System and method for detecting staphylococcus aureus based on nucleic acid aptamer and B-PER-CRISPR signal amplification and application

Abstract

The invention provides a system and a method for detecting staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification and application of the system and the method. On the basis of a B-PER technology, a nucleic acid aptamer is used as a recognition element, staphylococcus aureus is used as a target, magnetic microspheres are used as a probe carrier, CRISPR / Cas12a efficient trans-cleavage activity and the B-PER technology are combined, and a signal amplification system is constructed. The crRNA-Cas12a is specifically combined with a complementary sequence on a linear DNA nano-molecule, cis-cleavage is carried out on a basic group of the complementary sequence on the linear DNA molecule, a crRNA-Cas12a-cleavage chain trimer is released from the linear DNA molecule, and the trimer with trans-cleavage activity can non-specifically cleave a single-stranded DNA fluorescent probe, so that the crRNA-Cas12a-cleavage chain trimer is released from the linear DNA molecule. A fluorescence signal generated after the fluorescence probe is broken can be detected, so that the efficient detection of the staphylococcus aureus is realized.

Description

Technical Field

[0001] The present application relates to the field of label-free molecular probe colorimetric sensing technology, and specifically to a system, method, and application for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification. Background Art

[0002] Staphylococcus aureus (S. aureus) is a pathogen commonly found on the skin and mucous membranes of humans and animals. As one of the leading causes of foodborne illness and nosocomial infections, S. aureus has a wide range of pathogenicity, causing multisystem infections including those of the skin, respiratory tract, and bloodstream. S. aureus toxin A is a key toxin in food poisoning, which can cause severe symptoms and even death. Food contaminated with S. aureus and stored at temperatures between 20°C and 37°C for approximately 48 hours can produce enterotoxins. Studies have shown that while heating at 60°C for one hour effectively kills S. aureus itself, the enterotoxins it produces are heat-stable and remain active, potentially posing a threat to the human body.

[0003] Although plate counts, immunological methods, and molecular biology methods are currently the mainstream means of detecting Staphylococcus aureus, their inherent drawbacks, such as lengthy incubation processes, heavy testing workloads, and limited sensitivity and specificity, limit their applicability. Therefore, the development of rapid nucleic acid detection technologies with high sensitivity and specificity is urgently needed. However, the currently dominant PCR nucleic acid detection strategy has significant shortcomings: susceptibility to laboratory contamination, high cost, potential for false positives, susceptibility to sequence mismatches, and the requirement for highly purified nucleic acid samples to ensure polymerase activity, all of which restrict its widespread applicability. Summary of the Invention

[0004] To address the above technical issues, this proposal provides a system and method for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification, developing a new technology for simple, ultrasensitive, and highly specific detection of Staphylococcus aureus. Based on the bidirectional primer exchange reaction technology, this application uses nucleic acid aptamers as recognition elements, Staphylococcus aureus as the target, and magnetic microspheres as probe carriers. This innovative approach combines the efficient trans-cleavage activity of CRISPR / Cas12a with the bidirectional primer exchange reaction to construct a new signal amplification system.

[0005] Aptamers are single-stranded DNA or RNA molecules derived through ligand system evolution technology that can specifically recognize and bind to a variety of target molecules. Aptamers are typically oligonucleotide sequences with a length range of 60±20 nt. They fold into a specific three-dimensional structure, binding to target molecules with high affinity and further recognizing the target. Aptamers are essentially nucleic acids, offering advantages such as ease of synthesis and modification, excellent stability, and non-immunogenicity. Aptamers are classified into DNA aptamers and RNA aptamers, depending on the target type. The former are more stable, while the latter offer greater structural diversity. Aptamers have demonstrated significant advantages in the field of microbial detection. Compared to protein antibodies, aptamers offer a simpler preparation process, greater flexibility in chemical modification, and superior physicochemical stability. These properties enable the efficient integration of aptamers into biosensor platforms, enabling highly specific recognition and detection of microorganisms.

[0006] The bidirectional primer exchange reaction (B-PER) is an isothermal nucleic acid amplification technique involving the enzyme KF. It achieves target amplification through the autonomous cycling of two symmetrical primers (S1 and S2): the target triggers the extension of the forward primer (S1), generating a new strand T0 containing a binding site for the reverse primer (S2). After T0 binds to S2 and extends, it displaces and releases T1, which then binds to S2 to restart the cycle, forming a self-sustaining cascade reaction. This technique can complete amplification in a short period of time under constant temperature conditions.

[0007] CRISPR / Cas12a (formerly known as Cpf1, now commonly referred to as Cas12a or FnCpf1) is a representative system of the Type V-A subtype of the CRISPR-Cas family. Compared to other CRISPR systems (such as Cas9 and Cas13), its unique properties give it distinct advantages in applications such as gene editing and nucleic acid detection. Cpf1 consists of two components: the Cas12a protein and the CRISPR array. The CRISPR array contains spacer sequences and repeat sequences, the latter of which are derived from exogenous DNA fragments and serve to store immune memory. The Cas12a protein, a multi-domain endonuclease, recognizes and cleaves target DNA that has complementary bases to the spacer sequence. Unlike Cas9, Cas12a requires only a single-stranded guide RNA (sgRNA) for guidance. When cleaving DNA, it produces a 5' sticky overhang, facilitating gene knock-in. Its smaller protein size facilitates delivery, making it widely used in gene editing, pathogen detection, biosensing, and other fields.

