] Example 2

[0097] Feasibility verification of electrochemical biosensors:

[0098] To investigate the bare GCE prepared in Example 1;

[0099] MIL-101(Cr) / PPy@Au / GCE;

[0100] P-ssDNA / MIL-101(Cr) / PPy@Au / GCE;

[0101] PCN-222@AuPt / P-ssDNA / MIL-101(Cr) / PPy@Au / GCE;

[0102] The feasibility of Cas14a-sgRNA / PCN-222@AuPt / P-ssDNA / MIL-101(Cr) / PPy@Au / GCE was investigated, and the electrochemical behavior of the modified electrode was characterized using electrochemical impedance spectroscopy (EIS). Specific measurement parameters were as follows: the working solution was 5 mM [Fe(CN)6]. 3- / 4- The solution (containing 0.1 M KCl) was scanned at a frequency range of 0.1 Hz to 10000 Hz with an amplitude of 0.005 V. In the Nyquist plot, the diameter of the semicircle is equal to the electron transfer resistance (Ret). Figure 2As shown, the Ret of bare GCE is relatively small (curve a). After modifying bare GCE with MIL-101(Cr) / PPy@Au, the Ret is significantly reduced (curve b), which proves that MIL-101(Cr) / PPy@Au can effectively enhance the electron transfer capability of the electrode-electrolyte interface.

[0103] Subsequently, as P-ssDNA was immobilized on MIL-101(Cr) / PPy@Au / GCE, Ret increased slightly (curve c), indicating that the negatively charged P-ssDNA was successfully immobilized on the electrode.

[0104] After PCN-222@AuPt was immobilized on P-ssDNA / MIL-101(Cr) / PPy@Au / GCE, Ret further increased (curve d). However, when PCN-222@AuPt / ssDNA / MIL-101(Cr) / PPy@Au / GCE reacted with the CRISPR / Cas14a system, Ret significantly decreased. This may be because the trans-cleavage of the activated CRISPR / Cas14a system disrupted the integrity of P-ssDNA, leading to the release of the PCN-222@AuPt nanozyme from the electrode interface.

[0105] Meanwhile, the cyclic voltammetry (CV) measurement results Figure 2 Consistent with the EIS results, the CV parameters were: scan range -0.2 to 0.6 V, scan rate 50 mV / s, and scan environment 5 mM [Fe(CN)6]. 3- / 4- (Contains 0.1M KCl) solution.

[0106] In summary, the constructed sensor meets the detection requirements.

[0107] Example 3

[0108] Optimization of experimental conditions:

[0109] To obtain optimal analytical performance, the DPV method was used to optimize the experimental parameters. The current difference (ΔI) was used to evaluate the optimization results in the optimization analysis, and the calculation formula is ΔI = I before -I after , where I before and I after The values ​​represent the DPV current response before and after modification or reaction, respectively. The parameters for the DPV method are: measurement environment 5mM [Fe(CN)6]. 3- / 4- (Contains 0.1 MkCl) solution; measurement potential -0.2 to 0.6 V; pulse amplitude 0.05 V; pulse width 0.05 s; pulse period 0.5 s.

[0110] (1) The modification amount of MIL-101(Cr)-101 / PPy@Au nanocomposite material (concentration of 1.0 mg / mL) on GCE was optimized. Different volumes of MIL-101(Cr)-101 / PPy@Au were added to the surface of GCE electrode. After natural drying, the electrochemical signal at the electrode interface was measured at 37 °C using DPV technology.

[0111] The results are as follows Figure 3 As shown in Figure A, when the modification amount increases from 5 μL to 15 μL, ΔI gradually increases, and when the modification amount exceeds 15 μL, ΔI decreases slightly.

[0112] Therefore, the modification amount of the MIL-101(Cr)-101 / PPy@Au nanocomposite was 15 μL throughout the entire work.

[0113] (2) Different amounts of P-ssDNA were added to the electrode surface MIL-101(Cr)-101 / PPy@Au / GCE. After incubation at 37°C for 2 h, the electrochemical signal at the electrode interface was measured at 37°C using DPV technology.

[0114] The results are as follows Figure 3 As shown in Figure B, when the amount of P-ssDNA modification is 15 μL (concentration of 10 μM), ΔI reaches its maximum value. When the amount of modification exceeds 15 μL, ΔI remains almost unchanged, indicating that the amount of P-ssDNA modification on the electrode surface has reached saturation.

[0115] (3) In the detection of Staphylococcus aureus using an electrochemical biosensor, the effect of the concentration ratio of Cas14a to sgRNA (1:0.5, 1:1, 1:1.5, 1:2, and 1:3) on ΔI was studied, and the results are as follows: Figure 3 As shown in C;

[0116] ΔI reaches its maximum when the concentration ratio of CRISPR / Cas14a to sgRNA is 1:1.5;

[0117] Therefore, a concentration ratio of Cas14a (200 nM) to sgRNA (300 nM) of 1:1.5 was chosen.