[0008] The mechanism of action of Cas12a is divided into adaptation (integration of exogenous DNA fragments), expression (crRNA maturation), and interference (targeting and trans-cleavage) phases. In nucleic acid detection, its trans-cleavage activity is cleverly exploited: target DNA triggers Cas12a to cleave a reporter molecule (such as fluorescently labeled ssDNA), enabling visual signal amplification with sensitivity down to the single-molecule level. The CRISPR array transcribes pre-crRNA, which is then processed by nucleases to mature into sgRNA. This sgRNA binds to the Cas12a protein to form an RNAi (RNP) complex, which possesses targeted cleavage activity. The targeted cleavage (cis-cleavage) RNP complex specifically cleaves double-stranded DNA (dsDNA) at a specific position upstream of the PAM (PAM) by means of complementary base pairing between the sgRNA and the target DNA. Following target DNA cleavage, the Cas12a protein randomly cleaves surrounding ssDNA (single-stranded DNA). This "incidental cleavage" effect (trans-cleavage) can be exploited for signal amplification and is the core principle of nucleic acid detection technology using the Cpf1 system. Its technical advantages include low off-target risk, multi-target editing capabilities, and flexibility to adapt to diverse application scenarios.

[0009] To achieve the above objectives, the present application first provides a system for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification, comprising: an arch-bridge DNA probe, a ternary complex, crRNA, Cas12a protein, dNTPs, a fluorescent probe, and KF polymerase; The arch-bridge DNA probe is formed by base pairing between DNA single strands S1 and S2 through partial sequence complementarity. The ternary complex is formed by the sequential combination of nucleic acid aptamers, connecting strands T, and magnetic microspheres. The nucleic acid aptamers can specifically bind to Staphylococcus aureus. The crRNA / Cas12a binary complex is formed by the combination of Cas12a protein and crRNA. dNTPs are selected from dATP, dCTP, and dTTP. The gene sequence of the nucleic acid aptamer is shown in SEQ ID NO.1, the gene sequence of the connecting chain T is shown in SEQ ID NO.2, the gene sequence of the DNA single chain S1 is shown in SEQ ID NO.3, the gene sequence of the DNA single chain S2 is shown in SEQ ID NO.4, the gene sequence of the crRNA is shown in SEQ ID NO.5, and the gene sequence of the fluorescent probe is TCCCCCCT.

[0010] Preferably, the partial sequences 5'-TTACCC-3', 5'-GGGTAA-3' in the DNA single strand S1 and the partial sequences 5'-TTACCC-3', 5'-GGGTAA-3' in the DNA single strand S2 are base complementary; a convex loop structure is formed between the partial sequence 5'-TTTTTT-3' in the DNA single strand S1 and the partial sequence 5'-TTTTTT-3' in the DNA single strand S2, so that the DNA single strand S1 and the DNA single strand S2 combine to form an arch bridge-shaped DNA probe, i.e., a DAB arch bridge structure.

[0011] Based on a general inventive concept, the present application also provides a method for detecting Staphylococcus aureus for non-diagnostic purposes, comprising the following steps 1 to 6: Step 1. Preparation of arch-bridge DNA probe: Lyophilized powders of single-stranded DNA S1 and single-stranded DNA S2 were prepared into solutions using DEPC water; the two solutions were then mixed, NEB buffer was added, and the mixture was reacted at 90°C to 100°C for 3 to 7 minutes. The mixture was then cooled to room temperature to obtain an arch-bridge DNA probe.

[0012] Preferably, the step of preparing the lyophilized powders of DNA single strand S1 and DNA single strand S2 into solutions using DEPC water comprises: first preparing the lyophilized powders of DNA single strand S1 and DNA single strand S2 into 100 μM stock solutions using DEPC water, and then diluting the stock solutions into 20 μM dilutions using DEPC water. The dilutions are then mixed and reacted to obtain the arch-bridge DNA probe. Furthermore, if the prepared dilutions are not used immediately, they should be stored at -25°C to -18°C for future use.

[0013] The room temperature in this application refers to 20°C to 30°C.

[0014] Step 2, preparation of nucleic acid aptamer dilution solution and nucleic acid aptamer dilution solution: prepare the nucleic acid aptamer lyophilized powder of nucleic acid aptamer into solutions respectively with HEPES buffer, and then add Buffer I buffer respectively to obtain the nucleic acid aptamer dilution solution of nucleic acid aptamer and nucleic acid aptamer dilution solution.

[0015] Step 3, preparation of ternary complex: the dilution of the connecting chain T is mixed with the washed streptavidin magnetic microspheres, and incubated on a magnetic separation stand at 20°C~30°C and an oscillation speed of 280r / min~320r / min for 25min~35min, followed by magnetic separation and discarding the supernatant; the dilution of the nucleic acid aptamer is added, and the mixture is continued to be incubated at 20°C~30°C and an oscillation speed of 280r / min~320r / min for 25min~35min, and then DEPC water is added, vortexed to mix, magnetically separated and the supernatant discarded; DEPC water is then added to resuspend the complex to obtain a ternary complex solution.

[0016] Preferably, before mixing the diluted solution of the connecting chain T with the washed streptavidin magnetic microspheres, the following steps are included: Resuspend the streptavidin magnetic microspheres with a vortex mixer for 25s to 35s, then pipette a portion of the resuspended solution containing the streptavidin magnetic microspheres into an EP tube, place the EP tube on a magnetic separation rack, let it stand, and after the streptavidin magnetic microspheres are adsorbed, discard the supernatant; then add Buffer I buffer solution, use a vortex mixer to fully mix again, separate again with the help of a magnetic separation rack and discard the supernatant; add Buffer I buffer solution again, use a vortex mixer to fully mix, separate with the help of a magnetic separation rack and discard the supernatant to obtain washed streptavidin magnetic microspheres.

[0017] Preferably, in step 3, the molar ratio of the connecting chain T, the nucleic acid aptamer and the streptavidin magnetic microspheres is 1:(1.8-2.0):(0.9-1.2).

[0018] Preferably, the ternary complex solution can be stored at 2-5°C before use.

[0019] Step 4, bidirectional primer exchange reaction: the sample to be tested is mixed with the ternary complex solution and DEPC water, and the supernatant is removed after magnetic separation; then, the arch bridge DNA probe, KF enzyme, dCTP, dTTP, dATP and 10 times the concentration of KF enzyme buffer are added and mixed, first reacted at 35°C to 40°C for 60min to 120min, and then reacted at 70°C to 80°C for 25min to 35min to inactivate the KF enzyme; washed with PBS buffer 3 to 4 times to obtain a solution after the bidirectional primer exchange reaction.

[0020] Preferably, in step 4, the concentration of the ternary complex in the reaction solution is 480 nM to 520 nM, more preferably 500 nM.

[0021] Preferably, the molar ratio of the ternary complex to the arch-bridge-shaped DNA probe is 1:(0.8-1.2).

[0022] Preferably, the molar ratio of dCTP, dTT and dATP is 1:1:1.

[0023] Preferably, the concentration of KF enzyme in the reaction solution is 0.02 U / μL to 0.04 U / μL.

[0024] Preferably, the concentration of Staphylococcus aureus in the sample to be tested is ≥13 CFU / mL.

[0025] Preferably, the reaction time at 35°C to 40°C is 85 min to 95 min.

[0026] More preferably, the reaction is first carried out at 37° C. for 90 min, and then at 75° C. for 30 min.

[0027] Step 5, Cas12a protein cis-cleavage product chain trans-cleavage single-stranded fluorescent probe: DEPC water, Cas12a protein, crRNA, fluorescent probe and 10 times the concentration of Cas12a Buffer buffer were added to the solution after the bidirectional primer exchange reaction, and the reaction was carried out at 35 ° C ~ 40 ° C for 55 min ~ 65 min, and then heated to 70 ~ 80 ° C for 4 min ~ 6 min to inactivate Cas12a protein.

[0028] Preferably, DEPC water, Cas12a protein, crRNA, fluorescent probe and 10 times the concentration of Cas12a Buffer buffer are added to the solution after the bidirectional primer exchange reaction, reacted at 37 ° C for 60 minutes, and then heated to 75 ° C for 5 minutes.

[0029] Step 6: Fluorescence spectrophotometer detection and analysis: DEPC water was added, and the fluorescence spectrophotometer was used for detection to collect the fluorescence emission spectrum at a specific excitation wavelength.

[0030] Based on a general inventive concept, the present application also provides an application of a system for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification in detecting Staphylococcus aureus in food.

[0031] Preferably, the food comprises milk.

[0032] The detection mechanism of this application is as follows: like Figure 1As shown, the arch bridge DNA probe (DAB) is formed by mixing two single-stranded DNAs, S1 and S2. Under specific conditions, the S1 sequence (partial a sequence 5'-TTACCC-3' and part a sequence 5'-GGGTAA-3') and the S2 sequence (partial a sequence 5'-TTACCC-3' and part a sequence 5'-GGGTAA-3') are base-complementary paired, forming a convex loop structure (partial c sequence 5'-TTTTTT-3') in the middle, thereby obtaining the arch bridge DNA probe (DAB); both ends of the obtained product DAB (partial b sequence 5'-CACCGTTA-3' and part a sequence 5'-GGGTAA-3') can undergo primer exchange reaction (PER).

[0033] The magnetic microspheres are grafted with streptavidin, a high-affinity biotin that captures the linker T. Linker T then base-pairs with the aptamer, forming a ternary complex of aptamer / linker T / streptavidin magnetic microspheres. This complex also blocks the target recognition region of the aptamer. In the absence of target, the aptamer-linker T duplex is stable, blocking the subsequent bidirectional primer exchange reaction (B-PER). In the presence of Staphylococcus aureus, the aptamer preferentially competitively binds to the bacterial surface target, dissociating from linker T and releasing free linker T. Free linker T can then initiate the B-PER cascade. The base sequence of linker T (5'-TAACGGTG-3') complements the b sequence (5'-CACCGTTA-3') at the right end of DAB and serves as a primer for the right PER. When KF polymerase and dNTPs are present, the connecting chain T is extended and stops at the designed termination sites (5'-GGGCC-3', 5'-CCCCC-3', 5'-GGGGG-3', 5'-GGCCC-3'), achieving sequence amplification, and the original connecting chain T is extended into a new trigger subchain T0.