[0118] Meanwhile, the study also found that when the activated CRISPR / Cas14a system trans-cleaved P-ssDNA for 45 min (see 3D), ΔI reached its maximum, and then tended to stabilize. Therefore, 45 min was chosen for subsequent experiments.

[0119] Example 4

[0120] Analytical performance of electrochemical biosensors:

[0121] Under the above optimal conditions (the modification amount of MIL-101(Cr)-101 / PPy@Au nanocomposite was 15 μL; when the modification amount of P-ssDNA was 15 μL, the concentration ratio of Cas14a (200 nM) and sgRNA (300 nM) was 1:1.5; and the trans-cleavage time of P-ssDNA by the activated CRISPR / Cas14a system was 45 min), different concentrations of Staphylococcus aureus were detected using the DPV measurement method (same as in Example 1).

[0122] like Figure 4 As shown in A, in 5×10 1 -5×10 7 Within the CFU / mL concentration range, the DPV current response signal decreases with increasing Staphylococcus aureus concentration, and the response value of the current signal shows a certain linear relationship with the logarithm of the Staphylococcus aureus concentration. The linear regression equation ( Figure 4 B) is Ip = -3.603lgC + 51.560(R) 2 =0.995), and based on LOD=3S / N, the detection limit of this experiment is 10 CFU / mL.

[0123] Compared with other detection methods reported in the literature, this electrochemical biosensor has a lower LOD and a wider linear range.

[0124] Example 5

[0125] Specificity, repeatability, and stability of electrochemical biosensors:

[0126] Although electrochemical biosensors have high sensitivity and a wide detection range for Staphylococcus aureus, interference from other bacteria cannot be ignored to prevent false positives.

[0127] (1) To verify the specificity of this method, five non-target foodborne pathogens were used, all at a concentration of 1×10⁻⁶. 8 The experiment was conducted at CFU / mL, and the experimental conditions were the same as in Example 4.

[0128] Test results as follows Figure 5 As shown, compared with the blank, only the target bacteria showed a significant change in current response, indicating that the method has good detection specificity.

[0129] (2) Repeatability is one of the important indicators for evaluating the performance of electroactive biosensors. The experimental conditions were the same as in Example 4, and the detection results were as follows: Figure 6 As shown in (A), six parallel experiments were conducted, and the RSD was 1.09%, indicating that the method has good repeatability.

[0130] (3) In addition, the stability of the electrochemical biosensor was also evaluated under the same test conditions as in Example 4;

[0131] The results are as follows Figure 6 As shown in (B), after being stored at 4°C for 8 days, the response current of the electrochemical biosensor showed a slow decreasing trend, and the response current on the 8th day was 92.42% of the initial response current, showing good stability.

[0132] Example 6

[0133] Detection of Staphylococcus aureus in food samples:

[0134] To evaluate the practical applicability and accuracy of the method, 25 mL of sterile milk sample was centrifuged at 10,000 rpm for 20 min. After removing the upper fat layer, the aqueous phase in the centrifuge tube was filtered through a 0.45 μm sterile filter membrane and diluted with sterile PBS at a ratio of 1:20.

[0135] Then, Staphylococcus aureus was inoculated into the milk sample to prepare spiked samples with different amounts of Staphylococcus aureus (5×10⁻⁶). 2 5×10 3 and 5×10 4 (CFU / mL).

[0136] The test bacterial solutions obtained from food samples were detected using plate counting and electrochemical biosensors, and compared with pure bacterial solutions. The results of the two methods were analyzed using the T-test, and the results are shown in Table 2.

[0137] Table 2

[0138]

[0139] It can be seen that there is no significant difference in the detection results between the electrochemical biosensor and the plate counting method (P>0.05). Overall, this method can be used for the detection of actual samples and has certain application value and promotion significance in the detection of Staphylococcus aureus.

[0140] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for detecting Staphylococcus aureus in milk based on CRISPR-Cas14a, characterized in that: include, Preparation of MIL-101(Cr) / PPy@Au nanocomposite suspension; Preparation of PCN-222@AuPt nanozymes; The glassy carbon electrode (GCE) surface was polished, and the MIL-101(Cr) / PPy@Au suspension was drop-coated onto the polished GCE surface to obtain MIL-101(Cr) / PPy@Au / GCE. P-ssDNA was added to MIL-101(Cr) / PPy@Au / GCE and incubated to obtain the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor, wherein the sequence of the P-ssDNA is SH-(CH2)6-TTTTTTTTTTTT-PO4 3- P-ssDNA is fixed to MIL-101(Cr) / PPy@Au / GCE via Au-S bonds; The cDNA and aptamer solutions were mixed, hybridization buffer was added, and the mixture was incubated, annealed to room temperature, and incubated again to obtain the aptamer / cDNA complex. The nucleotide sequence of the aptamer is shown in SEQ ID NO.1, and the nucleotide sequence of the cDNA is shown in SEQ ID NO.