[0034] The termination site set in this application is controlled by three nucleotides: dATP, dCTP, and dTTP. Specifically, the DNA chain extension is terminated because the raw material lacks guanine (G), and the cytosine (C) on the corresponding template chain (S1 or S2) cannot be amplified accordingly, and the DNA chain cannot continue to extend; and the 3′ end of DAB is modified with reverse dT to prevent its amplification or cutting of the DNA chain. In addition, the released trigger subchain T0 can pair with a part of the left end of DAB and participate in the primer exchange reaction as a primer, thereby promoting the growth of the corresponding repetitive sequence, and DAB can be recycled. Ultimately, a single-stranded DNA chain (product chain) containing a large number of repetitive sequences is obtained.

[0035] The product chain formed by B-PER contains numerous repeats, with each three repeats (a total of 18 bases) capable of complementary pairing with the designed crRNA. Magnetic microspheres on the product chain also facilitate magnetic separation and purification. When the Cas12a protein and crRNA are added to the product chain, the crRNA recognizes the three repeats in the product chain (three repeats form base-pairing with one crRNA), triggering the Cas12a-crRNA complex and activating its cis-cleavage activity, specifically cleaving the activation strand (ssDNA). The cleavage product forms a crRNA / Cas12a / cleavage strand trimer. This active complex, through allosteric interaction, activates trans-cleavage, nonspecifically degrading the fluorescent reporter molecule. Trans-cleavage separates the fluorophore (F) and quencher (Q), restoring the fluorescence signal.

[0036] Compared with the prior art, this application has the following beneficial effects: This system builds a one-tube constant temperature system based on nucleic acid aptamer specific recognition, B-PER amplification and CRISPR / Cas12a signal amplification to detect Staphylococcus aureus in food, achieving high sensitivity and high specificity. Specifically: (1) Achieving high sensitivity: Construct an amplification system based on the efficient trans-cleavage activity of CRISPR / Cas12a and bidirectional primer exchange reaction amplification. The product chain containing a large number of repeat sequences generated by the bidirectional primer exchange reaction can bind to the complementary sequence of the crRNA / Cas12a dimer (the binding mode is the complementary pairing of three repeat sequences with one crRNA sequence). After binding, the nuclease domain of Cas12a is activated, and the single-stranded DNA bound to the crRNA / Cas12a protein is cis-cleaved. After cleavage, the crRNA / Cas12a / cleavage chain trimer is released and the trans-cleavage activity is activated. Then, the DNA fluorescent probe is cut off by the trans-cleavage action, the fluorescent group F is away from the quenching group Q, and the fluorescence signal is restored.

[0037] (2) Achievement of high specificity: Nucleic acid aptamers have the ability to specifically recognize targets. The nucleic acid aptamer (5'-CACCGCCACCGTGCTACAAC-3') can be complementary paired with the connecting chain T (5'-GTTGTAGCACGGTAACGGTG-3') on the surface of the magnetic microspheres. The active region of the nucleic acid aptamer is blocked to prevent the generation of high background in the absence of targets. The S1 part of the DNA arch bridge structure (5'-CACCGTTA-3') can bind to the connecting chain T part (5'-TAACGGTG-3'). After the nucleic acid aptamer is separated from the connecting chain T, the DNA arch bridge structure binds to the connecting chain T and performs a B-PER reaction, so that the connecting chain T is amplified into a product chain. The product chain on the surface of the magnetic microspheres can be obtained by magnetic separation and then subjected to subsequent CRISPR reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. Obviously, the drawings described below are only some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without paying any creative work.

[0039] Figure 1 This is a diagram of the detection principle of the Staphylococcus aureus detection system in this application.

[0040] Figure 2 This is a diagram showing the feasibility analysis results of the Staphylococcus aureus detection method of Example 2.

[0041] Figure 3 This is a graph showing the specificity of the Staphylococcus aureus detection system for bacterial species in Example 3.

[0042] Figure 4 This is a graph showing the detection results under different KF enzyme concentration test conditions in Example 4.

[0043] Figure 5 This is a graph showing the detection results under different B-PER reaction time conditions in Example 5.

[0044] Figure 6 Graph showing the test results under different aptamer-linker chain T modified magnetic microsphere concentration conditions in Example 6.

[0045] Figure 7 This is the fluorescence spectrum analysis curve of Example 7 for detecting Staphylococcus aureus at different concentrations.

[0046] Figure 8 This is the signal saturation curve for detecting Staphylococcus aureus at different concentrations in Example 7.

[0047] Figure 9 This is the calibration curve for different concentrations of Staphylococcus aureus in Example 7. DETAILED DESCRIPTION

[0048] The embodiments described in this specification are only for explaining the present application and are not intended to limit the present application.

[0049] For simplicity, this application only explicitly discloses certain numerical ranges. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, although not explicitly stated, each point or individual value between the endpoints of a range is included in the range. Thus, each point or individual value may serve as its own lower limit or upper limit and be combined with any other point or individual value, or with other lower limits or upper limits, to form an unspecified range.

[0050] The percentage sign "%" involved in this application, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0051] The weight parts recorded in this application can be weight units known in the art such as μg, mg, g, kg, etc., or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0052] The present application will be further described below with reference to the examples. It should be understood that these examples are intended to be illustrative only, as various modifications and variations will be apparent to those skilled in the art within the scope of the present disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by mass, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0053] The oligonucleotide chains and related chemical reagents involved in this application were purchased from Shanghai Sangon Biotechnology Co., Ltd., as shown in Table 1.

[0054]

[0055] Among them, the underlined and tilted nucleotide sequence in the sequence of the connecting chain T is combined with the partial sequence of the DAB arch bridge structure; in S1 and S2, the bold sequence represents the convex loop domain that forms the DAB arch bridge; after the DAB arch bridge is formed, the underlined and tilted nucleotide sequence in S1 is combined with the connecting chain T; the underlined partial sequence in crRNA is the product chain binding part formed with B-PER.

[0056] Example 1 Detection of Staphylococcus aureus by a signal amplification system based on nucleic acid aptamers and B-PER-CRISPR (1) DNA sequence and structure preprocessing Preparation of S1 and S2 solutions: Prepare 100 µM concentrated solutions of the lyophilized powders of S1 and S2 sequences using diethyl pyrocarbonate (DEPC). Transfer 10 µL of each to a PCR tube and dilute to a 20 µM solution by adding 40 µL of DEPC water. Store at -20°C.

[0057] Connector T and aptamer probes: Prepare 100 µM solutions of each of the aptamer and 100 µM lyophilized powders in 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (20 mM HEPES, 20 mM NaCl, 8 mM MgCl₂·6H₂O, 1 mM KCl, pH 7.5). Transfer 10 µL of the aptamer T solution to a PCR tube and dilute to a 10 µM solution with 90 µL of Buffer I (1 M NaCl, 1 mM Na₂EDTA, 10 mM Tris-HCl, 0.01%–0.1% Tween-20, pH 7.5). Transfer 10 µL of the aptamer solution to a PCR tube and dilute to a 20 µM solution with 40 µL of Buffer I. Store at -20°C until use.

[0058] Preparation of crRNA and fluorescent probe solutions: Use DEPC water to prepare 100µM concentrated solutions of crRNA and fluorescent probe lyophilized powder, respectively. Take 10µL of the crRNA concentrated solution in a PCR tube, add 90µL of DEPC water to dilute it to a 10µM solution, and store it at -20°C. Take 10µL of the fluorescent probe concentrated solution and add 90µL of DEPC water in a light-proof centrifuge tube to prepare a 10μM fluorescent probe solution. Store it at -20°C in the dark.

[0059] (2) Pretreatment of Staphylococcus aureus Resuscitated strains were inoculated into beef extract peptone broth (5g NaCl, 3g beef extract, 10g peptone) and cultured at 37°C with shaking at 220 rpm. One mL of the culture was centrifuged at 300 rpm for 5 minutes. The supernatant was discarded, and the pellet was washed twice with 1× PBS buffer (137mM NaCl₂, 7mM KCl, 10mM Na₂HPO₄, 1.8mM KH₂PO₄, pH=7.4). The pellet was resuspended in 1mL of 1× PBS and stored at 4°C until further use. Viable bacteria were counted using a 10-fold serial dilution method. 100μL of the dilution was spread onto beef extract peptone plates. After incubation at 37°C for 24 hours, the colonies were counted and expressed as CFU / mL.

[0060] (3) Formation of DNA arch loop structure 5 μL of S1 solution (20 μM), 5 μL of S2 solution (20 μM), and 10 μL of NEB buffer 2 (10×, 10 mM MgCl 、 Add 1mM DTT, 50mM NaCl, 10mM Tris-HCl, pH 7.9 to 80μL of DEPC water. Heat this mixture to 95°C in a water bath and maintain this temperature for 5 minutes, then cool to room temperature to obtain DAB. Store DAB at -20°C for subsequent use.

[0061] (4) Aptamer-linker chain T modified magnetic microsphere solution Resuspend the streptavidin magnetic microspheres using a vortex mixer for 30 seconds, then pipette 50 µL (10 µM) into an EP tube. Place the tube on a magnetic separation rack and wait for a while until the magnetic microspheres are adsorbed, then discard the supernatant. Add 100 µL of Buffer I, mix thoroughly again using a vortex mixer, separate the tube using a magnetic separation rack, and discard the supernatant. Repeat this wash step once. Next, add 50 µL (10 µM) of Connector T (diluted in Buffer I), incubate at 25°C with shaking at 300 rpm for 30 minutes, then magnetically separate and discard the supernatant. Add 100 µL of DEPC water, vortex to mix, magnetically separate, and discard the supernatant. Repeat this wash step once. Add 50 µL (20 µM) of the aptamer (diluted in Buffer I) and continue incubating at 25°C with shaking at 300 rpm for 30 minutes. Afterwards, repeat the steps of adding 100 µL of DEPC water, vortexing, magnetic separation, and discarding the supernatant once for a final wash. Finally, add 50 µL of DEPC water to resuspend the magnetic microspheres to obtain the aptamer-linker T-modified magnetic microsphere solution, which was stored at 4°C for subsequent experiments.