2. The test sample containing live Staphylococcus aureus was added to the aptamer / cDNA complex to form a mixture. The mixture was centrifuged, and the supernatant was added to the Cas14a / sgRNA complex. The mixture was incubated to obtain an activated CRISPR / Cas14a system, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO.

4. After the activated CRISPR / Cas14a system was added to the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor and reacted, the PCN-222@AuPt suspension was added to the biosensor surface. After incubation, the surface was rinsed with sterile, enzyme-free water, and the resulting electrode was immersed in a PBS solution containing 1.0 mM H2O2. The electrochemical signal of the electrode was measured by the DPV method. By controlling the concentration of viable Staphylococcus aureus, the electrochemical signal response value of the electrode was measured using the above method, and a linear relationship between the current signal response value and the logarithm of the Staphylococcus aureus concentration was established. After pretreatment of the milk to be tested, the electrochemical signal of the electrode is measured by the above treatment method. Substituting this signal into the linear relationship, the concentration of Staphylococcus aureus in the milk can be detected.

2. The method as described in claim 1, characterized in that: The preparation of the MIL-101(Cr) / PPy@Au nanocomposite suspension includes, Mix 5.0 g of MIL-101 (Cr), 0.85 g of PVP and 40 mL of ethanol with 210 mL of double-distilled water, sonicate at room temperature for 10 min, add 0.3 mL of pyrrole monomer, and stir at room temperature for 20 min. Then, 3.29 g of (NH4)2S2O8 was added and the reaction was continued for 4 h to obtain a mixed solution; Add 50 mL of AuNPs to the above mixture, stir at 25 °C for 10-12 h, centrifuge, and wash to obtain MIL-101(Cr) / PPy@Au nanocomposite material.

3. The method as described in claim 1 or 2, characterized in that: The preparation of the PCN-222@AuPt nanozyme includes, Disperse 200 mg of ZrOCl2·8H2O and 50 mg of TCPP in a mixture of 50 mL of DMF and 4 mL of acetic acid. The resulting mixture was stored at 120°C for 48 h, then washed three times with DMF, double-distilled water and ethanol, and vacuum dried for 12 h to obtain purple powder PCN-222. 20 mg of PCN-222 was ultrasonically dispersed in 20 mL of double-distilled water, and 20 mL of 0.05 mol / L NaBH4 solution was added. The mixture was stirred at room temperature for 20 min to obtain the stirred mixture. Add 10 mL of H2PtCl6·6H2O solution and 10 mL of HAuCl4·3H2O solution dropwise to the stirred mixture, stir at room temperature for 20-30 min, add 4 mL of NaBH4 solution, and stir at room temperature for 30 min. The resulting mixture was centrifuged, washed, and dried to obtain PCN-222@AuPt nanozyme.

4. The method as described in claim 1, characterized in that: The process involves drop-coating the MIL-101(Cr) / PPy@Au suspension onto the polished GCE surface, wherein the concentration of the MIL-101(Cr) / PPy@Au suspension is 1.0 mg / mL and the addition amount is 15 μL.

5. The method as described in claim 1, characterized in that: The step involves adding P-ssDNA to MIL-101(Cr) / PPy@Au / GCE, wherein the concentration of P-ssDNA is 10 μM and the amount of P-ssDNA modification is 10~15 μL.

6. The method as described in claim 1, characterized in that: The Cas14a / sgRNA complex, wherein the concentration ratio of Cas14a to sgRNA is 1:1.

5.

7. The method as described in claim 1, characterized in that: The activated CRISPR / Cas14a system was added to the P-ssDNA / MIL-101(Cr) / PPy@Au / GCE biosensor for reaction, wherein the reaction temperature was 37℃ and the reaction time was 45 min.

8. The method as described in claim 1, characterized in that: The electrode is immersed in a PBS solution, wherein the PBS concentration is 0.1M, contains 1.0mM H2O2, and has a pH of 7.2; the PCN-222@AuPt suspension is added to the biosensor surface, wherein the concentration of the PCN-222@AuPt suspension is 1.0mg / mL.

9. The method as described in claim 1, characterized in that: The electrochemical signal of the electrode is measured by the DPV method, wherein the DPV measurement parameters are: The test solution was a 0.1M PBS solution with pH 7.2 containing 1.0 mM H2O2; The measured potential is -0.2 to 0.6V, the pulse amplitude is 0.05V, the pulse width is 0.05s, and the pulse period is 0.5s.

10. The method as described in claim 1, characterized in that: The milk to be tested includes cow's milk and goat's milk.

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