[0062] (5) B-PER reaction Take 20 μL of the sample to be tested and 5 μL of nucleic acid aptamer-linker chain T modified magnetic microsphere solution (1 μM) and add them to an EP tube. Add 7.5 μL of DEPC water. After magnetic separation, remove 20 μL of supernatant, then add 5 μL of DAB (1 μM), 2 μL of KF enzyme (0.5 U / μL), 2.5 μL of KF enzyme buffer (10×), and 3 μL of dNTPs (10 mM each of dCTP, dTTP, and dATP) to the tube. React at 37°C for 90 min, then incubate in a water bath at 75°C for 30 min, inactivate the KF enzyme, and wash three times with PBS 1× buffer to obtain 10 μL of reaction solution.

[0063] (6) Cas12a protein cis-cleavage product chain trans-cleavage single-stranded fluorescent probe and result fluorescence analysis 9 μL of DEPC water, 3 μL of Cas12a (1 μM), 3 μL of Cas12a Buffer (10×), 3 μL of crRNA (10 μM), and 2 μL of fluorescent probe (10 μM) were added to the EP tube containing the above reaction solution (10 μL). The reaction was incubated at 37°C in the dark for 60 minutes. Afterwards, the Cas protein was inactivated in a 75°C water bath for 5 minutes. 170 μL of DEPC water was added to the EP tube. The reaction system was vortexed to mix. The fluorescence emission spectrum was collected using a fluorescence spectrophotometer at an excitation wavelength of 480 nm. The maximum absorption peak at F520 nm was obtained and the value was recorded.

[0064] Example 2 Feasibility Analysis of Staphylococcus aureus Detection Method In order to verify the feasibility of nucleic acid aptamers and B-PER-CRISPR system for detecting Staphylococcus aureus, 5 groups of key control experiments (controlled by a single variable) were set up. The feasibility analysis results are as follows: Figure 2 As shown. Figure 2 In the figure, the curves are from top to bottom: Curve 1 is the normal experimental group, that is, the method of Example 1 is used, and the Staphylococcus aureus solution is directly used instead of the sample to be tested; Curve 2 is the lack of Cas12a protein, and other conditions and factors remain unchanged except for the addition of the corresponding volume of DEPC water; Curve 3 is the case where KF enzyme is not added, the corresponding volume of DEPC water is added, and other conditions and factors are not changed; Curve 4 does not add crRNA, and the corresponding volume of DEPC water is added, and other conditions and factors remain unchanged; Curve 5 is the blank group, that is, Staphylococcus aureus solution is not added, and the corresponding volume of DEPC water is used instead, and the experimental system is complete.

[0065] Curve 1 can normally identify the target, trigger the aptamer to release the connecting chain T, enter B-PER, and the resulting product chain is complementary to the crRNA base pairing, which can trans-cut the fluorescent probe and restore the fluorescent signal. Curve 2 has no Cas12a protein, cannot form an active cleavage complex, loses the trans-cleavage function, and has no fluorescent signal. Curve 3 has no KF enzyme and cannot carry out a large amount of B-PER reaction, resulting in insufficient product chain generation and no large amount of fluorescent signal. Curve 4 is unable to target Cas12a due to the lack of crRNA, causing trimer assembly to fail and no fluorescent signal to be generated. Curve 5 does not have a target added, and the connecting chain T is blocked by the nucleic acid aptamer. Therefore, the DNA arch bridge structure cannot bind to the connecting chain T, resulting in the inability to cut the fluorescent probe and low fluorescence intensity.

[0066] Example 3 Analysis of the Specificity of the Staphylococcus aureus Detection System for Targets To evaluate the specificity of the detection system for Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella, a mixture of three microorganisms and blank were used as controls, and the fluorescence signal was detected at F520nm under the optimal conditions. The results are shown in Figure 2. Figure 3 As shown in the figure, the fluorescence values ​​of Salmonella and Escherichia coli were close to the blank control level, while Staphylococcus aureus and its mixture showed a significantly enhanced signal. This result shows that the aptamer-based detection system has good specificity and recognition ability for the target bacteria (Staphylococcus aureus).

[0067] Example 4 Exploration of the optimal concentration of KF enzyme To ensure that the single-stranded DNA (ssDNA) product generated by the B-PER reaction has sufficient length to meet the requirements of the downstream CRISPR / Cas12a system for effective recognition and cleavage of the target sequence. This example systematically optimizes the concentration of the key extension enzyme Klenow Fragment (exo-) (KF enzyme). Taking into account that the concentration of KF enzyme directly affects the efficiency of primer extension and the length distribution of the product chain during isothermal amplification, this example sets up a KF enzyme concentration gradient experimental group covering different activity units (0.01U / μL, 0.02U / μL, 0.04U / μL, 0.06U / μL), and observes the fluorescence signal detected at F520nm in each experiment; the results are as follows Figure 4 As shown. Figure 4 It can be seen that when the KF enzyme concentration is 0.04 U / μL, the fluorescence signal reaches the maximum. Therefore, the KF enzyme concentration under the optimal reaction conditions is 0.04 U / μL.

[0068] Example 5 Exploration of the Optimal Reaction Time of B-PER Reaction Reaction time is a key kinetic parameter affecting the efficiency of the B-PER reaction. To ensure sufficient nucleic acid binding and achieve optimal detection performance, this example systematically conducted a reaction time parameter optimization experiment. By setting up a gradient experiment group covering different B-PER reaction time lengths (30 min, 60 min, 90 min, 120 min, 150 min), the fluorescence signal at F520nm was observed in each experiment. The results are shown in Figure 2. Figure 5 As shown in the figure, when the reaction time is 90 minutes, the fluorescence signal is the largest. Therefore, the optimal reaction time is determined to be 90 minutes.

[0069] Example 6 Exploration of the Optimal Concentration of the Aptamer-Linker T Modified Magnetic Microsphere Solution To optimize the efficiency of the aptamer-linker T-modified magnetic microspheres (i.e., probes) and reduce unnecessary loss, this example systematically optimized the key parameter of probe concentration. By setting up multiple sets of experimental conditions covering different probe concentration gradients (250nM, 500nM, 1000nM, 2000nM), the aim was to accurately explore the influence of probe concentration on its performance in the target. The results are shown in Figure 2. Figure 6 As shown in Figure 2, when the probe concentration is 500 nM, the fluorescence signal reaches its maximum value. It can be concluded that the probe concentration under the optimal reaction conditions is 500 nM.

[0070] Example 7 Detection and Analysis of Different Concentrations of Bacteria Under the optimized system of optimal KF enzyme concentration, B-PER reaction time, and aptamer-linker chain T modified magnetic microspheres, a signal amplification system based on aptamers and B-PER-CRISPR was set up to amplify the concentrations of Staphylococcus aureus suspension (10 2 ~10 10 CFU / mL) and observed the fluorescence signal of the experimental group. Figure 7 This is a fluorescence spectrum analysis curve diagram of Staphylococcus aureus suspension with different concentrations. The corresponding Staphylococcus aureus suspension concentrations from top to bottom are: 10 10 CFU / mL, 10 9 CFU / mL, 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL; the fluorescence spectrum intensity increases with the increase of bacterial concentration. Figure 8The saturation curve of the detection signal of Staphylococcus aureus at different concentrations at 520 nm is shown. Figure 8 It can be seen that when the concentration of Staphylococcus aureus is lower than 1.0×10 6 CFU / mL, the fluorescence signal at 520 nm was positively correlated with the bacterial concentration (R 2 =0.998). Figure 9 The calibration curves of Staphylococcus aureus at different concentrations are shown in Figure 2. Figure 9 It can be seen that the concentration of Staphylococcus aureus is 10 2 ~10 6 There was a significant linear correlation between CFU / mL, and the regression equation was: ΔF 520 nm =571.3logC+3210.4(R²=0.9976), where ΔF 520nm represents the relative fluorescence intensity at 520 nm; logC (CFU / mL) is the logarithm of the concentration of Staphylococcus aureus. 2 to 10 6 Within the range of CFU / mL, the relative fluorescence intensity value was linearly correlated with the logarithm of the Staphylococcus aureus concentration, and the detection limit was 13 CFU / mL (calculation standard: LOD=3σ / S; σ is the standard deviation of the blank solution, and S is the linear slope).

[0071] Example 8 Detection of Staphylococcus aureus in milk To investigate the detection performance of this method in actual sample detection, 10-fold diluted milk was used as the matrix and Staphylococcus aureus was added to evaluate the detection performance of the aptamer and B-PER-CRISPR system in actual samples.

[0072] Containing 1.0×10 2 , 1.0×10 3 and 1.0×10 4 The actual sample was prepared by using a Staphylococcus aureus bacterial solution with a CFU / mL as the mother solution and adding it into sterilized pure milk.

[0073] The experimental results are shown in Table 2. The average recovery rate of actual sample detection ranged from 98.9% to 100.7%, indicating that this method has good detection performance for Staphylococcus aureus in milk samples.

[0074]

[0075] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present application, and such modifications or substitutions should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A system for detecting Staphylococcus aureus based on nucleic acid aptamers and B-PER-CRISPR signal amplification, characterized in that: include: Arch-bridge DNA probe, ternary complex, crRNA, Cas12a protein, dNTPs, fluorescent probe, and KF polymerase; The arch bridge DNA probe is formed by base pairing between a DNA single strand S1 and a DNA single strand S2 through partial sequence complementarity; the ternary complex is formed by sequentially combining a nucleic acid aptamer, a connecting strand T, and a magnetic microsphere; the nucleic acid aptamer can specifically bind to Staphylococcus aureus; and the dNTPs are selected from dATP, dCTP, and dTTP. The gene sequence of the nucleic acid aptamer is shown in SEQ ID NO.1, the gene sequence of the connecting chain T is shown in SEQ ID NO.2, the gene sequence of the DNA single strand S1 is shown in SEQ ID NO.3, the gene sequence of the DNA single strand S2 is shown in SEQ ID NO.4, the gene sequence of the crRNA is shown in SEQ ID NO.5, and the gene sequence of the fluorescent probe is TCCCCCCT.

2. A method for detecting Staphylococcus aureus for non-diagnostic purposes, characterized in that: The following steps are involved: Step 1, preparation of an arch-bridge-shaped DNA probe: freeze-dried powders of DNA single-strand S1 and DNA single-strand S2 are prepared into solutions using DEPC water; the two solutions are then mixed, NEB buffer is added, and the mixture is reacted at 90°C to 100°C for 3 minutes to 7 minutes, and cooled to room temperature to obtain the arch-bridge-shaped DNA probe; Step 2, preparation of nucleic acid aptamer dilution solution and nucleic acid aptamer dilution solution: using HEPES buffer to prepare solutions of nucleic acid aptamer lyophilized powder and nucleic acid aptamer lyophilized powder, and then adding Buffer I buffer to obtain nucleic acid aptamer dilution solution and nucleic acid aptamer dilution solution; Step 3, preparation of ternary complex: the dilution of the connecting chain T is mixed with the washed streptavidin magnetic microspheres, and incubated on a magnetic separation stand at 20°C to 30°C at an oscillation speed of 280r / min to 320r / min for 25min to 35min, followed by magnetic separation and discarding the supernatant; the dilution of the nucleic acid aptamer is added, and the mixture is continued to be incubated at 20°C to 30°C at an oscillation speed of 280r / min to 320r / min for 25min to 35min, and then DEPC water is added, vortexed to mix, magnetically separated and the supernatant discarded; DEPC water is then added to resuspend the complex to obtain a ternary complex solution; Step 4, bidirectional primer exchange reaction: the sample to be tested is mixed with the ternary complex solution and DEPC water, and the supernatant is removed after magnetic separation; then the arch bridge DNA probe, KF enzyme, dCTP, dTTP, dATP and 10 times the concentration of KF enzyme buffer are added and mixed, first reacted at 35°C to 40°C for 60min to 120min, and then reacted at 70°C to 80°C for 25min to 35min to inactivate the KF enzyme; washed with PBS buffer 3 to 4 times to obtain a solution after bidirectional primer exchange reaction; Step 5, Cas12a protein cis-cleavage product chain trans-cleavage single-stranded fluorescent probe: DEPC water, Cas12a protein, crRNA, fluorescent probe and 10 times the concentration of Cas12a Buffer buffer were added to the solution after the bidirectional primer exchange reaction, and the reaction was carried out at 35 ° C ~ 40 ° C for 55 min ~ 65 min, and then heated to 70 ° C ~ 80 ° C for 4 min ~ 6 min to inactivate Cas12a protein; Step 6: Fluorescence spectrophotometer detection and analysis: DEPC water was added, mixed evenly, and detected using a fluorescence spectrophotometer to collect the fluorescence emission spectrum at a specific excitation wavelength; The gene sequence of the nucleic acid aptamer is shown in SEQ ID NO.1, the gene sequence of the connecting chain T is shown in SEQ ID NO.2, the gene sequence of the DNA single strand S1 is shown in SEQ ID NO.3, the gene sequence of the DNA single strand S2 is shown in SEQ ID NO.4, the gene sequence of the crRNA is shown in SEQ ID NO.5, and the gene sequence of the fluorescent probe is TCCCCCCT.

3. The method according to claim 2, characterized in that In step 3, before mixing the dilution solution of the connecting chain T with the washed streptavidin magnetic microspheres, the following steps are included: Resuspend the streptavidin magnetic microspheres with a vortex mixer for 25s to 35s, then pipette a portion of the resuspended solution containing the streptavidin magnetic microspheres into an EP tube, place the EP tube on a magnetic separation rack, let it stand, and after the streptavidin magnetic microspheres are adsorbed, discard the supernatant; then add Buffer I buffer solution, use a vortex mixer to fully mix again, separate again with the help of a magnetic separation rack and discard the supernatant; add Buffer I buffer solution again, use a vortex mixer to fully mix, separate with the help of a magnetic separation rack and discard the supernatant to obtain the washed streptavidin magnetic microspheres.

4. The method according to any one of claims 2 to 3, characterized in that In step 4, the molar ratio of the ternary complex to the arch-bridge-shaped DNA probe is 1:(0.8-1.2).

5. The method according to any one of claims 2 to 3, characterized in that: In step 4, the concentration of the KF enzyme in the reaction solution is 0.02 U / μL to 0.04 U / μL.

6. The method according to any one of claims 2 to 3, characterized in that: In step 4, the concentration of Staphylococcus aureus in the test sample is ≥13 CFU / mL.

7. The method according to any one of claims 2 to 3, characterized in that: In step 4, the reaction time at 35°C to 40°C is 85 min to 95 min.

8. The method according to any one of claims 2 to 3, characterized in that: In step 4, the concentration of the ternary complex in the reaction solution is 480 nM to 520 nM.

9. The method according to any one of claims 2 to 3, characterized in that: The excitation wavelength is 480 nm.

10. Use of the system for detecting Staphylococcus aureus according to claim 1 or the method for detecting Staphylococcus aureus for non-diagnostic purposes according to any one of claims 2 to 9 in detecting Staphylococcus aureus in food, characterized in that: The food includes milk.

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